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The contribution of endogenous cellulase to the cellulose digestion in the gut of earthworm (Pheretima hilgendorfi: Megascolecidae)
Abstract
Cellulase activity has been detected in the digestive tract of earthworms. However, it has not been well clarified whether the origin of those cellulases are the earthworm themselves or the symbionts. In our study, zymogram analysis suggests that one cellulase (endo-β-1,4-glucanase, EC3.2.1.4) mainly works to digest cellulose in Pheretima hilgendorfi. To identify the cellulase in P. hilgendorfi, we carried out cDNA cloning of the cellulase gene from the digestive tract. A novel cellulase gene was identified from the gut of earthworm. The cDNA encoding cellulase of P. hilgendorfi (phhEG) is 1606 bp with an open reading frame encoding a protein of 449 amino acid residues. The deduced amino acid sequence of P. hilgendorfi cellulase showed higher homology to invertebrate cellulases than bacterium cellulases belonging to the glycosyl hydrolase family (GHF) 9. The phhEG gene was detected in intestinal epithelium cell of midforegut using Northern blot and in situ hybridization. Similarly, specific cellulase activity against carboxymethyl cellulose (CMC) was significantly higher in midforegut tissue. Recombinant phhEG produced by wheat germ cell-free protein synthesis system had a cellulase activity which degrade CMC. In zymogram analysis, the molecular size of cellulase was detected as a single band of 51 kDa from the whole gut contents extracts of P. hilgendorfi, and was very similar to the predicted molecular size of the mature phhEG protein. These results strongly suggested that the earthworm has the capacity to produce the endogenous and functional cellulase around the midforegut, and use this cellulase for their cellulose digestion with the support of intestinal caecum.
... This robust dataset underpins our phylogenetic inferences, providing insights into the evolutionary relationships of these enzymes. Reciprocal BLASTp analyses of endo-β-1,4-glucanase I/II protein sequences primarily obtained from Clitellata revealed significant similarity to previously characterized and closely related cellulases (GH9 family) from earthworms such as Metaphire hilgendorfi [40] and Eisenia spp. [41,42]. ...
... Therefore, this hypothesis was rejected. Phylogenetic and in silico structural analyses revealed that E. albidus endoβ-1,4-glucanases are homologous to a few previously described endo-β-1,4-glucanases (cellulases) from earthworm species such as Metaphire hilgendorfi, Eisenia fetida, and E. andrei [40][41][42]. Moreover, transcriptomics data derived from other clitellate species and integrated into phylogenetic analysis demonstrated that, in addition to the aforementioned earthworm species, which provided initial evidence for endogenous cellulase production in clitellates, GH9 endo-β-1,4-glucanases are present in other members of Clitellata, including Capilloventridae, Phreodrilidae, Naididae, Lumbriculidae, and Randiellidae. ...
To assess the impact of Enchytraeidae (potworms) on the functioning of the decomposer system, knowledge of the feeding preferences of enchytraeid species is required. Different food preferences can be explained by variations in enzymatic activities among different enchytraeid species, as there are no significant differences in the morphology or anatomy of their alimentary tracts. However, it is crucial to distinguish between the contribution of microbial enzymes and the animal’s digestive capacity. Here, we computationally analyzed the endogenous digestive enzyme genes in Enchytraeus albidus. The analysis was based on RNA-Seq of COI-monohaplotype culture (PL-A strain) specimens, utilizing transcriptome profiling to determine the trophic position of the species. We also corroborated the results obtained using transcriptomics data from genetically heterogeneous freeze-tolerant strains. Our results revealed that E. albidus expresses a wide range of glycosidases, including GH9 cellulases and a specific digestive SH3b-domain-containing i-type lysozyme, previously described in the earthworm Eisenia andrei. Therefore, E. albidus combines traits of both primary decomposers (primary saprophytophages) and secondary decomposers (sapro-microphytophages/microbivores) and can be defined as an intermediate decomposer. Based on assemblies of publicly available RNA-Seq reads, we found close homologs for these cellulases and i-type lysozymes in various clitellate taxa, including Crassiclitellata and Enchytraeidae.
... Earthworms also rely on dual digestive mechanisms involving both endogenous and microbial cellulase for lignocellulose degradation. Few reports demonstrate that earthworms possess complete enzymatic machinery for glycosidic enzymes [131][132][133][134]. Glycolytic activities in the gut have been detected in the earthworm species Pontoscolex corethrums [118], Millsonia anomala [132], Polypheretima elongata [133], Hormogaster elisae [134,135], Hyperiodrilus africaizus, Dichogaster terrae nigrae [135], Pheretima hilgendorfi [136]. Nacetylglucosaminase, laminarinase, laminaribiase activities are found to be most potent within the gut of these earthworm species except Pheretima hilgendorfi. ...
... Week activities of other glycolytic enzymes within the gut of earthworms reflect their dependency on microbial cellulases for degradation of substrates like mannan and cellulose [134]. In the case of Pheretima hilgendorfi, endo-β-1, 4-glucanase contributes to the degradation of cellulose, and a novel cellulase gene (phhEg) has been detected from this species [136]. ...
Cellulosic plant materials are a reliable source of renewable energy. Cellulose-based plant materials are now being used for bioenergy production as alternatives to fossil fuels. The traditional way of converting lignocellulosic materials to ethanol and other bioenergy is an expensive and environmentally unsafe process. Several research works have been conducted to find outsource of low-cost cellulolytic enzymes. Initially, fungal species were considered as sources of cellulolytic enzymes. Later on, several studies showed that bacterial species are a more potent source of cellulose-degrading enzymes. Phytophagous lower invertebrates are a good source of cellulolytic gut bacteria. They utilize a wide variety of plant materials as their food source. In this review, thorough literature studies have been made to explore the invertebrate groups that are novel sources of cellulolytic gut bacteria with high efficacy for enzyme production. This study also encompasses a brief description of cellulose, the activity, and cellulase enzyme application in industrial aspects.
... pruriens, corn stubble, and control), i.e., the invasive species displayed faster growth than the native species when nutritional quality improved. This suggests that P. corethrurus consumes and degrades a greater variety of organic materials given its greater ability (efficiency), evidenced by: a) producing endogenous cellulases [59][60][61][62]; b) its association with the gut microbiota [63][64][65][66]; c) gene expression (transcriptome) that contribute to the adaptation of its digestive system [65]; d) improving its digestion efficiency according to the type of cecum [59,67]; and e) its association with nephridial bacteria [50,68,69]. ...
... pruriens, corn stubble, and control), i.e., the invasive species displayed faster growth than the native species when nutritional quality improved. This suggests that P. corethrurus consumes and degrades a greater variety of organic materials given its greater ability (efficiency), evidenced by: a) producing endogenous cellulases [59][60][61][62]; b) its association with the gut microbiota [63][64][65][66]; c) gene expression (transcriptome) that contribute to the adaptation of its digestive system [65]; d) improving its digestion efficiency according to the type of cecum [59,67]; and e) its association with nephridial bacteria [50,68,69]. ...
The presence of earthworm species in crop fields is as old as agriculture itself. The earthworms Pontoscolex corethrurus (invasive) and Balanteodrilus pearsei (native) are associated with the emergence of agriculture and sedentism in the region Amazon and Maya, respectively. Both species have shifted their preference from their natural habitat to the cropland niche. They contrast in terms of intensification of agricultural land use (anthropic impact to the symbiotic soil microbiome). P. corethrurus inhabits conventional agroecosystems, while B. pearsei thrives in traditional agroecosystems, i.e., P. corethrurus has not yet been recorded in soils where B. pearsei dwells. The demographic behavior of these two earthworm species was assessed in the laboratory over 100 days, according to their origin (OE; P. corethrurus and B. pearsei) food quality (FQ; soil only, maize stubble, Mucuna pruriens), and soil moisture (SM; 25, 33, 42%). The results showed that OE, FQ, SM, and the OE x FQ interaction were highly significant for the survival, growth, and reproduction of earthworms. P. corethrurus showed a lower survival rate (> mortality). P. corethrurus survivors fed a diet of low-to-intermediate nutritional quality (soil and stubble maize, respectively) showed a greater capacity to grow and reproduce; however, it was surpassed by the native earthworm when fed a high-quality diet (M. pruriens). Besides, P. corethrurus displayed a low cocoon hatching (emergence of juveniles). These results suggest that the presence of the invasive species was associated with a negative interaction with the soil microbiota where the native species dwells, and with the absence of natural mutualistic bacteria (gut, nephridia, and cocoons). These results are consistent with the absence of P. corethrurus in milpa and pasture-type agricultural niches managed by peasants (agroecologists) to grow food regularly through biological soil management. Results reported here suggest that P. corethrurus is an invasive species that is neither wild nor domesticated, that is, its eco-evolutionary phylogeny needs to be derived based on its symbionts.
... However, it should be noted that the 1-2-year-old Calluna used in Latter's experiment was pretreated with enzymes (cellulase and pectinase, for details see: Latter, 1977;Latter and Howson, 1978), in contrast to 2-5-year-old plant material, which was naturally exposed to microbial activity longer period. The capacity to produce endogenous and functional cellulase in the gut has been shown in the earthworm Pheretima hilgendorfi (Nozaki et al., 2009). In the case of Enchytraeidae, further research is needed at the molecular level. ...
... Molecular methods such as DNA barcoding (Hebert et al., 2003) could be applied to improve the identification of species (Schlegel et al., 1991;Erséus et al., 2010;Martinsson and Erséus, 2014;Vivien et al., 2015). Techniques such as RT-PCR (Reverse Transcription-Polymerase Chain Reaction) may also help to distinguish the origin of enzymes that are active in the worm gut (Nozaki et al., 2009). ...
... However, the existence of cellulase encoding genes in the genome of Chlamydomonas is of particular scientific interest, since it cannot be explained by the requirement of cell wall rearrangement, because the cell wall of this organism is solely composed of hydroxyproline-rich glycoproteins (Adair and Snell, 1990;Horne et al., 1971;Miller et al., 1972). In silico analysis of amino acid sequences of the identified hydrolytic enzymes (CrCel9B/ CrCel9C) [3, Fig. 1B, C] revealed that these proteins belong to glycoside hydrolase family 9 (GHF9/subgroup E2) and their catalytic domains show a high homology to metazoan endo-β-1,4-glucanases (Nozaki et al., 2009). There are two distantly related families of the GHF9 gene: subgroup E1 is confined to bacteria (Tomme et al., 1995), ...
... Endogenous GHF9 genes were reported in the animal phyla Arthropoda (Dismukes et al., 2008;Nozaki et al., 2009;Watanabe et al., 1998), the Mollusca (Suzuki et al., 2003) and ...
... However, it should be noted that the 1-2-year-old Calluna used in Latter's experiment was pretreated with enzymes (cellulase and pectinase, for details see: Latter, 1977;Latter and Howson, 1978), in contrast to 2-5-year-old plant material, which was naturally exposed to microbial activity longer period. The capacity to produce endogenous and functional cellulase in the gut has been shown in the earthworm Pheretima hilgendorfi (Nozaki et al., 2009). In the case of Enchytraeidae, further research is needed at the molecular level. ...
... Molecular methods such as DNA barcoding (Hebert et al., 2003) could be applied to improve the identification of species (Schlegel et al., 1991;Erséus et al., 2010;Martinsson and Erséus, 2014;Vivien et al., 2015). Techniques such as RT-PCR (Reverse Transcription-Polymerase Chain Reaction) may also help to distinguish the origin of enzymes that are active in the worm gut (Nozaki et al., 2009). ...
... The mucus is a necessary highly energetic investment for the earthworm that facilitates the transport of the ingested material through the alimentary canal, protects the gut tissue from damage by sharp-edged soil or organic particles, and activates ingested soil microbes [95,109]. Hydrolysis of organic polymers is supposed to be conducted by exoenzymes (e.g., cellulases, chitinases, lipases, and proteases) that may be excreted by ingested microbes and/or the earthworm [35,234,316,339]. Soluble organics and inorganic nutrients are absorbed by the earthworm primarily at the midgut and hindgut region, and undigested materials are excreted as casts [32,109]. ...
... However, the earthworm needs a positive energy balance to survive and cannot exclusively feed on its own mucus and organic acids that are derived from mucus-fermenting microbes. Organic acids, on which the earthworm could feed on, might also be produced from microbes that ferment plant litter-derived biopolymers or organics derived from disrupted microbial cells that were grinded in the gizzard [35,74,316,339,442]. This symbiosis between the earthworm that provides a high water content, anoxia, mucus, grinded microbial and plant cells and the ingested microbes that feed the earthworm (e.g., with organic acids, amino acids, and nucleotides) is called the mutualistic digestive system of the earthworm [16,35,236,442]. ...
H2 is a central intermediate of the complex anaerobic microbioal degradation of plant biomass and in situ concentrations of the gas are generally low because of its ongoing consumption. In contrast, high H2 concentrations were determined in the gut of the earthworm Lumbricus terrestris. These observations raised the question why the anaerobic microbial community in peatlands is poised to effectively scavenge H2 whereas H2 production by ingested soil anaerobes exceeds H2 consumption in the gut of L. terrestris. To address this question, H2-producing and H2-consuming processes were analyzed in peat soil slurries (soil microcosms) and diluted L. terrestris gut contents (gut content microcosms). In order to identify active H2 metabolizing taxa, gene marker analyses were intended. Hydrogenases are the key enzymes of the H2 metabolism and therefore represent suitable gene marker for H2-metabolizing microorgansims. Thus, PCR-primers for the amplification of hydrogenase gene sequences from environmental samples were designed. Furthermore, a sequence similarity cut-off of 80% for the clustering of environmental hydrogenase gene sequences on the family level was established by comparative 16S rRNA-hydrogenase gene analyses. Cellulose is a major constituent of sedges, which is the dominant vegetation and the major source of organic carbon in the investigated peatland. The polymer was readily degraded mainly to propionate, acetate, and CO2, whereas an accumulation of H2 was not observed in peat soil microcosms. Based on process data and thermodynamic calculations, methano¬genesis and acetogenesis could be excluded as abundant sinks for cellulose-derived H2. Propionate fermenters might have cometabolized H2 and cellulose hydrolysis products. Fibrobacter-related unclassified Bacteria, Prolixibacteraceae, Porphyro¬monanda¬ceae, Clostridiaceae, Ruminococcaceae, Acidobacteriaceae, Holo¬phagaceae, and Spiro-chaetaceae were identified as active assimilators of cellulose-derived carbon by 16S rRNA SIP (stable isotope probing). However, the H2 metabolism and the hydrolytic capabilities especially of the novel taxa remain unresolved. In contrast to the cellulose-supplemented soil microcosms, considerably higher concentrations of H2 were observed in microcosms with washed roots of Carex rostrata (an abundant sedge in the investigated peatland). Hydrogenase gene analyses revealed that several families within the Firmicutes (e.g., Clostridiaceae, Ruminococcaceae, and Lachnospiraceae) dominated H2 production in unsupplemented root microcosms. Formate, which can be excreted by roots, was converted into H2 and CO2 by formate hydrogenlyase-containing taxa (e.g., Betaproteobacteria and Acidobacteria). H2, derived from the fermentation of endogenous sources or supplemented formate, was primarily consumed by acetogens (e.g., Clostridiaceae und Veillonellaceae). These finding reinforced the assumption that the rhizosphere of sedges is a hotspot for H2-evolving fermenters and H2-consuming acetogens. In addition to primary fermenters, secondary syntrophic fermenters (syntrophs) are considered as major H2 producers in peatlands. 16S rRNA transcript analyses identified (i) a novel strain of Pelobacter propionicus as syntrophic ethanol oxidizer, (ii) Syntrophomonas and Telmatospirillum-related taxa as syntrophic butyrate oxidizers, and (iii) Syntrophobacter, Smithella, unclassified Bacteroidetes, and unclassified Fibrobacteres as potential syntrophic propionate oxidizers in soil microcosms. CH4 and CO2 were the only accumulating endproducts of the propionate, butyrate, and ethanol degradation, suggesting that H2, formate, and acetate (the fermentation products of the syntrophs) were effectively scavenged by methanogens. Aceticlastic methanogens (Methanosarcina and Methano¬saeta) outnumbered hydrogenotrophic methanogens (Methanoregula and Methanocella). This might indicate that acetogens were active and competed with hydrogenotrophic methanogens for available H2. In a previous study, in which L. terrestris gut content microcosms were supplemented with glucose, Clostridiaceae and Enterobacteriaceae were identified as important primary fermenters and potential producers of H2 whereas syntrophs, methanogens, and acetogens were not crucial for the H2 turnover. Hydrogenase transcript analyses corroborated these findings. Aeromonadaceae and Peptostreptococcaceae were determined as abundant H2-evolving taxa in addition to Clostridiaceae and Enterobacteriaceae. However, the former two families were not involved in the degradation of glucose and might have fermented endogenous carbon compounds. Proteins, nucleic acids, and carbohydrates derived from disrupted microbial cells represent potential endogenous substrates that are available in the earthworm gut. Aeromonas sp. and Clostridium bifermentans (phylogenetically belongs to the Peptostreptococcaceae) were indeed stimulated within a few hours after the supplementation of yeast cell lysates to gut content microcosms. Subsequently, proteolytic Clostridiaceae, saccharolytic Enterobacteriaceae, and unclassified Lachnospiraceae partially replaced the initially dominating fermenters. The acetogens Clostridium glycolicum and Clostridium magnum were also abundant. They probably utilized formate rather than H2, underscoring the assumption that acetogens are not an important sink for H2 in the gut of L. terrestris. The collective data indicated that at the oligotrophic conditions prevailing in peatlands (i) H2, is produced by primary and secondary fermenters and is effectively scavenged by methanogens, acetognes, and propionate fermenters, (ii) the rhizosphere of sedges is a hotspot for H2 metabolizers, and (iii) novel microbial taxa are involved in the complex anaerobic degradation of plant biomass. In contrast to the oligotrophic peatland soils, huge amounts of readily degradable carbon sources are available for the anaerobic microorganisms in the gut of earthworms. Because of the short gut passage, the anaerobes do not form an interwoven foodweb and consequently, primary and secondary fermentation products are not completely scavenged. Thus, fermentation-derived organic acids can be absorbed by the earthworm whereas H2 diffuses out of the worm and becomes available for H2 oxidizers in the soil.
... However, evidence is emerging that at least some invertebrates, such as molluscs, crustaceans and phytophagous insects, can synthesize cellulase enzymes themselves (Busch et al., 2019;Chang & Lai, 2018;Cragg et al., 2015;Griffiths et al., 2021;Han et al., 2022;Kern et al., 2013;King et al., 2010;Shelomi et al., 2014;Watanabe et al., 1998). Scattered evidence also exists for the expression of active endogenous cellulases by distantly related soil invertebrates, for example, the earthworm Pheretima hilgendorfi (Nozaki et al., 2009), the Antarctic springtail Cryptopygus antarcticus (Hong et al., 2014), as well as few oribatid mites and other springtails (Busch et al., 2019). Based on these individual findings, we hypothesize that a larger fraction of soil invertebrates than previously thought may be directly contributing to decomposition of lignocellulose in dead plant matter in soils using endogenous cellulases, with or without relying on a microbiome. ...
Lignocellulose is a major component of vascular plant biomass. Its decomposition is crucial for the terrestrial carbon cycle. Microorganisms are considered primary decomposers, but evidence increases that some invertebrates may also decompose lignocellulose. We investigated the taxonomic distribution and evolutionary origins of GH45 hydrolases, important enzymes for the decomposition of cellulose and hemicellulose, in a collection of soil invertebrate genomes. We found that these genes are common in springtails and oribatid mites. Phylogenetic analysis revealed that cellulase genes were acquired early in the evolutionary history of these groups. Domain architectures and predicted 3D enzyme structures indicate that these cellulases are functional. Patterns of presence and absence of these genes across different lineages prompt further investigation into their evolutionary and ecological benefits. The ubiquity of cellulase genes suggests that soil invertebrates may play a role in lignocellulose decomposition, independently or in synergy with microorganisms. Understanding the ecological and evolutionary implications might be crucial for understanding soil food webs and the carbon cycle.
... Examples of these enzymes are cellulases, chitinases, proteases, and lipases. 117 Earthworm's digestive system and gut microflora generate the enzymes amylase, cellulase, xylanase, endoglucase, alkaline phosphatase, acid phosphatase, and nitrate reductase, which support soil organic matter's humification and digestion. The enzymes amylase, xylanase, cellulase, cellulase, xylanase, endoglucanase, Amylase, and cellobiase are the enzymes that degrade starch, cellulose, xylan, and cellodextrins. ...
Our planet is really lovely, and there are plenty of living organisms here. Among these organisms, human beings are also contributing to the planet. However with a growing population of human beings, there is a growing need for food, which is entirely dependent on agriculture, several strategies have been employed to boost agricultural output, including inorganic fertilizers and synthetic pesticides. Inorganic fertilizers have largely enhanced productivity as well, but they also have several negative health impacts. However, continued use and reliance on these inputs has reduced crop productivity and deteriorated the quality of natural resources and the ecosystem. So, to overcome these problems and for sustainable development, long-term agricultural expansion may be achieved with organic farming while also preserving the environment. It may take time to switch from chemical to organic farming, which could reduce the farmer's profits. Advancements in organic farming, using Eisenia fetida to promote the vermicomposting of water hyacinth (wastewater weed) and employing more organic fertilizer while decreasing inorganic fertilizer usage in farming. However, the farmer will only make the changeover if he is persuaded that organic farming has long-term advantages over chemical farming. Vermicompost is a type of organic fertilizer, prepared from earthworms using various unused waste organic materials. It is an alternative source for increasing agriculture production. Many works have shown that vermicompost has beneficial effects on the environment. Various organic waste materials are used by earthworm species to form vermicompost. Water hyacinth poisons water bodies, turning pleasant water into unappealing water by emitting a foul smell. Most times, water hyacinths thrive in sewage water. Controlling this aquatic weed is a major issue, but it is easily solved through vermitechnology. Extremely, in our review study, we are going to give our concentrate on development of organic farming using vermicomposting.
... To date, digestive enzyme activities in enchytraeids have been studied based on traditional biochemical [4e9] or histochemical methods [10]. However, in many mesofaunal invertebrates the contribution of microbial enzymatic apparatus cannot be excluded, and the activity of the microflora can even conceal the endogenous digestive capacity of animals [3,7,129]. While earlier studies had indicated that enchytraeids are capable of digesting proteins, disaccharides, and some polysaccharides, no attempt has been made to distinguish whether the origin of digestive enzymes is the enchytraeids themselves or microorganisms [6e9]. ...
Although enchytraeids have gained popularity in scientific research, fundamental questions regarding their feeding ecology and biology remain largely unexplored. This study investigates α-amylases, major digestive enzymes responsible for hydrolyzing starch and similar polysaccharides into sugars, in Enchytraeus albidus. Genetic data related to α-amylases is currently lacking for the family Enchytraeidae but also for the entire Annelida. To detect and identify coding sequences of the expressed α-amylase genes in COI-monohaplotype culture (PL-A strain) of E. albidus, we used classical “gene fishing” and transcriptomic approaches. We also compared coding sequence variants of α-amylase retrieved from transcriptomic data related to freeze-tolerant strains. Our results reveal that E. albidus possesses two distinct α-amylase genes (Amy I and Amy II) that are homologs to earthworm Eisenia fetida Ef-Amy genes. Different strains of E. albidus possess distinctive alleles of α-amylases with unique SNP patterns specific to a particular strain. Unlike Amy II, Amy I seems to be a highly polymorphic and multicopy gene. The domain architecture of the putative Amy proteins was found the same as for classical animal α-amylases with ABC-domains. A characteristic feature of Amy II is the lack of GHGA motif in the flexible loop region, similarly to many insect amylases. We identified “Enchytraeus-Eisenia type” α-amylase homologs in other clitellates and polychaetes, indicating the ancestral origin of Amy I/II proteins in Annelida. This study provides the first insight into the endogenous non-proteolytic digestive enzyme genes in potworms, discusses the evolution of Amy α-amylases in Annelida, and explores phylogenetic implications.
... It is estimated that they decompose more deadwood in 61 tropical forests than free-living microorganisms (Griffiths et al., 2019). Nevertheless, the 62 mechanisms behind decomposition performed by soil invertebrates remains obscure and the 63 ability of soil animals to degrade composite polysaccharides without relying on gut symbionts 64 remains a long-standing debate in soil ecology (Berg et al., 2004;Cragg et al., 2015). of active endogenous cellulases by distantly-related soil invertebrates, e.g. the earthworm 72 Pheretima hilgendorfi (Nozaki et al., 2009), the Antarctic springtail Cryptopygus antarcticus 73 (Hong et al., 2014), as well as few oribatid mites and other springtails (Busch et al., 2019). ...
Lignocellulose is a major component of plant biomass. Its decomposition is crucial for the terrestrial carbon cycle. Microorganisms are considered as primary decomposers and evidence increases that some invertebrates may also decompose lignocellulose. We investigated the taxonomic distribution and evolutionary origins of GH45 cellulases in a collection of soil invertebrate genomes and found that these genes are common in springtails and oribatid mites. Phylogenetic analysis revealed that cellulase genes were acquired early in the evolutionary history of these groups. Domain architectures and predicted 3D enzyme structures indicate that these cellulases are functional. Patterns of presence and absence of these genes across different lineages prompt further investigation into their evolutionary and ecological benefits. The ubiquity of cellulase genes suggests that soil invertebrates may play a role in lignocellulose decomposition, independently from microorganisms. Understanding the ecological and evolutionary implications might be crucial for understanding soil food webs and the carbon cycle.
... For instance, it has been shown that N-cycling enzyme activities increase with C availability, while C-cycling enzyme activities increase with N availability (Bowles et al., 2014). Therefore, the significant decrease in β-glucosidase activity observed in casts from polluted soils could reflect a reduction in N availability and/or a decrease in cellulose content following organic matter breakdown during soil ingestion (Nozaki et al., 2009). ...
Pesticides exert important effects on the soil fauna and health. However, little is known about the interactions of soil, microorganisms and earthworms in the presence of pesticides and about their respective roles in the soil biological activity. The aim of this study was to evaluate the effect of the soil type on enzyme activities, measured in bulk soil and in casts of two earthworm species, after exposure to the organophosphorus pesticide parathion. To this aim, two endogeic earthworm species (Apporectodea caliginosa and Allolobophora chlorotica) were cross-acclimated in two different soil textures (each representing the most favorable soil environment for that species). Enzyme activities were measured as a soil quality indicator in samples of bulk soil (collected at day 4 and day 7 of exposure to parathion) and in earthworm casts (collected at day 7). A short exposure (4 days) to parathion significantly (ANOVA, p < 0.001) inhibited carboxylesterases (25-43 % of inhibition) and alkaline phosphatase (~23 % of inhibition). At day 7 of exposure, parathion impact on the overall soil enzyme activities mainly depended on the soil texture. Indeed, activity inhibition was higher (ANOVA, p < 0.001) in silt-clay soil (decrease by 37 %) than in silt-loamy soil (decrease by ~18 %). Conversely, parathion effect was not influenced by earthworm presence/absence and earthworm species. However, after soil exposure to parathion, earthworms (both species) improved enzyme activity restoration in their casts.
... The endo-β-1, 4-glucanases of gut termites (i.e. UniProtKB_Q64I76) and earthworms (UniProtKB_O77045; UniProtKB_B9A7E3) were the most potent and effective cellulase enzymes toward the degradation of lignocellulosic biomass [44]. These potential enzymes were belonging to the GH protein family 9 (GHF9). ...
Human civilization faces a huge energy crisis due to climate alterations and reduction in fossil fuel reserves. Biofuels
are sustainable platform that can combat this burning issue currently. Biodiesel is a potential fossil fuel alternative
from cooking, soybean, or plant-derived oil resources. Existing biodiesel production processes are directly competing
with food chain. Algae are impressive alternatives for biodiesel generation as algae grow without competition with
food chain, have an inherently faster growth rate, and have higher lipid content under diverse nutrient-limiting
environments. Lignocellulosic waste (LCW) accumulation has been increasing through anthropogenic activities.
Accumulation of LCW causes ill effects on environment and human health. Thus, a faster paradigm shift needs to
be required for establishing a single platform to perform lignocellulosic biomass degradation along with higher lipid
production as biodiesel precursor. Few studies show algal endoglucanase or cellulase activities for degrading LCW
to generate monomeric constituents which can further be channelized to produce higher biomass and lipid contents
as biodiesel precursor. Thus, the present study focuses on a feasibility attempt to show that mangrove algal isolates
could be a potential microbial cell factory for cellulose degradation as a major constituent of LCW and higher lipid
production as potential precursor for biodiesel production in near future.
... In the first stage (active stage), earthworms grind the consumed material in gut section, the gut-secreted enzymes crack the complex substances, and in the second stage (maturation stage), the earthworm releases biologically modified biochar with humus like substance (Gomez-Brandon et al. 2011;Gomez-Brandon and Domínguez 2014;Lores et al. 2006). During the active stage, the consumed organic material gets ground in gizzard, gut epithelium releases multiple enzymes, and induces biochemical reactions (Nozaki et al. 2009) for different enzymes, microbes, beneficial nutrients, and biologically modified biochar Zhu et al. 2017a, b;Quilliam et al. 2013;Ennis et al. 2012;Jeffery et al. 2011). The gut enzymes have various biomolecules which can be used as catalyst and affect soil pollutants (Burns et al. 2013;Gianfreda et al. 2016). ...
The expanding demand for environmental treatment increasingly requires different types of engineered char with high performance. In this context, new trends in pyrolysis methods have emerged and contributed to the sustainable development of pyrolysis technologies, namely microwave-assisted pyrolysis, co-pyrolysis, pyrolysis under non-inert ambiances, hydrothermal carbonization (wet pyrolysis), and integrated pyrolysis techniques. The outstanding advantages of these technologies over conventional pyrolysis include: increase in biomass conversion efficiency of the process, use of nonconventional raw material, increase in adsorption capacity of biochar by enhanced activated oxygen species, porosity, and functional groups, and removal or immobility of contaminants. The biochar products can be widely applied in various environmental fields, such as carbon capture and sequestration, soil amendment, adsorption of contaminants in soil, water, and air, and energy production. Nevertheless, challenges remain for these new trends, such as the increase in cost for the installation and operation, the lack of knowledge of the mechanism involved during pyrolysis, the difficulty in scaling up, etc. Further studies are recommended to facilitate the application of these new trends, such as pilot tests or field experiments to evaluate the real effects of biochar products prior to large-scale applications or their long-term risk during use, or prediction of properties of biochar and their impacts on environmental applications using modeling or machine learning approaches.KeywordsAdvantagesBiocharLimitationNew trendsOpportunitiesPyrolysis
... In the first stage (active stage), earthworms grind the consumed material in gut section, the gut-secreted enzymes crack the complex substances, and in the second stage (maturation stage), the earthworm releases biologically modified biochar with humus like substance (Gomez-Brandon et al. 2011;Gomez-Brandon and Domínguez 2014;Lores et al. 2006). During the active stage, the consumed organic material gets ground in gizzard, gut epithelium releases multiple enzymes, and induces biochemical reactions (Nozaki et al. 2009) for different enzymes, microbes, beneficial nutrients, and biologically modified biochar Zhu et al. 2017a, b;Quilliam et al. 2013;Ennis et al. 2012;Jeffery et al. 2011). The gut enzymes have various biomolecules which can be used as catalyst and affect soil pollutants (Burns et al. 2013;Gianfreda et al. 2016). ...
Biochar is a prominent adsorbent for environmental remediation. The physicochemical properties responsible for adsorption can be significantly enhanced by using physical, chemical, and biological treatments of biochar. The biological treatment methods are advantageous in terms of cost-effectiveness and reduced secondary pollutants. The present chapter summarizes the need, methodology, modification mechanism of biological modification of biochar, and its implementation for environmental remediation. The biologically modified biochar can be obtained by either ex situ (pyrolysis of anaerobically digested residue) or in situ (using extracellular enzymes) technologies. The process includes colonization and biofilm formation by microorganisms on biochar surface and attachment of microbes. Biologically modified biochar metabolizes organic/inorganic contaminants and helps in adsorption, biodegradation, and bio-adsorption simultaneously.
... Notably, cellulase plays a major role in gut epithelial cells. In an artificial soil test, the gut absorbs environmental pollutants under controlled conditions (Nozaki et al. 2009). Increases in cellulase activity were induced by IMI and ACE exposures of 1 day and recovered to normal levels at 14 days. ...
Mixed pesticides have been broadly used in agriculture. However, assessing the combined effects of pesticides in the environment is essential for potential risk assessment, though the task is far from complete. Median lethal concentrations of pesticides as well as acetylcholinesterase (AChE) levels and cellulose activities were measured in earthworms (Eisenia fetida) individually and jointly exposed to pesticides imidacloprid (IMI), acetamiprid (ACE), chlorpyrifos (CRF), and abamectin (ABM)). A 3:1 mixture of CRF and IMI had additive effects, while a 3:1 mixture of CRF and ACE had synergic effects. The joint effects of ABM with IMI or with ACE were synergistic. As CRF concentration increased, AChE activities were significantly decreased. For high concentrations of IMI, AChE activities under combined CRF and IMI applications were significantly inhibited following increased exposure time. Moreover, the cellulase activities under combined applications of CRF with IMI or with ACE had similar effects. This study provides basic data for scientifically evaluating the environmental risk and safety of combined uses of pesticides.
Graphical abstract
... It has long been suggested that most earthworm species are not capable of secreting the full set of enzymes that are required for the depolymerization of plant-derived polymers. Whilst the possession of endogenous endocellulase genes by some earthworm species has been reported [19], indicating the ability to digest cellulose, it is thought that even when earthworms can produce endocellulase, their ability to digest and acquire nutrients from plant litter lies fundamentally in their relationship with microorganisms [20]. This is because efficient degradation of a complex polymer such as lignocellulose requires the synergistic action of suites of enzymes, such as hemicellulase, endocellulase, lignin peroxidase and exocellulase, that are primarily secreted by microorganisms [21]. ...
Earthworms are an integral part of soil ecosystems, especially for their role in soil functions such as organic matter (OM) decomposition and nutrient cycling. Earthworms and microorganisms are interdependent, and a considerable portion of the contribution earthworms make to influencing OM fate is through interactions with microorganisms. However, the importance of the earthworm-associated microbiome is not fully understood, because it is difficult to separate the direct influence of the earthworms from the indirect influence of their microbiome. Here, we evaluated an antibiotic-based procedure to suppress the microbiome of individuals of ecologically-contrasting earthworm species (Eisenia fetida, Lumbricus terrestris, Allolobophora chlorotica) as the first step towards soil studies aimed at understanding the importance of the earthworm microbiome for host health and function. Individual earthworms were exposed to antibiotics: cycloheximide (150 μg ml⁻¹), ampicillin (100 μg ml⁻¹), ciprofloxacin (50 μg ml⁻¹), nalidixic acid (50 μg ml⁻¹), and gentamicin (50 μg ml⁻¹) either singly or in a cocktail via culture (96 h) in a semi-solid agar carrier. Compared to the non-antibiotic treated control, the cocktail (for all three species) and ciprofloxacin (for E. fetida and A. chlorotica) treatments significantly reduced (P < 0.05) culturable microbial abundance on nutrient agar and potato dextrose agar. The microbial counts were reduced to below detection (<50 CFU individual⁻¹) for E. fetida and A. chlorotica receiving the cocktail. Illumina 16S rDNA amplicon sequence analysis of culturable L. terrestris -associated bacteria showed that antibiotic treatment influenced community composition revealing putative sensitive (Comomonas, Kosakonia and Sphingobacterium) and insensitive (Aeromonas, Pseudochrobactrum) taxa. Overall, we report a rapid, with minimal earthworm-handling, process of creating suppressed-microbiome E. fetida, A. chlorotica and L. terrestris as a tool to be used in future ecological studies of earthworm microbial interactions affecting host health and function.
... Even though several studies have been conducted to better understand the bioconversion process of organic material during vermicomposting, knowledge concerning hemicellulolytic (and xylanolytic in particular) enzymes and intestinal microorganisms in earthworms remains fairly limited. The cellulolytic activity, on the other hand, of several enzymes and bacteria in earthworm gut has already been reported by various groups (Nozaki et al., 2009;Beloqui et al., 2010;Fujii et al., 2012;Jyotsna and Rao, 2015). ...
Tropical agriculture produces large amounts of lignocellulosic residues that can potentially be used as a natural source of value-added products. The complexity of lignocellulose makes industrial-scale processing difficult. New processing techniques must be developed to improve the yield and avoid this valuable resource going to waste. Hemicelluloses comprise a variety of polysaccharides with different backbone compositions and decorations (such as methylations and acetylations), and form part of an intricate framework that confers structural stability to the plant cell wall. Organisms that are able to degrade these biopolymers include earthworms (Eisenia fetida), which can rapidly decompose a wide variety of lignocellulosic substrates. This ability probably derives from enzymes and symbiotic microorganisms in the earthworm gut. In this work, two substrates with similar C/N ratios but different hemicellulose content were selected. Palm fibre and coffee husk have relatively high (28%) and low (5%) hemicellulose contents, respectively. A vermicomposting mixture was prepared for the earthworms to feed on by mixing a hemicellulose substrate with organic market waste. Xylanase activity was determined in earthworm gut and used as a selection criterion for the isolation of hemicellulose-degrading bacteria. Xylanase activity was similar for both substrates, even though their physicochemical properties principally pH and electrical conductivity, as shown by the MANOVA analysis) were different for the total duration of the experiment (120 days). Xylanolytic strains isolated from earthworm gut were identified by sequence analysis of the 16S rRNA gene. Our results indicate that the four Actinobacteria, two Proteobacteria, and one Firmicutes isolated are active participants of the xylanolytic degradation by microbiota in the intestine of E. fetida. Most bacteria were more active at pH 7 and 28 °C, and those with higher activities are reported as being facultatively anaerobic, coinciding with the microenvironment reported for the earthworm gut. Each strain had a different degradative capacity.
... Notably, cellulase plays a major role in gut epithelial cells. In an arti cial soil test, the gut absorbs environmental pollutants under controlled conditions (Nozaki et al. 2009). Increases in cellulase activity were induced by IMI and ACE exposures of 1 d and recovered to normal levels at 14 d. ...
Mixed pesticides have been broadly used in agriculture. However, assessing the combined effects of pesticides in the environment is essential for potential risk assessment, though the task is far from complete. Median lethal concentrations of pesticides as well as acetylcholinesterase (AChE) levels and cellulose activities were measured in earthworms ( Eisenia fetida ) individually and jointly exposed to pesticides imidacloprid (IMI), acetamiprid (ACE), chlorpyrifos (CRF), and abamectin (ABM)). A 3:1 mixture of CRF and IMI had additive effects, while a 3:1 mixture of CRF and ACE had synergic effects. The joint effects of ABM with IMI or with ACE were synergistic. As CRF concentration increased, AChE activities were significantly decreased. For high concentrations of IMI, AChE activities under combined CRF and IMI applications were significantly inhibited following increased exposure time. Moreover, the cellulase activities under combined applications of CRF with IMI or with ACE had similar effects. This study provides basic data for scientifically evaluating the environmental risk and safety of combined uses of pesticides.
... During the active phase, earthworms consume the organic matter through the mouth and grind in the gut region. The gut epithelium of the earthworms and symbionts secrete a wide variety of enzymes (see discussion section) that enhance the biochemical reactions (Garvín et al., 2000;Nozaki et al., 2009) resulting in the production of end materials that contain different components including enzymes, beneficial microbial communities, and plant essential nutrients (Balachandar et al., 2021;Domínguez et al., 2019). ...
The industrial revolution and indiscriminate usage of a wide spectrum of agrochemicals account for the dumping of heavy metals in the environment. In-situ/ex-situ physicochemical, and bioremediation strategies with pros and cons have been adopted for recovering metal contaminated soils and water. Therefore, there is an urgent requirement for a cost-effective and environment-friendly technique to combat metal pollution. Biochar combined with earthworms and vermifiltration is a suitable emerging technique for the remediation of metal-polluted soils and water. The chemical substances (e.g., sodium hydroxide, zinc chloride, potassium hydroxide, and phosphoric acid) have been used to activate biochar, which also faces several shortcomings. Studies reveal that extracellular enzymes have been used to activate biochar which is produced by earthworms and microbes that can alter the surface of the biochar. The present review focuses on the global scenario of metal pollution and its remediation through biochar activation using earthworms. The earthworms and biochar can produce “vermibiochar” which is capable of reducing the metal ions from contaminated water and soils. The vermifiltration can be a suitable technology for metal removal from wastewater/effluent. Thus, the biochar has a trick of producing entirely new options at a time when vermifiltration and other technologies are least expected. Further attention to the biochar-assisted vermifiltration of different sources of wastewater is required to be explored for the large-scale utilization of the process.
... Earthworms are known as the intestine of the soil as they helps in increasing the fertility and aeration of the soil by their turning action [5] . They helps in the decomposition of the materials by enhancing the activity of the microbial population present in their gut and thus the microbes behaves as a bioreactor [6] . Most of the earthworms exhibit a symbiotic relation with their gut microbes for the decomposition of organic matter by producing enzymes such as cellulase, amylase etc. ...
... In the earthworm, endogenous cellulase activity was identified primarily for the contribution to cellulose digestion [10]. Interestingly, earthworm cellulase mRNA was shown to be expressed not only in gut epithelial cells but also in non-digestive tissues, such as the epidermis and coelomic cell-aggregates. ...
To investigate whether earthworm cellulases contribute to the innate immune system, the responsiveness of cellulase activity and mRNA expression to bacterial challenge was examined by zymography and RNA sequencing. A zymographic analysis revealed that the activity levels of earthworm cellulases were upregulated in response to either a bacterial (Bacillus subtilis or Escherichia coli) or LPS challenge. After the challenge, significant increases in cellulase 1 and cellulase 2 activity levels were observed within 8-16 and 16-24 h, respectively. In the coelomic fluid, both activities were significantly upregulated at 8 h post-injection with B. subtilis. Based on RNA sequencing, cellulase-related mRNAs encoding beta-1,4-endoglucanases were upregulated by 3-fold within 6 h after B. subtilis injection. Our results clearly demonstrated that earthworm cellulases are upregulated by bacterial challenge at the mRNA and protein levels. These results support the view that earthworm cellulases act as inducible humoral effectors of innate immunity against bacterial infection.
... According to Domínguez (2011), vermicomposting can be described as a two-phase decomposing process operating simultaneously: the earthworm gut-associated processes (GAPs) and the castassociated processes (CAPs). During GAPs, the organic matter ingested by earthworms undergoes physical transformations; grinding and mixing of the ingested material, and complex biochemical transformations facilitated by the action of digestive enzymes originated from both the microbial symbionts and the earthworm gut epithelium (Garvín et al., 2000;Drake and Horn, 2007;Nozaki et al., 2009). Moreover, changes in the chemical composition of the ingested organic matter imply absorption of nutrients mainly in the foregut (or anterior intestine), and secretion of compounds such as mucus, urea, ammonia and enzymes. ...
Intensive use of agrochemicals is considered one of the major threats for soil quality. In an attempt to mitigate their side-effects on non-target organisms and soil functioning, many engineering and biological remediation methodologies are currently available. Among them, the use of biochar, a carbonaceous material produced from pyrolysing biomass, represents an attractive option enhancing both remediation and soil carbon storage potentials. Currently, activation of biochar with chemical or physical agents seeks for improving its remediation potential, but most of them have some undesirable drawbacks such as high costs and generation of chemical wastes. Alternatively, the use of biological procedures to activate biochar with extracellular enzymes is gaining acceptance mainly due to its eco-friendly nature and cost-effectiveness. In these strategies, microorganisms play a key role as a source of extracellular enzymes, which are retained on the biochar surface. Recently, several studies point out that soil macrofauna (earthworms) may act as a biological vector facilitating the adsorption of enzymes on biochar. This paper briefly introduces current biochar bioactivation methodologies and the mechanisms underlying the coating of biochar with enzymes. We then propose a new conceptual model using earthworms to activate biochar with extracellular enzymes. This new earthworm-biochar model can be used as a theoretical framework to produce a new product “vermichar”, vermicompost produced from blended feedstock, earthworms, and biochar that can be used to improve soil quality and remove soil. This model can also be used to develop innovative in-situ “vermiremediation” technologies utilizing the beneficial effects of both earthworms and biochar. Since biochar may contain toxic chemicals generated during its production stages or later concentrated when applied to polluted soils, this paper also highlights the need for an ecotoxicological knowledge around earthworm-biochar interaction, promoting further discussion on suitable procedures for assessing the environmental risk of this conceptual model application in soil bioremediation.
... doi: bioRxiv preprint first posted online May. 2, 2019; 13 control), i.e., the invasive species displayed a faster growth than the native species when nutritional quality improved. This suggests that P. corethrurus consumes and degrades a greater variety of organic materials given its greater ability (efficiency), evidenced by: a) producing endogenous cellulases: Ean-Eg, EF-EG2, and GHF9 (Nozaki et al. 2009;Shweta 2012;Ueda et al. 2014;Park et al. 2017); b) its association with the gut microbiota: Protobacteria, Firmicutes, Actinobacteria, Chloroflexi, and Bacteroidete (Thakuria et al. 2010;Liu et al. 2017;Gong et al. 2018;Liu et al. 2018); c) expressing genes (transcriptome) that contribute to the adaptation of its digestive system: salivation, gastric acid, and pancreatic secretion (Liu et al. 2018); d) improving its digestion efficiency according to the type of cecum (Nozaki et al. 2013;Ikeda et al. 2017 America. In Mexico, it is unknown whether P. corethrurus arrived before or after the inmigration . ...
The presence of earthworm species in crop fields is as old as agriculture itself. The earthworms Pontoscolex corethrurus (invasive) and Balanteodrilus pearsei (native) are associated with the emergence of agriculture and sedentism in the region Amazon and Maya, respectively. Both species have shifted their preference from their natural habitat to the cropland niche; however, they contrast in terms of intensification of agricultural land use (anthropic impact to the symbiotic soil microbiome). P. corethrurus inhabits conventional agroecosystems (pesticides, herbicides, and fertilizers are applied to soil), while B. pearsei thrives in traditional agroecosystems (biological management of soil); that is, P. corethrurus has not yet been recorded in soils where B. pearsei dwells. The demographic behavior of these two earthworm species was assessed in the laboratory over 100 days, according to their origin (OE; P. corethrurus and B. pearsei) food quality (FQ; soil only, maize stubble, and Mucuna pruriens), and soil moisture (SM; 25, 33, and 42%). The results showed that OE, FQ, SM, and the OE x FQ interaction were highly significant for the survival, growth, and reproduction of earthworms. P. corethrurus showed a lower survival rate (> mortality). P. corethrurus survivors fed a diet of low-to-intermediate nutritional quality (soil and corn stalks, respectively) showed a greater capacity to grow and reproduce; however, it was surpassed by the native earthworm when fed a high-quality diet (M. pruriens). Besides, P. corethrurus displayed a low cocoon hatching (emergence of juveniles). These results suggest that the presence of the invasive species was associated with the absence of natural mutualistic bacteria (gut, nephridia, and cocoons), and with a negative interaction with the soil microbiota where the native species dwells. These results are consistent with the absence of P. corethrurus in milpa and pasture-type agricultural niches managed by peasants (agroecologists) to grow food regularly a biological management of soil. The results reported here and the published information jointly (e.g., designation of the neotype and ambiguity of the place of origin) suggest that P. corethrurus is an invasive species that is neither wild nor domesticated, that is, its eco-evolutionary phylogeny needs to be derived based on its symbionts.
... Past studies have demonstrated that the earthworm gastrointestinal tract can secrete glycolytic digestive enzymes, thus contributing, together with enzymes produced by gut symbionts, to the decomposition of ingested organic matter (Lattaud et al. 1998, Garvín et al. 2000, Nozaki et al. 2009. Interestingly, gastrointestinal secretion of an important group of pesticide-detoxifying enzymes called carboxylesterases (EC 3.1.1.1) ...
... Although the model structural polysaccharides cellulose, chitin, pectin and xylan were poorly utilized, it cannot be excluded that naturally occurring related polymers might be subject to more efficient utilization in the alimentary canal, a capacity that might be partially linked to the potential occurrence of certain digestive enzymes in the alimentary canal (Laverack, 1963;Edwards and Fletcher, 1988;Nozaki et al., 2009). In this regard, worm-derived hydrolases could be present in gut contents and therefore contribute to the activities observed in the present study. ...
The alimentary canal of the earthworm is representative of primitive gut ecosystems, and gut fermenters capable of degrading ingested biomass‐derived polysaccharides might contribute to the environmental impact and survival of this terrestrial invertebrate. Thus, this study evaluated the postulation that gut microbiota of the model earthworm Lumbricus terrestris ferment diverse biomass‐derived polysaccharides. Structural polysaccharides (e.g., cellulose, chitin) had marginal impact on fermentation in anoxic gut content treatments. In contrast, non‐structural polysaccharides (e.g., starch, glycogen) greatly stimulated (a) the formation of diverse fermentation products (e.g., H2, ethanol, fatty acids) and (b) the facultatively‐fermentative families Aeromonadaceae and Enterobacteriaceae. Despite these contrasting results with different polysaccharides, most saccharides derived from these biopolymers (e.g., glucose, N‐acetylglucosamine) greatly stimulated fermentation, yielding 16S rRNA gene‐based signatures of Aeromonadaceae‐, Enterobacteriaceae‐, and Fusobacteriaceae‐affiliated phylotypes. Roots and litter are dietary substrates of the earthworm, and as proof‐of‐principle, gut‐associated fermenters responded rapidly to root‐ and litter‐derived nutrients including saccharides. These findings suggest that (a) hydrolysis of certain ingested structural polysaccharides may be a limiting factor in the ability of gut fermenters to utilize them and (b) non‐structural polysaccharides of disrupted biomass are subject to rapid fermentation by gut microbes and yield fatty acids that can be utilized by the earthworm.
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... Many invertebrates express endogenous cellulases. Plantparasitic nematodes, cockroaches and termites were among the first to be proven to carry cellulase genes, but more recently these genes (mostly of the families GH5, 9 and 45) have also been unambiguously demonstrated in other taxa, such as other insects [44], Gastropoda [45], Crustacea [6,46,47] and Annelida [48]. The lack of large digestive gut chambers (as known from ruminants and termites) for cultivation of microbial gut symbionts in many insects or crustaceans argues that endogenous cellulases are needed in these herbivorous and detritivorous animals. ...
Organisms use diverse mechanisms involving multiple complementary enzymes, particularly glycoside hydrolases (GHs), to deconstruct lignocellulose. Lytic polysaccharide monooxygenases (LPMOs) produced by bacteria and fungi facilitate deconstruction as does the Fenton chemistry of brown-rot fungi. Lignin depolymerisation is achieved by white-rot fungi and certain bacteria, using peroxidases and laccases. Meta-omics is now revealing the complexity of prokaryotic degradative activity in lignocellulose-rich environments. Protists from termite guts and some oomycetes produce multiple lignocellulolytic enzymes. Lignocellulose-consuming animals secrete some GHs, but most harbour a diverse enzyme-secreting gut microflora in a mutualism that is particularly complex in termites. Shipworms however, house GH-secreting and LPMO-secreting bacteria separate from the site of digestion and the isopod Limnoria relies on endogenous enzymes alone. The omics revolution is identifying many novel enzymes and paradigms for biomass deconstruction, but more emphasis on function is required, particularly for enzyme cocktails, in which LPMOs may play an important role.
... Materials that have a high cellulose/lignin ratio contain more labile energy than materials that have a low cellulose/lignin ratio (McKendry, 2002). Earthworms can produce endogenous cellulase in their gut (Nozaki et al., 2009), which may be responsible for much of the straw degradation, and subsequent increase in L. terrestris biomass, observed in our microcosm experiments. ...
Earthworms benefit agriculture by providing several ecosystem services. Therefore, strategies to increase earthworm abundance and activity in agricultural soils should be identified, and encouraged. Lumbricus terrestris earthworms primarily feed on organic inputs to soils but it is not known which organic amendments are the most effective for increasing earthworm populations. We conducted earthworm surveys in the field and carried out experiments in single-earthworm microcosms to determine the optimum food source for increasing earthworm biomass using a selection of crop residues and organic wastes available to agriculture. We found that although farmyard manure increased earthworm populations more than cereal straw in the field, straw increased earthworm biomass more than manures when milled and applied to microcosms. Earthworm growth rates were positively correlated with the calorific value of the amendment and straw had a much higher calorific value than farmyard manure, greenwaste compost, or anaerobic digestate. Reducing the particle size of straw by milling to <3mm made the energy in the straw more accessible to earthworms. The benefits and barriers to applying milled straw to arable soils in the field are discussed.
... Recently, in the earthworm, endogenous cellulase activities as well as genes have been identified and characterized mainly focusing on their contribution to the cellulose digestion (Nozaki et al., 2009) and on potential application associated with biofuel production (Ueda et al., 2014). In the light of these significant findings, we have deposited a complete cDNA sequence of earthworm cellulase from common earthworm species, E. andrei, in GenBank database with the accession number of AY914054 (unpublished). ...
A new endogenous cellulose (Ean-EG) from the earthworm, Eisenia andrei and its expression pattern are demonstrated. Based on a deduced amino acid sequence, the open reading frame (ORF) of Ean-EG consisted of 1368 bps corresponding to a polypeptide of 456 amino acid residues in which is contained the conserved region specific to GHF9 that has the essential amino acid residues for enzyme activity. In multiple alignments and phylogenetic analysis, the deduced amino acid sequence of Ean- EG showed the highest sequence similarity (about 79%) to that of an annelid (Pheretima hilgendorfi) and could be clustered together with other GHF9 cellulases, indicating that Ean-EG could be categorized as a member of the GHF9 to which most animal cellulases belong. The histological expression pattern of Ean-EG mRNA using in situ hybridization revealed that the most distinct expression was observed in epithelial cells with positive hybridization signal in epidermis, chloragogen tissue cells, coelomic cell-aggregate, and even blood vessel, which could strongly support the fact that at least in the earthworm, Eisenia andrei, cellulose function must not be limited to digestive process but be possibly extended to the innate immunity.
... form longa (Ude) and that the earthworm gut microhabitat had a high microbial load as compared to the soil, initiating several investigations into the similarities and equivalence of earthworm gut to their soil habitats [15,16]. Few studies have concentrated on in-depth characterization of hydrolytic enzymes of microbial origin like carboxymethylcellulase (CMCase), b-glucosidase, b-1,3 glucanase, and b-xylosidase of microbes associated with earthworm gut such as E. foetida, Pheretima hilgendorfi, Aporrectodea caliginosa, Hormogaster elisae, etc. [17][18][19]. ...
Eisenia foetida and Perionyx excavatus are potent vermicomposting earthworms having immense importance in organic matter recycling under tropical conditions, particularly in India. Comparative assessment of the cultivable gut microbiome of these two epigeic earthworms after growth on lignocellulosic biomass, revealed populations of 3.2-8.3×109 CFU. Diversity analyses using 16S rDNA sequences revealed that the major dominating classes were Firmicutes (50-60%), followed by Actinobacteria (26.7-33%), and Alphaproteobacteria (5.6-6.7%). Despite exhibiting similar diversity indices and species richness, Betaproteobacteria (6.7%) and Gammaproteobacteria (11.1%) were solely present in E. foetida and P. excavatus, respectively. A set of 33 distinct morphotypes, including 18 from E. foetida and 15 from P. excavatus were selected. Carbohydrate utilization profiles generated using Hi-Carbo™ kits revealed that the isolates from the gut of P. excavatus - Arthrobacter pascens IARI-L13 and Bacillus subtilis IARIC were able to utilize 54 and 51.4% of the carbohydrates tested. Sorbose was not utilized, while unusual carbohydrates - adonitol and methyl-d-mannoside were utilized only by members from the gut of P. excavatus, while melizitose was utilized by those uniquely by E. foetida microbiome. Functional characterization revealed that β-glucosidase activity was most prevalent in the culturable microbial community. Alkaline and acid phosphatase activity was more widespread in the E. foetida gut microbiome. All the culturable gut bacterial isolates produced ammonia, but IAA was detected only in five cultures. The unique functional attributes of the two culturable microbiomes, grown on a similar diet, reveals the significance of proper selection of earthworm substrate combinations for effective vermicomposting.
... Garvin et al. (2000) and Zhang et al. (1993) reported that cellulase cannot be synthesized in the gut of Hormogaster elisae and Pontoscolex corethrurus to digest the cellulose. In contrast, Nozaki et al. (2009) identified a novel cellulose-encoding gene (phhEg) in the gut of Pheretima hilgendorfi and concluded that the earthworms themselves have the ability to produce the endogenous and functional cellulase. Occurrence of cellulase in the earthworms' gut denotes its role in the decomposition of plant litter and other cellulosic materials (Dash, 1987). ...
The purpose of the current study was to measure the ecotoxicity of silver nanoparticles (AgNPs) on Aporrectodea caliginosa earthworm. No adult earthworm mortality was observed at any treatment during the sub-chronic exposure period (28d). Biomass and cellulase levels reduced in a concentration-dependent manner in the exposed earthworms compared to those of the control. The hatched cocoons from the contaminated substrates were significantly (p<0.05) fewer than that of the control substrate. This finding provided further support for the conclusion that AgNPs may affect cocoon hatchability. Cocoon hatchability could therefore be a more reliable endpoint at a specific concentration than cocoon production. A significant increase in DNA damage was revealed in the earthworms treated with AgNPs compared to the untreated ones. The results denoted the effectiveness of measuring cellulase activity, biomass, reproduction and DNA damage and reinforced the application of the present methods in nanoparticles pollution biomonitoring studies.
... However, it has long been recognized that most earthworms and other animals living in the soil do not produce their own endogenous cellulase; instead they depend on the cellulase from their resident gut microorganism (Domínguez et al., 2005). However, endogenous cellulase genes have been recently reported from earthworm Pheretima hilgendor (Nozaki et al., 2009). Despite these newly discovered abilities, earthworm cannot assimilate lignocellulose by means of endocellulase alone, since efficient lignocellulose degradation requires synergetic action of a suite of other enzymes, including exocellulase, hemicellulase (xylanase) and lignin peroxidase (Lynd et al., 2002). ...
With advent of the nanotechnology era, the environmental risk has continuously been receiving engineered nanomaterials, as well as their derivatives. Our current understanding of the potential impact of nanomaterials and their effect on soil organism is limited. The present study fills the gap between effect of manufactured nanomaterials (NPs) and their available natural scavengers. In the study, earthworm Eisenia fetida (EW), which occupies 60 to 80% of the total biomass and well known for its contribution to cellulolytic degradation of organic wastes, was exposed to ZnO-NPs. Findings suggests that E. fetida can survive even at high exposure of ZnO-NPs (10 mg/kg) and can exhibit increase in bio-accumulation of Zn content in its body tissue with decreased NPs. Exposure of 35 and 10 nm ≥3.5 mg/kg sized NPs showed an increase in cellulase activity by 38 to 41%. This increase in cellulolytic activity in EWs’ gut may also be helpful in the bioconversion of lignocelluloses waste. Eighteen strains of cellulose hydrolytic bacteria capable of producing cellulase were obtained from the guts of EWs exposed to ZnO-NPs. The results of biochemical and 16SrRNA gene sequence examinations showed that six strains belongs to Bacillus sp.; five strains belongs to the sublines of Bacillus and others belongs to the Pseudomonas sp. The study advocates the application of ZnO-NPs enhance gut-associated microbial activity.
Key words: Cellulose hydrolytic bacteria, ZnO-NPs, E.fetida, Gut -flora.
... Many invertebrates express endogenous cellulases. Plantparasitic nematodes, cockroaches and termites were among the first to be proven to carry cellulase genes, but more recently these genes (mostly of the families GH5, 9 and 45) have also been unambiguously demonstrated in other taxa, such as other insects [44], Gastropoda [45], Crustacea [6,46,47] and Annelida [48]. The lack of large digestive gut chambers (as known from ruminants and termites) for cultivation of microbial gut symbionts in many insects or crustaceans argues that endogenous cellulases are needed in these herbivorous and detritivorous animals. ...
Organisms use diverse mechanisms involving multiple complementary enzymes, particularly glycoside hydrolases (GHs), to deconstruct lignocellulose. Lytic polysaccharide monooxygenases (LPMOs) produced by bacteria and fungi facilitate deconstruction as does the Fenton chemistry of brown-rot fungi. Lignin depolymerisation is achieved by white-rot fungi and certain bacteria, using peroxidases and laccases. Meta-omics is now revealing the complexity of prokaryotic degradative activity in lignocellulose-rich environments. Protists from termite guts and some oomycetes produce multiple lignocellulolytic enzymes. Lignocellulose-consuming animals secrete some GHs, but most harbour a diverse enzyme-secreting gut microflora in a mutualism that is particularly complex in termites. Shipworms however, house GH-secreting and LPMO-secreting bacteria separate from the site of digestion and the isopod Limnoria relies on endogenous enzymes alone. The omics revolution is identifying many novel enzymes and paradigms for biomass deconstruction, but more emphasis on function is required, particularly for enzyme cocktails, in which LPMOs may play an important role.
... In addition, they are among the most abundant soil animals in many terrestrial ecosystems and are important prey for many predators (Macdonald, 1983;Lee, 1985;Curry, 2004). Earthworms likely digest labile organic matter, such as carbohydrates and proteins, and possibly cellulose and simple phenolic compounds as well through enzymatic activities from ingested soil microbes and endogenous origin (Curry and Schmidt, 2007;Nozaki et al., 2009). ...
Natural abundance of radiocarbon (14C) has been applied to estimate the turnover time of soil carbon (C) across different climate regions. However, despite the important functional role played by soil animals in decomposition processes, little is known about variation in their 14C concentrations across different climate regions. In this study, we measured 14C concentrations of earthworms collected in three forests in Japan. In addition, we also reviewed 14C data on earthworms that were previously reported. We used these data to test whether the diet ages (defined as time elapsed since C in the diet of earthworms was fixed from atmospheric CO2 by photosynthesis) differed according to feeding habits and across study sites in various climate regions ranging from cool temperate forest to tropical savanna. Multiple regression analysis showed that the diet ages of earthworms were significantly affected by both feeding habits and study sites. The diet ages of endogeic (soil-feeding) earthworms (8.3 ± 0.4 years, mean ± SE) were significantly older than those of epigeic (litter-feeding) earthworms (2.6 ± 0.5 years), with anecic (litter-/soil-feeding) earthworms (5.7 ± 0.9 years) having intermediate diet ages. When mean diet age was compared for each feeding habit, only that of endogeic earthworms differed significantly across the sites. However, it did not necessarily become younger in warmer climate regions. These results either suggest that the degree of decomposition of soil organic matter used by earthworms differs among the study sites, or that the difference in the turnover time of soil organic C used by earthworms across the sites is relatively small and variable due to factors other than temperature, such as soil texture and vegetation.
... We chose an artificial soil microecosystem in our evaluations, since it is very difficult to control the environmental conditions in natural ecosystems. For earthworms there is an ongoing debate of whether the main cellolytic activity in the gut is host or microbial derived (9). This is the rationale for the choice to evaluate the effect of crystalline cellulose in our feeding experiments. ...
The interplay between diet, gut bacteria and health still remain enigmatic. Here, we addressed this issue through the investigation of the effect of crystalline cellulose on the earthworm Lumbricus terrestris gut microbiota composition and survival.
Earthworm gut contents were analyzed after 14 days of feeding using a mixed 16S rRNA gene sequencing approach, in addition to direct measurements of cellulase activity. The survival of earthworms was followed each week for 17 weeks.
We found a tendency that the crystalline cellulose fed earthworms survived better than the high energy fed earthworms (p=0.08). Independent of feeding we found that the bacterial group related to Ferrimonadaceae was correlated to an increased lifespan (p=0.01). We also found a positive correlation between Ruminococcaceae related bacteria and cellulase activity in the earthworm gut (p=0.05). Surprisingly, however, the cellulase activity was not correlated to the feeding regime.
Taken together, the interactions between diet, gut microbiota and lifespan seem complex.
... This activity was exclusively found in the culture supernatant (Fig. 1c), and identification of two highly similar (64% identity) CMCases CrCel9B (UniProtKB A8JFG8) and CrCel9C (UniProtKB A8JFH1) within the secretome of this alga was achieved by affinity-based enrichment on microcrystalline cellulose (Avicel) in conjunction with mass spectrometric analysis ( Fig. 1d and Supplementary Table S3). Both proteins belong to GH family 9 (GHF9) with high homology to endo-b-1,4-glucanases from lower (for example, UniProtKB Q64I76) and higher (UniProtKB O77045) termites, as well as from earthworms (UniProtKB B9A7E3) 24 . Homology-based modelling 25 identified the GHF9 endo/exocellulase CelE4 from T. fusca 26 as a structural homologue of CrCel9B and endoglucanase AaCel9A from A. acidocaldarius 27 as a homologue of CrCel9C (Supplementary Fig. S1 and Supplementary Table S1). ...
Plants convert sunlight to biomass, which is primarily composed of lignocellulose, the most abundant natural biopolymer and a potential feedstock for fuel and chemical production. Cellulose assimilation has so far only been described for heterotrophic organisms that rely on photosynthetically active primary producers of organic compounds. Among phototrophs, the unicellular green microalga Chlamydomonas reinhardtii is widely known as one of the best established model organisms. It occupies many habitats, including aquatic and soil ecosystems. This ubiquity underscores the versatile metabolic properties of this microorganism. Here we present yet another paradigm of adaptation for C. reinhardtii, highlighting its photoheterotrophic ability to utilize cellulose for growth in the absence of other carbon sources. When grown under CO(2)-limiting conditions in the light, secretion of endo-β-1,4-glucanases by the cell causes digestion of exogenous cellulose, followed by cellobiose uptake and assimilation. Phototrophic microbes like C. reinhardtii may thus serve as biocatalysts for cellulosic biofuel production.
Earthworms and their casts have been widely used for organic waste degradation and plant growth promotion. The microbial communities that reside in the guts and casts of earthworms markedly influence both applications. In the present study, next-generation sequencing was applied to identify the microbial communities in the guts and casts of three epigeic earthworm species, Eudrilus eugeniae, Perionyx excavatus, and Eisenia fetida, reared under two different feeding conditions. A total of 580 genera belonging to 43 phyla were identified. By comparing bacterial diversity among samples divided into groups based on the earthworm species, sample types, and conditions, the beta diversity analysis supported the impact of the sample type and suggested that there was significant dissimilarity of the bacterial diversity between the gut and cast. Besides, bacterial Phylum compositions within the group were compared. The result showed that the top three high relative frequency phyla found in the casts were the same regardless of earthworm species, while those found in the gut depended on both the condition and earthworm species. Focusing on the cellulolytic and plant growth-promoting bacteria, certain cellulolytic bacteria, Paenibacillus, Comamonas, and Cytophaga, were found only in the cast. Citrobacter and Streptomyces aculeolatus were detected only in the guts of earthworms reared in the bedding containing vegetables and bedding alone, respectively. Besides, Actinomadura and Burkholderia were detected only in the gut of E. eugeniae and E. fetida, respectively. The results proved that the microbial composition was affected by sample type, condition, and earthworm species. In addition, the proportion of these beneficial bacteria was also influenced by these factors. Hence, the information from this study can be used as a guide for selecting earthworm species or their casts for more efficient organic waste decomposition and plant growth promotion.
Cellulose is the most abundant polymer in the living world. Its enzymatic degradation is a crucial step in the decomposition of plant litter. Despite a wide variety of enzymatic mechanisms contributing to cellulose degradation, some general patterns driven by extracellular enzymes have emerged, allowing estimates of cellulolytic activities in environmental samples. The basic procedure described in this chapter is to incubate natural, crystalline cellulose fibres with an extract from environmental samples containing enzymes. The amount of glucose moieties being released is measured and attributed to cellulolysis. These measurements give an estimate of the potential for complete degradation of crystalline cellulose into glucose under optimal pH conditions.
Eisenia fetida earthworm is an ecotoxicologically important test species to monitor various pollutants. However, there is a little knowledge about the effects of cadmium (Cd) on earthworms at the transcriptional level. Firstly, we exposed E. fetida to soils supplemented with different concentrations (10, 30, 60 mg/kg soil) of Cd. Moreover, we depicted the characterization of gene expressions with E. fetida using high-throughput profiling of gene expression. In addition, a comparison of the gene expression profiles between each Cd treatment group and the control group suggested that differential expressional genes (DEGs) mainly enriched in enzyme activity, metabolism, oxidative stress, regeneration and apoptosis pathways. 8 DEGs from these pathways had been selected randomly to confirm the data of RNA-seq. Among these DEGs, six genes (metallothionein-2, phytochelatin synthase 1a, CuZn superoxide dismutase, sex determining region Y-box 2, sex determining region Y-box 4b, TP53-regulated inhibitor of apoptosis 1-like) up-regulated and 2 genes (beta-1,4-endoglucanase, apoptosis-stimulating of p53 protein 2-like) down-regulated in response to Cd exposure. The alteration of them indicated that earthworms could reduce the toxicity and bioavailability of Cd in polluted soil ecosystems through different pathways. This work lays an important foundation for linking earthworm transcriptional level with the ecological risk of Cd in soil ecosystem.
Introduction: Paper is one of the organic waste posing major environmental and disposal problems. Cellulose is the major constituent of paper and cellulase being the key enzyme involved in the degradation of paper is of due importance.
Aim: In this view, the effect of vermicomposting paper waste is dealt in the present study with cellulase activity and its quest of origin being observed with prime significance. Materials and Methods: The experiments were conducted in triplicates for each treatment taken, (T1)-newspaper waste and cow dung, (T2)-written paper and cow dung. The epigeic earthworm species, Eisenia foetida is used in the study. The earthworms were bred in both the treatments and their controls were devoid of worms. This setup was monitored over a period of 60 days.
Results and Conclusion: The study reveals a peak increase in the cellulase activity on the 45 th day of the experimental period in T1. Further, the Eisenia foetida gut isolate on the 60 th day was found to be of the Bacillus genera in both the treatments. The phylogenetic analysis of the retrieved cellulase gene of Eisenia foetida, Bacillus subtilis and Bacillus pumilus disclosed a close relation of Bacillus pumilus to Eisenia foetida for cellulase gene leading to an inference of horizontal gene transfer (HGT) that would have occurred between the prokaryote's (Bacteria) and eukaryote's (Earthworm) genome during the course of evolution. Thus, vermicomposting using Eisenia foetida can be regarded as an alternative method of degrading the cellulosic paper waste.
The living cell is the site of tremendous biochemical activity called metabolism. This is the process of chemical and physical change which goes on continually in the living organism. The greatest majority of these biochemical reactions do not take place spontaneously (Bennett and Frieden. Modern topics in biochemistry. Macmillan, London, pp 43–45, 1969). The phenomenon of catalysis makes possible biochemical reactions necessary for all life processes. The catalysts of biochemical reactions are enzymes and are responsible for bringing about almost all of the chemical reactions in living organisms (Holum. Elements of general and biological chemistry, 2nd edn. Wiley, New York, p 377, 1968). Without enzymes, these reactions take place at a rate far too slow for the pace of metabolism (Martinek. J Am Med Tech 31:162, 1969). All known enzymes are proteins. They are high molecular weight compounds made up principally of chains of amino acids linked together by peptide bonds (Pfeiffer. Enzymes, the physics and chemistry of life. Simon and Schuster, New York, pp 171–173, 1954). A protein molecule consists of one or more polypeptide chains which continue without interruption throughout the molecule folded into a uniquely defined configuration held together by hydrogen bonds between the peptide nitrogen and oxygen atoms also between the charged sidechains (Blow. Structure 8(4):R77–R81, 2000). It has long been assumed that most invertebrates do not possess the enzymatic complement to digest polysaccharides, but now the opposite is often shown for different groups of soil fauna, enabling us redefine species diets and therefore their ecological function. Enzymatic activities have been widely used as an index of soil fertility or ecosystem status because they are involved in biological transformation of native and foreign compounds in soils (Tate. Soil microbiology, 2nd edn. Wiley, New York, 2000). The digestive enzymes of the litter feeding animals, particularly oligochaetes, are responsible for decomposition and humification processes (Parthasarathi and Ranganathan. Trop Ecol 41(2):251–254, 2000). The interpretation of data arising from enzyme assay is complicated since enzyme activity depends on several factors and different locations of enzymes in the studied system (Nannipieri et al. Enzyme activities and microbiological and biochemical processes in soil. In: Burns RG, Dick R (eds) Enzymes in the environment. Marcel Dekker, New York, pp 1–33, 2002). So far only a few enzymatic studies on earthworm casts have been published, and they are limited to observations on soil only (Parthasarathi and Ranganathan. Trop Ecol 41(2):251–254, 2000). Some authors have described a direct role of earthworms in the decomposition of plant debris, and presume the existence of their own digestive enzymatic activities. Worms being hermaphrodites with simultaneous functioning gonads may require more energy and increased enzyme activities during this active phase of reproduction. Enzyme activity is influenced also by type of food. The differential enzyme-activity is perhaps related to the type of food and rate of eating of each species (Table 5.1). Earthworm which feed and depend on microbes, litter, and grit present in soil should contain battery of enzymes. Earthworm castings are known to be a rich source of plant growth promoting substances viz., growth hormones, enzymes and vitamins (Karthikeyan et al. AgroIndia 7:34–353, 2004). Earthworm castings also contains a number of beneficial microorganisms, nitrogen fixing, phosphorous solubilizing and cellulose decomposing organisms, which help in improving soil productivity. Earthworms have an in-house supply of enzymes like Nitrate reductase, acid phosphatase and alkaline phosphatase, which are involved in the metabolism of nitrogen and phosphate materials present in the compost. The earthworms speed up the composting process and transform wastes into nutrient rich castings with the help of the enzymes (Prabha et al. South Asian J Socio-Polit Stud 2:129–130–156, 2005).
Eisenia fetida and Eisenia andrei are vermicomposting species that are used as model animals for testing chemical material toxicology. Eisenia spp. are grown commercially in various fields in Japan. However, these two species have not been classified because it is difficult to distinguish them morphologically; thus, all bred earthworms are called E. fetida. However, it has been proposed that these two species have different expression regulation mechanisms. Here, we classified a sample of earthworms purchased from several farms, confirming that both E. fetida and E. andrei are present in Japanese earthworm breeding programs. We also characterized two highly active endoglucanases (EfEG1 and EfEG2) from the E. fetida Waki strain, which contained strong fibrinolytic enzymes for improving human health. We confirmed that EfEG1 is 1371 bp long and belongs to GHF9. Thus, E. fetida Waki may have commercial application for biomass utilization and as a dietary health supplement.
Bacteria and fungi encounter a large number of complex organic materials in soil, many of which represent a source of the energy, carbon and the nutrients that are required for cell maintenance and growth. Cellulose and lignin are the two most abundant plant polymers but contaminated soils may also contain recalcitrant xenobiotics including polycyclic aromatic hydrocarbons and polychlorinated biphenyls. However, all of these natural and anthropogenic structures are only potential substrates because bacteria and fungi cannot absorb insoluble macromolecules. Instead, they depend on the activities of extracellular enzymes to generate the soluble low molecular mass structures that are recognized by cell membrane receptors and transported into the cell. Some of the organic pollutants in soil are not only polymeric and poorly soluble but are also toxic and may require transformation prior to uptake. In this chapter the diversity of extracellular enzymes and some of the polymers that they degrade are reviewed as are the many locations and multiple fates of these enzymes once they have left the cytoplasm. We summarise the diversity of soil enzymes and describe some of the natural and synthetic macromolecules that they degrade. Aspects of the regulation of extracellular enzyme synthesis and secretion and the many locations and multiple fates of microbial enzymes after they are externalized will be outlined. The chemical, physical and biological properties of soil all affect enzyme diffusion, survival and substrate turnover, as well as the proportion of the product that is available to and assimilated by the producer cells. The ways in which microbes and their extracellular enzymes attempt to overcome the generally destructive, inhibitory and competitive properties of the soil matrix and the various strategies they adopt for effective substrate detection and utilization will be described.
Regarding to the urbanization and industrialization and population growth in the world, there isan urgent need to overcome waste materials recycling problems. Different approaches have been taken for waste evanescence. One of the successful approaches might be through vermitechnology. Earthworms are key soil invertebrates in organic matter decomposition and because of their interactions with soil microorganisms. They accelerate organic waste decomposition rates and removal of hydrocarbons especially recalcitrant ones and also petroleum derived hydrocarbons and its products. These animals use various approaches in this route, either by burrowing and feeding (direct effects) or cast ageing and aeration (indirect effects). Although, they require demanded conditions, like optimum pH, moisture, temperature, etc. Moreover, for efficient zoo remediation, a large number of worms are required. Several studies have investigated the effects of soil amended with different pollutants on earthworms such as polychlorinated bi-phenols, petroleum hydrocarbons and Polyaromatic hydrocarbons (PAH). Earthworms have the ability to degrade these materials through not well-known mechanisms. It is believed that the pollutants are degraded by the earthworms' cytochrome p450 enzymatic system activity. Hydrolases and dehydrogenase activities of earthworm enzymes as well as the earthworm microflora are responsible for organic matter decomposition. Earthworms contain beneficial microorganisms in different parts of their body such as nephridia, foregut, midgut and hindgut. So far, diverse microorganism capable of producing enzymes have been isolated from earthworms which are involved in the decomposition and degradation process including cellulase, xylanase, laccase, endo-β-1,4-mannanase. In this review, we will concentrate on the role of symbiont microorganisms besides the earthworm role in enhancing waste material degradation.
Biofuel production from plant-derived lignocellulosic material using fungal cellulases is facing cost-effective challenges related to high temperature requirements. The present study identified a cold-adapted cellulase named endo-1,4-β-glucanase (EF-EG2) from the earthworm Eisenia fetida. The gene was cloned in the cold-shock expression vector (pCold I) and functionally expressed in Escherichia coli ArcticExpress RT (DE3). The gene consists of 1368bp encoding 456 amino acid residues. The amino acid sequence shares sequence homology with the endo-1,4-β-glucanases of Eisenia andrei (98%), Pheretima hilgendorfi (79%), Perineresis brevicirris (63%), and Strongylocentrotus nudus (58%), which all belong to glycoside hydrolase family 9. Purified recombinant EF-EG2 hydrolyzed soluble cellulose (carboxymethyl cellulose), but not insoluble (powdered cellulose) or crystalline (Avicel) cellulose substrates. Thin-layer chromatography analysis of the reaction products from 1,4-β-linked oligosaccharides of various lengths revealed a cleavage mechanism consistent with endoglucanases (not exoglucanases). The enzyme exhibited significant activity at 10°C (38% of the activity at optimal 40°C) and was stable at pH 5.0-9.0, with an optimum pH of 5.5. This new cold-adapted cellulase could potentially improve the cost effectiveness of biofuel production.
Cellulose is utilized as a nutritional source by various organisms. For a long time it was believed that only protozoa, bacteria, and fungi, in addition to plants and photosynthetic bacteria, are able to synthesize cellulases encoded by their own genes. However, the widespread distribution of cellulases throughout the animal kingdom has recently been recognized. Conventionally, animals digest cellulose utilizing cellulases derived from symbiotic bacteria in the digestive organs. However, recent molecular biological studies have shown that some cellulase genes are actually encoded on animal chromosomes. In addition, the homologous primary structure of cellulases obtained from various invertebrate phyla indicates the possible vertical transfer of the cellulase gene from ancient organisms that are now extinct. The results of studies on cellulases with unique enzymatic properties are expected to be applied to bioethanol production and aquaculture. In the present review, we describe cellulases, focusing primarily on aquatic invertebrates in which both endogenous and exogenous cellulases are involved in the breakdown of cellulose in the digestive organs.
Earthworms ingest various materials in addition to food items, such as soil particles. Most earthworms of the family Megascolecidae, a dominant family in Japan, have intestinal caeca connected directly to the intestinal tract. The function of the caeca has not been demonstrated, although it is thought to be associated with digestion. We investigated the activity of the digestive enzymes amylase, phosphatase, cellulase, and protease in different regions of the gut, including the intestinal caeca, in three species of megascolecid earthworms, Pheretima heteropoda, Pheretima hilgendorfi, and Pheretima sieboldi. Activities of several enzymes were high in the intestinal caeca; in particular, protease activity was higher in the caeca than that in the anterior gut, foregut, midgut, and hindgut in all three species. Moreover, the ratio of enzyme activities in the intestinal caeca to whole-gut tended to be higher in manicate intestinal caeca than in simple intestinal caeca. These results suggest that the digestive system of earthworms relies on the intestinal caeca.
The ability of earthworms to decompose lignocellulose involves the assistance of microorganisms in their digestive system. While many studies have revealed a diverse microbiota in the earthworm gut, including aerobic and anaerobic microorganisms, it remains unclear which of these species contribute to lignocellulose digestion. In this study, aerobic microorganisms with cellulolytic activity isolated from the gut of two endogeic earthworms, Amynthas heteropoda (Megascolecidae) and Eisenia fetida (Lumbricidae) were isolated by solid culture of gut homogenates using filter paper as a carbon source. A total of 48 strains, including four bacterial and four fungal genera, were isolated from two earthworm species. Characterization of these strains using enzyme assays showed that the most representative ones had exocellulase and xylanase activities, while some had weak laccase activity. These findings suggest that earthworms digest lignocellulose by exploiting microbial exocellulase and xylanase besides their own endocellulase. Phylogenetic analysis showed that among the cellulolytic isolates in both earthworm species Burkholderia and Chaetomium were the dominant bacterial and fungal members.
Polychaetes constitute most of the benthic macroinvertebrates in estuarine and coastal environments. We investigated the utilization
of organic matter in two polychaete species, Capitella sp. I and Perinereis nuntia brevicirris, living in different coastal habitats. The protease activity of Capitella sp. I (89.7μgmg−1) was about 10 times that of P. nuntia brevicirris (8.0μgmg−1). High cellulase (endo-β-1,4-glucanase) activity was detected in P. nuntia brevicirris (3.2μgmg−1), whereas scarcely any was detected in Capitella sp. I. We isolated cDNA clones of protease mRNA from Capitella sp. I and of cellulase mRNA from P. nuntia brevicirris. The high protease activity of Capitella sp. I enabled it to survive in the sediment under a fish farm, where it degrades organic matter. In contrast, the high cellulase
activity of the estuary-dwelling P. nuntia brevicirris allowed it to degrade organic matter originating from terrestrial areas.
Earthworms are recognized to play an important role in the decomposition of organic materials. To test the use of earthworms as an indicator of plant litter decomposition, we examined the abundance and biomass of earthworms in relation to plant litter decomposition in a tropical wet forest of Puerto Rico. We collected earthworms at 0-0.1 m and 0.1-0.25 m soil depths from upland and riparian sites that represent the natural variation in soils and decomposition rates within the forest. Earthworms were hand-sorted and weighed for both fresh and dry biomass. Earthworms were dominated by the exotic endogeic species Pontoscolex corethrurus Müller; they were more abundant, and had higher biomasses in the upland than in riparian sites of the forest. Plant leaf litter decomposed faster in the upland than riparian sites. We found that earthworm abundance in the upper 0.1 m of the soil profile positively correlated with decomposition rate of plant leaf litter. Ground litter removal had no effect on the abundance or biomass of endogeic earthworms. Our data suggest that earthworms can be used to predict decomposition rates of plant litter in the tropical wet forest, and that the decomposition of aboveground plant litter has little influence on the abundance and biomass of endogeic earthworms.
β-1,4-Endoglucanases (EGases, EC 3.2.1.4) degrade polysaccharides possessing β-1,4-glucan backbones such as cellulose and xyloglucan and have been found among extremely variegated taxonomic groups. Although many animal species depend on cellulose as their main energy source, most omnivores and herbivores are unable to produce EGases endogenously. So far, all previously identified EGase genes involved in the digestive system of animals originate from symbiotic microorganisms. Here we report on the synthesis of EGases in the esophageal glands of the cyst nematodes Globodera rostochiensis and Heterodera glycines. From each of the nematode species, two cDNAs were characterized and hydrophobic cluster analysis revealed that the four catalytic domains belong to family 5 of the glycosyl hydrolases (EC 3.2.1, 3.2.2, and 3.2.3). These domains show 37–44% overall amino acid identity with EGases from the bacteria Erwinia chrysanthemi, Clostridium acetobutylicum, and Bacillus subtilis. One EGase with a bacterial type of cellulose-binding domain was identified for each nematode species. The leucine-rich hydrophobic core of the signal peptide and the presence of a polyadenylated 3′ end precluded the EGases from being of bacterial origin. Cyst nematodes are obligatory plant parasites and the identified EGases presumably facilitate the intracellular migration through plant roots by partial cell wall degradation.
• endoglucanase
• Globodera
• Heterodera
• in situ hybridization
• secretory protein
Endoglucanase D (EC 3.2.1.4; EGD) from Clostridium thermocellum is rapidly (k = 216 M-1.min-1) and almost completely (greater than 95%) inactivated with Woodward's reagent K (N-ethyl-5-phenylisoxazolium-3'-sulphonate). Spectrophotometric analysis at 340 nm reveals that eight carboxy residues react, whereas specific ligands protect one residue against modification. The enzyme retains it full activity under the latter conditions. The kinetics and pH-dependence of inactivation point towards the involvement of one or more essential carboxy groups with a pKa of 5.7-5.8. Samples modified in the absence or presence of ligand were analysed by reversed-phase liquid chromatography after proteolysis with subtilisin. Dual-wavelength monitoring at 214 and 340 nm during this fractionation leads to the identification of a putatively active-site peptide (Gly-508-Ala-562) which was further characterized by amino acid and partial N-terminal sequence analyses. Asp-546 and Glu-555 are postulated as possible active-site residues. This follows from alignments using ten endoglucanase sequences belonging to the same family. Strong local conservation suggests that this C-terminal sequence is structurally and/or functionally important.
Diethylpyrocarbonate modification of endoglucanase D from Clostridium thermocellum, cloned in Escherichia coli, resulted in a rapid but partial (maximally 70-80%) loss of activity. The second-order rate constant of inactivation proved to be exceptionally high (3210 M-1.min-1). A 3-fold reduction of the kcat and a 2-fold increase of the Km for 2'-chloro-4'-nitrophenyl beta-cellobioside were observed. Spectrophotometric analysis indicate the presence of one rapidly (k = 0.45 min-1) and two slower (k = 0.23 min-1) reacting histidyl residues. In the presence of 50 mM methyl beta-cellotrioside, the rate of inactivation was reduced 16-fold, and the kinetics of modification were compatible with the protection of 1 histidyl residue. Since peptide analysis was inconclusive, identification of the critical residue was attempted by site-directed mutagenesis. Each of the 12 histidyl residues present in the endoglucanase D sequence was mutated into either Ala or Ser. Seven of the mutant enzymes had specific activities lower than 50% of the wild-type. Only in the case of the Ser-516 mutant, however, was the residual activity not affected by diethyl pyrocarbonate. These findings suggest an important functional or structural role for His-516 in the wild-type enzyme.
We have developed a new method for the identification of signal peptides and their cleavage sites based on neural networks
trained on separate sets of prokaryotic and eukaryotic sequence. The method performs significantly better than previous prediction
schemes and can easily be applied on genome-wide data sets. Discrimination between cleaved signal peptides and uncleaved N-terminal
signal-anchor sequences is also possible, though with lower precision. Predictions can be made on a publicly available WWW
server.
Cellulase E4 from Thermomonospora fusca is unusual in that it has characteristics of both exo- and endo-cellulases. Here we report the crystal structure of a 68K M(r) fragment of E4 (E4-68) at 1.9 A resolution. E4-68 contains both a family 9 catalytic domain, exhibiting an (alpha/alpha)6 barrel fold, and a family III cellulose binding domain, having an antiparallel beta-sandwich fold. While neither of these folds is novel, E4-68 provides the first cellulase structure having interacting catalytic and cellulose binding domains. The complexes of E4-68 with cellopentaose, cellotriose and cellobiose reveal conformational changes associated with ligand binding and allow us to propose a catalytic mechanism for family 9 enzymes. We also provide evidence that E4 has two novel characteristics: first it combines exo- and endo-activities and second, when it functions as an exo-cellulase, it cleaves off cellotetraose units.
beta-1,4-Endoglucanases (EGases, EC 3.2.1.4) degrade polysaccharides possessing beta-1,4-glucan backbones such as cellulose and xyloglucan and have been found among extremely variegated taxonomic groups. Although many animal species depend on cellulose as their main energy source, most omnivores and herbivores are unable to produce EGases endogenously. So far, all previously identified EGase genes involved in the digestive system of animals originate from symbiotic microorganisms. Here we report on the synthesis of EGases in the esophageal glands of the cyst nematodes Globodera rostochiensis and Heterodera glycines. From each of the nematode species, two cDNAs were characterized and hydrophobic cluster analysis revealed that the four catalytic domains belong to family 5 of the glycosyl hydrolases (EC 3.2.1, 3.2.2, and 3.2.3). These domains show 37-44% overall amino acid identity with EGases from the bacteria Erwinia chrysanthemi, Clostridium acetobutylicum, and Bacillus subtilis. One EGase with a bacterial type of cellulose-binding domain was identified for each nematode species. The leucine-rich hydrophobic core of the signal peptide and the presence of a polyadenylated 3' end precluded the EGases from being of bacterial origin. Cyst nematodes are obligatory plant parasites and the identified EGases presumably facilitate the intracellular migration through plant roots by partial cell wall degradation.
A single injection of human chorionic gonadotropin (HCG) can induce complete spermatogenesis in immature Japanese eel (Anguilla japonica) testes consisting of only premitotic spermatogonia. Proliferation of spermatogonia, meiosis and spermiogenesis begin on 3, 12 and 18 days after HCG injection, respectively. To isolate the genes responsible for regulating the initiation of meiosis, differential mRNA display using poly (A)+ RNA extracted from testes of eels at different times after HCG treatment was carried out. Five cDNA clones in which expression was initiated before the onset of meiosis were obtained. Northern blot analysis showed that one clone, which encoded activin betaB subunit, was expressed in the initial phase of spermatogenesis (1-6 days after HCG treatment), in agreement with the previous suggestion that activin B induces the initiation of spermatogenesis in the Japanese eel. The remaining four were expressed in the testes during the following time frames: 3-18 days (two clones), 6-18 days (one clone) and 9-18 days (one clone) after HCG treatment. One of the two clones expressed on day 3 exhibited strong expression on days 12 and 15, just at the initiation period of meiosis. This clone was selected as a candidate gene responsible for initiating meiosis, and its full-length cDNA isolated. The cDNA contained an open reading frame of 1571 nucleotides encoding a protein of 260 amino acid residues, which showed high homology with the proliferating cell nuclear antigen (PCNA) of human, mouse and Xenopus. Northern blot analysis using eel PCNA cDNA showed that a 1.6 kb transcript first appeared on day 3 and became abundant, reaching maximum levels on days 12-15. In situ hybridization analysis revealed that PCNA mRNA was expressed strongly in late type B spermatogonia before the sixth mitotic division. It has already been shown that spermatogonia have a regulatory point to enter meiosis between the fifth and sixth mitotic division. The coincidence of PCNA expression and this regulatory point suggests an involvement of PCNA in the progression of mitotic germ cells into meiosis during HCG-induced spermatogenesis in the eel.
Current cell-free protein synthesis systems can synthesize proteins with high speed and accuracy, but produce only a low yield because of their instability over time. Here we describe the preparation of a highly efficient but also robust cell-free system from wheat embryos. We first investigated the source of the instability of existing systems in light of endogenous ribosome-inactivating proteins and found that ribosome inactivation by tritin occurs already during extract preparation and continues during incubation for protein synthesis. Therefore, we prepared our system from extensively washed embryos that are devoid of contamination by endosperm, the source of tritin and possibly other inhibitors. In a batch system, we observed continuous translation for 4 h, and sucrose density gradient analysis showed formation of large polysomes, indicating high protein synthesis activity. When the reaction was performed in a dialysis bag, enabling the continuous supply of substrates together with the continuous removal of small byproducts, translation proceeded for >60 h, yielding 1-4 mg of enzymatically active proteins, and 0.6 mg of a 126-kDa tobacco mosaic virus protein, per milliliter of reaction volume. Our results demonstrate that plants contain endogenous inhibitors of translation and that after their elimination the translational apparatus is very stable. This contrasts with the common belief that cell-free translation systems are inherently unstable, even fragile. Our method is useful for the preparation of large amounts of active protein as well as for the study of protein synthesis itself.
Previous dogma has maintained that cellulose, ingested by xylophagous or herbivorous animals, is digested by cellulolytic symbiotes. The first evidence in conflict with this contention involved the demonstration of cellulolytic activities in symbiote-free secreting organs (e.g., the salivary glands of termites) or defaunated guts. Following these demonstrations, possible endogenous cellulase components were purified from several cellulose-digesting invertebrates, but this research did little to change the general view concerning animal cellulose digestion. Thanks to recent developments in molecular biology, the existence of cellulases of animal origin has been firmly established. To date, cellulase genes have been reported from arthropods (insects and a crayfish) and nematodes. This paper describes and discusses the presence and nature of endogenous cellulases in higher animals.
Contrary to conventional wisdom, it has been shown recently that termites do not necessarily depend on symbiotic bacteria to process cellulose. They secrete their own cellulases, mainly endo-beta-1,4-glucanase and beta-1,4-glucosidase. Here, the first structure of an endogenous endoglucanase from the higher termite Nasutitermes takasagoensis (NtEgl) is reported at 1.40 A resolution. NtEgl has the general folding of an (alpha/alpha)(6) barrel, which is a common folding pattern for glycosyl hydrolase family 9. Three-dimensional structural analysis shows that the conserved Glu412 is the catalytic acid/base residue and the conserved Asp54 or Asp57 is the base. The enzyme has a Ca(2+)-binding site near its substrate-binding cleft. Comparison between the structure of the Ca(2+)-free enzyme produced by reducing the pH of the soaked crystal from 5.6 (the pH of optimum enzyme activity) to 2.5 with that of the Ca(2+)-bound enzyme did not show significant differences in the locations of the C(alpha) atoms. The main differences are in the conformation of the residue side chains ligating the Ca(2+) ion. The overall structure of NtEgl at pH 6.5 is similar to that at pH 5.6. The major change observed was in the conformation of the side chain of the catalytic acid/base Glu412, which rotates from a hydrophobic cavity to a relatively hydrophilic environment. This side-chain displacement may decrease the enzyme activity at higher pH.
The relictual Mastotermes darwiniensis is one of the world's most destructive termites. Like all phylogenetically basal termites, it possesses protozoa in its hindgut,
which are believed to help it digest wood. L. Li, J. Frohlich, P. Pfeiffer, and H. Konig (Eukaryot. Cell 2:1091-1098, 2003) recently cloned the genes encoding cellulases from the protozoa of M. darwiniensis; however, they claimed that these genes are essentially inactive, not contributing significantly to cellulose digestion.
Instead, they suggested that the protozoa sequester enzymes produced by the termite in its salivary glands and use these to
degrade cellulose in the hindgut. We tested this idea by performing gel filtration of enzymes in extracts of the hindgut,
as well as in a combination of the salivary glands, foregut, and midgut. Three major cellulases were found in the hindgut,
each of which had a larger molecular size than termite-derived salivary gland enzymes. N-terminal amino acid sequencing of
one of the hindgut-derived enzymes showed that it was identical to the putative amino acid sequence of one mRNA sequence isolated
by Li et al. (Eukaryot. Cell 2:1091-1098, 2003). The overall activity of the hindgut cellulases was found to be of approximately equal magnitude to the
termite-derived cellulases detected in the mixture of salivary gland, foregut, and midguts. Based on these results, we conclude
that, contrary to Li et al. (Eukaryot. Cell 2:1091-1098, 2003), the hindgut protozoan fauna of M. darwiniensis actively produce cellulases, which play an important role in cellulose digestion of the host termite.
The intestinal flagellates of termites produce cellulases that contribute to cellulose digestion of their host termites. However, 75% of all termite species do not harbour the cellulolytic flagellates; the endogenous cellulase secreted from the midgut tissue has been considered a sole source of cellulases in these termites. Using the xylophagous flagellate-free termites Nasutitermes takasagoensis and Nasutitermes walkeri, we successfully solubilized cellulases present in the hindgut pellets. Zymograms showed that the hindguts of these termites possessed several cellulases and contained up to 59% cellulase activity against crystalline cellulose when compared with the midgut. Antibiotic treatment administered to N. takasagoensis significantly reduced cellulase activity in the hindgut, suggesting that these cellulases were produced by symbiotic bacteria.
Higher cellulase activities are found in the gut wall of the epigeic than of the endogeic earthworms, except for epigeic Lumbricus rubellus, which shows a very low enzymatic activity. -from Author
Cellulolytic microorganisms play an important role in the biosphere by recycling cellulose, the most abundant carbohydrate produced by plants. Cellulose is a simple polymer, but it forms insoluble, crystalline microfibrils, which are highly resistant to enzymatic hydrolysis. All organisms known to degrade cellulose efficiently produce a battery of enzymes with different specificities, which act together in synergism. The study of cellulolytic enzymes at the molecular level has revealed some of the features that contribute to their activity. In spite of a considerable diversity, sequence comparisons show that the catalytic cores of cellulases belong to a restricted number of families. Within each family, available data suggest that the various enzymes share a common folding pattern, the same catalytic residues, and the same reaction mechanism, i.e. either single substitution with inversion of configuration or double substitution resulting in retention of the β-configuration at the anomeric carbon. An increasing number of three-dimensional structures is becoming available for cellulases and xylanases belonging to different families, which will provide paradigms for molecular modeling of related enzymes. In addition to catalytic domains, many cellulolytic enzymes contain domains not involved in catalysis, but participating in substrate binding, multi-enzyme complex formation, or possibly attachment to the cell surface. Presumably, these domains assist in the degradation of crystalline cellulose by preventing the enzymes from being washed off from the surface of the substrate, by focusing hydrolysis on restricted areas in which the substrate is synergistically destabilized by multiple cutting events, and by facilitating recovery of the soluble degradation products by the cellulolytic organism. In most cellulolytic organisms, cellulase synthesis is repressed in the presence of easily metabolized, soluble carbon sources and induced in the presence of cellulose. Induction of cellulases appears to be effected by soluble products generated from cellulose by cellulolytic enzymes synthesized constitutively at a low level. These products are presumably converted into true inducers by transglycosylation reactions. Several applications of cellulases or hemicellulases are being developed for textile, food, and paper pulp processing. These applications are based on the modification of cellulose and hemicellulose by partial hydrolysis. Total hydrolysis of cellulose into glucose, which could be fermented into ethanol, isopropanol or butanol, is not yet economically feasible. However, the need to reduce emissions of greenhouse gases provides an added incentive for the development of processes generating fuels from cellulose, a major renewable carbon source.
The tropical endogeic earthworms have developed strong mutualism with the ingested microflora to digest soil organic matter. In order to determine the role of the intestinal mucus in these mutualistic relationships, glycolytic enzymatic activities have been assayed in the gut contents of adult #Millsonia anomala$ from Lamto (Côte d'Ivoire). Twenty-one tested substrates selected for their plant origin were broken down, which indicates that this species may use root and fungal substrates available in soils. With the aim of comparing the origins of these glycolytic enzymes found in the gut, the intestinal wall tissues were cultured in vitro and enzymatic activities were measured in both cultured tissues and culture media. #M. anomala$ cannot produce cellulase and mannanase and use instead the digestive enzymatic capabilities of the ingested microflora which synthesize extracellular glycolytic enzymes. These enzyme capabilities are similar to those of #Pontoscolex corethrurus$, and inferior to #Polypheretima elongata$ that produces cellulase and mannanase. (Résumé d'auteur)
The gut mucus production of different earthworms species has been measured in order to verify the mutualistic hypothesis between earthworms and soil microflora. Earthworms of different origin (tropical and temperate) and ecological category (endogeic, anecic and epigeic), individuals of the same species occuping different soils, and earthworms of different species from the same soil, have been studied. Intestinal mucus production suggests the existence of the mutualistic digestion system. The mucus percentage in the gut was greater in the epigeic than in both anecic and endogeic. Nevertheless, the relative enrichment was higher in the endogeic species. The organic matter content of the substratum seems to play a regulating role in the mutualistic system, because the greater the substratum organic matter content the lower the relative enrichment. Generally, in temperate zones more mucus was added to the substratum, suggesting that the low temperature do necessary a greater microflora stimulation.
Endogeic geophagous earthworms from tropical areas seem to digest soil organic matter through a mutualist earthworm microflora-digestion system and the intestinal mucus produced by earthworms was supposed to play a central role in the process of digestion. A large range of glucosidic substrates characteristic of plant material was used to reveal the activities of digestive enzymes in the gut (wall and contents) of Polypheretima elongata. This worm consumes some plant substrates tested and is able mainly to degrade root and fungal substrates. It corroborates that tropical endogeic earthworms feed on litter debris and soils poor in organic matter. These glucosidic activities were higher than those found previously in Pontoscolex corethrurus. The in vitro tissue culture of gut wall allowed us to infer that P. elongata can synthesize by itself all its extra and intracellular enzymes, contrary to P. corethrurus which requires the microflora of the soil ingested in order to hydrolyse some substrates such as cellulose and mannan. It should be interesting to compare cellulases and mannanases of both earthworms after extraction and purification and to study the mechanisms by which P. corethrurus may enhance microbial activities.
Some of the enzymes found in the gut contents of endogeic geophagous earthworms are produced by ingested microflora. This study compares the origin and activities of glucosidic enzymes present in the gut contents of adult Polypheretima elongata from Sainte Anne (Martinique), Pontoscolex corethrurus from Palma Sola, Veracruz (Mexico) and Millsonia anomala from Lamto (Côte d'Ivoire). Substrates characteristic of plant material were used to compare enzymatic capabilities of gut contents and isolated gut tissues. All substrates tested were digested which indicates that all three species may use root and fungal substrates available in soils. In vitro tissue culture of P. elongata produced all the glucosidic enzymes unlike cultures of the other two species which required microbial activity to synthetize mannanase and cellulase; moreover, glucosidic activities measured in P. elongata were higher than those found in P. corethrurus and M. anomala.
The water extract of the crude high-protein (68·80%)-containing dry powder of Lumbricus rubellus (USA) was found to contain cellulase, chitinase, protease and amylase. Cellulase activity was present at high levels (940 units/ml) and prompted comparative studies with cellulase from the culture filtrate of the cellulase-rich Trichoderma viride BRC-25, which was present at 2390 units/ml. A partial purification of the cellulase from both these sources by acetone precipitation followed by ammonium sulphate precipitation at different saturation levels was carried out: 40–80% ammonium sulphate precipitates of both sources had an optimum pH of 6 and temperature of 37°C. Filter paper was the best substrate for both the enzymes. The Km for 40–80% ammonium sulphate fraction for L. rubellus (USA) and T. viride BRC-25 enzymes were 9 and 6·75 mg/ml, respectively.
SUMMARY Microbiological, physical and chemical changes were followed in worm casts ageing in the field. Filamentous fungi and yeasts increased in number rapidly after the cast was produced, but not bacteria or actinomycetes which were initially numerous. Measurements of hyphal length confirmed the increased growth of fungi. Ageing casts showed a declining respiratory activity, possibly because the bacteria formed resting stages. Aggregate stability increased rapidly as casts age, probably due to increasing amounts of fungal hyphae. Polysaccharide content of casts was much greater than that of soil, but did not vary with changes in stability. Total and mineral nitrogen levels of casts were greater than those of soil; the major part of the inorganic nitrogen occurred as ammonia which was rapidly converted to nitrate.
Soil-engineering organisms (earthworms, termites and ants) affect the soil and litter environment indirectly by the accumulation of their biogenic structures (casts, pellets, galleries, crop sheetings nests.). An enzymatic typology was conducted on six types of biogenic structures: casts produced by two earthworms (Andiodrilus sp. and Martiodrilus sp.), a nest built by a soil-feeding termite (Spinitermes sp.), crop galleries built by another soil-feeding termite (Ruptitermes sp.) and soil pellets produced by two species of leaf-cutting ant (Acromyrmex landolti and Atta laevigata) and an control soil from a natural Colombian savanna. A total of 10 enzymes (xylanase, amylase, cellulase, a-glucosidase, b-glucosidase, b-xylosidase, N-acteyl-glucosaminidase, alkaline and acid phosphatases and laccase) were selected to characterize the functional diversity of the biogenic structures. Our results showed that (i) Martiodrilus casts were characterized by a broad enzymatic profile that was different from that of the soil. (ii) A. laevigata pellets and termite structures had a profile broadly similar to the soil only with some enzymes (iii) Andiodrilus casts had an enzyme profile very similar to that of the soil. These results suggest that the functional diversity of these structures is related to differences between species and not to differences between taxonomic groups. For the first time, we evaluated differences in enzyme typology between biogenic structures collected on the same site but produced by different organisms. These differences suggested species dependent pathways for the decomposition of organic matter.
Rochelle salt, normally present in the dinitrosalicylic acid reagent for reducing sugar, interferes with the protective action of the sulfite, but is essential to color stability. The difficulty may be resolved either by eliminating Rochelle salt from the reagent and adding it to the mixture of reducing sugar and reagent after the color is developed, or by adding known amounts of glucose to the samples of reducing sugar to compensate for the losses sustained in the presence of the Rochelle salt. The optimal composition of a modified dinitrosalicylic acid reagent is given.
Cellulose digestion by Corbicula japonica was investigated according to the hypothesis that without any symbiotic aid, this organism can utilize cellulose as a carbon source. Enzymatic studies revealed the complete cellulase activity of this species, and molecular cloning resulted in the isolation of cDNA with an ORF encoding a 596-amino-acid protein that shares significant homology with abalone and termite cellulases with an amino acid identity of 52.2% and 50.5%, respectively. The isolated cellulase had a carbohydrate-binding module at the N-terminal region that was also reportedly present in abalone cellulase, and its mRNA were specifically expressed in the digestive gland. These findings strongly support the assumption that C. japonica has an endogenous cellulose, as well as abalones and termites. It is further believed that C. japonica plays an important roll in decomposing cellulose, and consequently contributes to the carbon-cycle in the aquatic environment, as termites do in terrestrial forests.
Termites, ants and earthworms are considered as soil engineers because of their effects on soil properties and their influence on the availability of resources for other organisms, including microorganisms and plants. However, the links between their impacts on the soil environment and the resulting modification of natural selection pressures on engineer as well as on other organisms have received little attention.We suggest that the strategy evolved by species, or functional groups to control their environment can explain the difference between extended phenotype and accidental engineers. Extended phenotype engineers concentrate their activities on the building of biogenic structures in order to maintain optimal conditions for their growth. Conversely, accidental engineers expend energy in moving through the soil to be as close as possible to their optimal environment. We discuss in this paper why termites and ants are best viewed as extended phenotype engineers, and particularly how their impacts on ecosystem functioning can be considered as results of their requirements and how their activities could induce feedback loops affecting themselves. We also focus on the engineering activities of earthworms and discuss why some species can be considered as extended phenotype while others are more accidental engineers.Finally, we discuss links between the strategies developed by engineers and ecosystem functioning. Although both types of engineer create “hot-spots” in soil, we argue that extended phenotype engineers have a greater effect on the maintenance of ecosystem heterogeneity since they concentrate their activities at a few points, as compared to accidental engineers which may move through the soil and thus contribute to homogenisation of nutrient distribution throughout the whole ecosystem.
Bands that have cellulolytic activity are visualized after polyacrylamide gel electrophoresis by laying the slab gel on top of a thin sheet of 2% agar containing 0.1% carboxymethylcellulose. After a suitable incubation time, zones of carboxymethylcellulose hydrolysis are revealed by staining the agar replica with Congo red.
Invertebrates play significant, but largely ignored, roles in the delivery of ecosystem services by soils at plot and landscape scales. They participate actively in the interactions that develop in soil among physical, chemical and biological processes. We show that soils have all the attributes of self-organized systems as proposed by Perry (Trends Ecol. Evol. 10 (1995) 241) and detail the scales at which invertebrates operate and the different kinds of ecosystem engineering that they develop. This comprehensive analysis of invertebrate activities shows that they may be the best possible indicators of soil quality. They should also be considered as a resource that needs to be properly managed to enhance ecosystem services provided by agro-ecosystems.
1. Soil organisms, such as earthworms, accelerate mineralization of soil organic matter and are thought to be beneficial for plant growth. This has been shown in short-term microcosm experiments. It is thus legitimate to ask whether these increases in plant growth are due to brief pulses of mineralization or whether these increases are long-lasting.
2. This question was addressed using a system of differential equations modelling the effects of decomposers on nutrient cycling via trophic (nutrient assimilation) and nontrophic effects (through their ecosystem engineering activities).
3. The analytical study of this model showed that these processes increase primary production in the long term when they recycle nutrients efficiently, allowing a small fraction of the recycled nutrients to be leached out of the ecosystem.
4. Mineralization by the ecosystem engineering activities of decomposers seems to deprive them of a resource. However, it was shown that a decomposer may increase its own biomass, through its ecosystem engineering activities, provided the created recycling loop is efficient enough.
5. Mechanisms through which earthworms may modify the efficiency of nutrient cycling are discussed. The necessity of measuring the effect of earthworms on the nutrient input–output balance of ecosystems under field conditions is emphasized.
The glycolytic enzymatic activities in the gut of the endogeic earthworm #Hormogaster elisae$ from El Molar (Madrid, Spain) were studied in order to determine its digestive capacity and to assess its alimentary regime. Most endogeic earthworms have weak enzymatic complement and they usually establish mutualistic relationships with soil microflora to digest some organic compounds. Therefore, the intestinal wall tissues were cultured in vitro to assess the origin of the glycolytic enzymes found in the gut and enzymatic activities were measured in both cultured tissues and culture media. #H. elisae$ had a wide but not very strong enzyme complement, since all substrates were degraded but most of them at a low rate. This species cannot produce cellulase and mannamase, so for the digestion of these substrates it probably uses the digestive enzymatic capabilities of the ingested microflora. (Résumé d'auteur)
The sensitivity of the commonly used progressive multiple sequence alignment method has been greatly improved for the alignment
of divergent protein sequences. Firstly, individual weights are assigned to each sequence in a partial alignment in order
to downweight near-duplicate sequences and up-weight the most divergent ones. Secondly, amino acid substitution matrices are
varied at different alignment stages according to the divergence of the sequences to be aligned. Thirdly, residue-specific
gap penalties and locally reduced gap penalties in hydrophilic regions encourage new gaps in potential loop regions rather
than regular secondary structure. Fourthly, positions in early alignments where gaps have been opened receive locally reduced
gap penalties to encourage the opening up of new gaps at these positions. These modifications are incorporated into a new
program, CLUSTAL W which is freely available.
Cellulolytic microorganisms play an important role in the biosphere by recycling cellulose, the most abundant carbohydrate produced by plants. Cellulose is a simple polymer, but it forms insoluble, crystalline microfibrils, which are highly resistant to enzymatic hydrolysis. All organisms known to degrade cellulose efficiently produce a battery of enzymes with different specificities, which act together in synergism. The study of cellulolytic enzymes at the molecular level has revealed some of the features that contribute to their activity. In spite of a considerable diversity, sequence comparisons show that the catalytic cores of cellulases belong to a restricted number of families. Within each family, available data suggest that the various enzymes share a common folding pattern, the same catalytic residues, and the same reaction mechanism, i.e. either single substitution with inversion of configuration or double substitution resulting in retention of the beta-configuration at the anomeric carbon. An increasing number of three-dimensional structures is becoming available for cellulases and xylanases belonging to different families, which will provide paradigms for molecular modeling of related enzymes. In addition to catalytic domains, many cellulolytic enzymes contain domains not involved in catalysis, but participating in substrate binding, multi-enzyme complex formation, or possibly attachment to the cell surface. Presumably, these domains assist in the degradation of crystalline cellulose by preventing the enzymes from being washed off from the surface of the substrate, by focusing hydrolysis on restricted areas in which the substrate is synergistically destabilized by multiple cutting events, and by facilitating recovery of the soluble degradation products by the cellulolytic organism. In most cellulolytic organisms, cellulase synthesis is repressed in the presence of easily metabolized, soluble carbon sources and induced in the presence of cellulose. Induction of cellulases appears to be effected by soluble products generated from cellulose by cellulolytic enzymes synthesized constitutively at a low level. These products are presumably converted into true inducers by transglycosylation reactions. Several applications of cellulases or hemicellulases are being developed for textile, food, and paper pulp processing. These applications are based on the modification of cellulose and hemicellulose by partial hydrolysis. Total hydrolysis of cellulose into glucose, which could be fermented into ethanol, isopropanol or butanol, is not yet economically feasible. However, the need to reduce emissions of greenhouse gases provides an added incentive for the development of processes generating fuels from cellulose, a major renewable carbon source.
The traditional view of cellulose digestion in animals is that they cannot produce their own cellulase, and so rely on gut microorganisms to hydrolyse cellulose. A classic example of this symbiosis is that between phylogenetically lower termites and the unicellular organisms (protists) that colonize their hindguts: cellulose fermented to acetate by the protists can be used as an energy source by the termite. There is evidence for the production of endogenous cellulase components by termites and other wood-feeding insects; however, an unambiguous origin for such enzymes has not been established, to our knowledge, until now. Here we describe the first insect cellulase-endoding gene to be identified, RsEG, which encodes an endo-beta-1,4-glucanase (EC 3.2.1.4) in the termite Reticulitermes speratus.
Amino acid sequences of cellulases have been determined in insects, nematodes, plants, slime moulds and bacteria but not in crustaceans. However, cellulase activity has been demonstrated in the hepatopancreas of the red claw crayfish, Cherax quadricarinatus. In order to obtain information on the nature of this cellulase, a C. quadricarinatus hepatopancreas cDNA library was screened with a PCR product generated using degenerate oligonucleotide primers derived from conserved regions of known cellulases. Two identical 1.56kb cDNAs with sequence similarities to known cellulases, particularly the termite endoglucanases, were identified and sequenced. The clones contain the complete cDNA open reading frame for an endo-1, 4-beta-glucanase of 469 amino acids termed Cherax quadricarinatus endoglucanase (CqEG). The endogenous origin of the gene was confirmed by PCR amplification and sequencing of a 1012bp PCR product from genomic DNA. This fragment contains four exon sequences identical to the cDNA and is interrupted by three introns of 371, 102, 194bp respectively, with one intron exhibiting typical eukaryotic splice sites. The isolation of an endo-1,4-beta-glucanase encoding cDNA from the crayfish C. quadricarinatus provides the first endogenous cellulase sequence in a crustacean species.
Using polymerase chain reaction, cloning and sequencing techniques, a complementary DNA encoding a low molecular mass cellulase (endo-1,4-beta-D-glucanase, EC 3.2.1.4) has been identified in the digestive gland of the marine mussel, Mytilus edulis. It contains a 5' untranslated region, a 633-nucleotide ORF encoding a 211 amino-acid protein, including a 17 amino-acid signal peptide and a complete 3' untranslated region. At the C-terminal end of the purified mature protein, a 13 amino-acid peptide is lacking in comparison to the protein sequence deduced from the ORF. This peptide is probably removed as a consequence of post-translational amidation of the C-terminal glutamine. The endoglucanase genes have been isolated and sequenced from both Swedish and French mussels. The coding parts of these two sequences are identical. Both genes contain two introns, the positions of which are conserved. However the length of the introns are different due to base substitutions, insertions or deletions showing the existence of interspecies length polymorphism. The percentage of similarity for the introns of the two gene sequences is 96.9%. This is the first time a molluscan cellulase is characterized at DNA level. Amino acid sequence-based classification has revealed that the enzyme belongs to the glycosyl hydrolase family 45 [B. Henrissat (Centre de Recherches sur les Macromolecules Végétales, CNRS, Joseph Fourier Université, Grenoble, France), personal communication]. There is no cellulose binding domain associated with the sequence.
The distribution of endo-beta-1,4-glucanase (EG) components in the digestive system of the wood-feeding termite, Coptotermes formosanus Shiraki, was investigated by zymogram analysis using polyacrylamide gel electrophoresis, followed by N-terminal protein sequencing. EG components similar to glycoside hydrolase family (GHF) 9 members were restricted to the salivary glands, the foregut, and the midgut, whereas components similar to GHF7 members were confined to the hindgut where numerous cellulolytic flagellates were harbored. RT-PCR experiments revealed that five GHF9 EG mRNAs (1348 bp) homologous to other termite EGs were expressed in the salivary glands and the midgut. The crude extract prepared from the midgut as well as that from the hindgut produced glucose from crystalline cellulose. These data suggest that C. formosanus has two independent cellulose-digesting systems: one in the midgut where cellulose digestion is accomplished by endogenous cellulases and the other in the hindgut which makes use of other cellulases possibly from symbiotic flagellates.
We report a cell-free system for the high-throughput synthesis and screening of gene products. The system, based on the eukaryotic translation apparatus of wheat seeds, has significant advantages over other commonly used cell-free expression systems. To maximize the yield and throughput of the system, we optimized the mRNA UTRs, designed an expression vector for large-scale protein production, and developed a new strategy to construct PCR-generated DNAs for high-throughput production of many proteins in parallel. The resulting system achieves high-yield expression and can maintain productive translation for 14 days. Additionally, in the integration of a PCR-directed system for template creation, at least 50 genes can be translated in parallel, yielding between 0.1 and 2.3 mg of protein by one person within 2 days. Assessment of correct protein folding by the products of this high-throughput protein-expression system were performed by enzymatic assays of kinases and by NMR spectroscopic analysis. The cell-free system, reported here, bypasses many of the time-consuming cloning steps of conventional expression systems and lends itself to a robotic automation for the high-throughput expression of proteins.
A cellulase [endo-beta-1,4-D-glucanase (EC 3.2.1.4)] was isolated from the hepatopancreas of abalone Haliotis discus hannai by successive chromatographies on TOYOPEARL CM-650M, hydroxyapatite and Sephacryl S-200 HR. The molecular mass of the cellulase was estimated to be 66 000 Da by SDS/PAGE, thus the enzyme was named HdEG66. The hydrolytic activity of HdEG66 toward carboxymethylcellulose showed optimal temperature and pH at 38 degrees C and 6.3, respectively. cDNAs encoding HdEG66 were amplified by the polymerase chain reaction from an abalone hepatopancreas cDNA library with primers synthesized on the basis of partial amino-acid sequences of HdEG66. By overlapping the nucleotide sequences of the cDNAs, a sequence of 1898 bp in total was determined. The coding region of 1785 bp located at nucleotide position 56-1840 gave an amino-acid sequence of 594 residues including the initiation methionine. The N-terminal region of 14 residues in the deduced sequence was regarded as the signal peptide as it was absent in HdEG66 protein and showed high similarity to the consensus sequence for signal peptides of eukaryote secretory proteins. Thus, matured HdEG66 was thought to consist of 579 residues. The C-terminal region of 453 residues in HdEG66, i.e. approximately the C-terminal three quarters of the protein, showed 42-44% identity to the catalytic domains of glycoside hydrolase family 9 (GHF9)-cellulases from arthropods and Thermomonospora fusca. While the N-terminal first quarter of HdEG66 showed 27% identity to the carbohydrate-binding module (CBM) of a Cellulomonas fimi cellulase, CenA. Thus, the HdEG66 was regarded as the GHF9-cellulase possessing a family II CBM in the N-terminal region. By genomic PCR using specific primers to the 3'-terminal coding sequences of HdEG66-cDNA, a DNA of 2186 bp including three introns was amplified. This strongly suggests that the origin of HdEG66 is not from symbiotic bacteria but abalone itself.
A cellulase (endo-beta-1,4-glucanase, EC 3.2.1.4) was purified from the gut of larvae of the yellow-spotted longicorn beetle Psacothea hilaris by acetone precipitation and elution from gels after native PAGE and SDS/PAGE with activity staining. The purified protein formed a single band, and the molecular mass was estimated to be 47 kDa. The purified cellulase degraded carboxymethylcellulose (CMC), insoluble cello-oligosaccharide (average degree of polymerization 34) and soluble cello-oligosaccharides longer than cellotriose, but not crystalline cellulose or cellobiose. The specific activity of the cellulase against CMC was 150 micro mol.min-1.(mg protein)-1. TLC analysis showed that the cellulase produces cellotriose and cellobiose from insoluble cello-oligosaccharides. However, a glucose assay linked with glucose oxidase detected a small amount of glucose, with a productivity of 0.072 micro mol.min-1.(mg protein)-1. The optimal pH of P. hilaris cellulase was 5.5, close to the pH in the midgut of P. hilaris larvae. The N-terminal amino-acid sequence of the purified P. hilaris cellulase was determined and a degenerate primer designed, which enabled a 975-bp cDNA clone containing a typical polyadenylation signal to be obtained by PCR and sequencing. The deduced amino-acid sequence of P. hilaris cellulase showed high homology to members of glycosyl hydrolase family 5 subfamily 2, and, in addition, a signature sequence for family 5 was found. Thus, this is the first report of a family 5 cellulase from arthropods.
LITTLE is known of the digestive enzymes of the earthworm. The presence of protease, amylase and lipase has been reported in the gut contents in isolated instances. Since up to 10 per cent of the top four inches of soil in grassland may pass through the intestines of worms in a year1, their digestive abilities are of a relevance to transformation of soil organic matter.
While it is widely accepted that most animals (Metazoa) do not have endogenous cellulases, relying instead on intestinal symbionts for cellulose digestion, the glycosyl hydrolase family 9 (GHF9) cellulases found in the genomes of termites, abalone, and sea squirts could be an exception. Using information from expressed sequence tags, we show that GHF9 genes (subgroup E2) are widespread in Metazoa because at least 11 classes in five phyla have expressed GHF9 cellulases. We also demonstrate that eukaryotic GHF9 gene families are ancient, forming distinct monophyletic groups in plants and animals. As several intron positions are also conserved between four metazoan phyla then, contrary to the still widespread belief that cellulases were horizontally transferred to animals relatively recently, GHF9 genes must derive from an ancient ancestor. We also found that sequences isolated from the same animal phylum tend to group together, and in some deuterostomes, GHF9 genes are characterized by substitutions in catalytically important sites. Several paralogous subfamilies of GHF9 can be identified in plants, and genes from primitive species tend to arise basally to angiosperm representatives. In contrast, GHF9 subgroup E2 genes are relatively rare in bacteria.
Glycoside-hydrolase-family 9 (GHF9) cellulases are known to be widely distributed in metazoa. These enzymes have been appreciably well investigated in protostome invertebrates such as arthropods, nematodes, and mollusks but have not been characterized in deuterostome invertebrates such as sea squirts and sea urchins. In the present study, we isolated the cellulase from the Japanese purple sea urchin Strongylocentrotus nudus and determined its enzymatic properties and primary structure. The sea urchin enzyme was extracted from the acetone-dried powder of digestive tract of S. nudus and purified by conventional chromatographies. The purified enzyme, which we named SnEG54, showed a molecular mass of 54kDa on SDS-PAGE and exhibited high hydrolytic activity toward carboxymethyl cellulose with an optimum temperature and pH at 35 degrees C and 6.5, respectively. SnEG54 degraded cellulose polymer and cellooligosaccharides larger than cellotriose producing cellotriose and cellobiose but not these small cellooligosaccharides. From a cDNA library of the digestive tract we cloned 1822-bp cDNA encoding the amino-acid sequence of 444 residues of SnEG54. This sequence showed 50-57% identity with the sequences of GHF9 cellulases from abalone, sea squirt, and termite. The amino-acid residues crucial for the catalytic action of GHF9 cellulases are completely conserved in the SnEG54 sequence. An 8-kbp structural gene fragment encoding SnEG54 was amplified by PCR from chromosomal DNA of S. nudus. The positions of five introns are consistent with those in other animal GHF9 cellulase genes. Thus, we confirmed that the sea urchin produces an active GHF9 cellulase closely related to other animal cellulases.
Termites play an important role in degradation of dead plant materials in nature. Over the last century, many researchers have investigated the mechanisms of their lignocellulose digesting system. A recent publication by Zhou et al. (Zhou, X., Smith, J.A., Oi, F.M., Koehler, P.G., Bennett, G.W., Scharf, M.E., 2007. Correlation of cellulase gene expression and cellulolytic activity throughout the gut of the termite Reticulitermes flavipes. Gene 395, 29-39) dealt with the cellulolytic system of the flagellate-harboring termite R. flavipes and suggested "the presence of a single unified cellulose digestion system" in the termite, as an alternative hypothesis of a "dual (i.e. endogenous and symbiotic) cellulose digesting system" proposed by Nakashima et al. (Nakashima, K., Watanabe, H., Saitoh, H., Tokuda, G., Azuma, J.-I., 2002. Dual cellulose-digesting system of the wood-feeding termite, Coptotermes formosanus Shiraki. Insect Biochem. Mol. Biol. 32, 777-784). Here we show that their results actually support a dual cellulose digesting system rather than "a single unified cellulose digestion system". In addition, potential problems with their results are highlighted.
We previously reported on the endogenous cellulase gene of Corbicula japonica, CjCel9A. In this study, the tissue localization of the mRNA and translated products of CjCel9A was investigated in order to understand how this gene is physiologically involved in cellulose decomposition by C. japonica. Antiserum against recombinant CjCel9A protein was prepared. Multiple bands were observed mainly on western blot analysis of the crystalline style, and the band sizes partially corresponded to the active bands detected using zymographic analysis. In situ hybridization and immunohistochemical analyses clarified the exclusive production and secretion of this cellulase by the secretory cells localized in the epithelium of the digestive tubules in the digestive gland. These data strongly support our previous assumption that the endogenous cellulase of C. japonica is produced in the digestive gland and transported to the crystalline style to act as a component of its cellulolytic activity.
The Physiology of Earthworms
- M S Laverack
Laverack, M.S., 1963. The Physiology of Earthworms. Pergamon Press, Oxford, 206 pp.
Earthworms and potworms as keystone functional animals in pedospheres
- Nakamura