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The relationship between instability of H-2 production and compositions of bacterial communities within a dark fermentation fluidized-bed bioreactor
Abstract
Microbial community composition dynamics was studied during H(2) fermentation from glucose in a fluidized-bed bioreactor (FBR) aiming at obtaining insight into the H(2) fermentation microbiology and factors resulting in the instability of biofilm processes. FBR H(2) production performance was characterised by an instable pattern of prompt onset of H(2) production followed by rapid decrease. Gradual enrichment of organisms increased the diversity of FBR attached and suspended-growth phase bacterial communities during the operation. FBR bacteria included potential H(2) producers, H(2) consumers and neither H(2) producers nor consumers, and those distantly related to any known organisms. The prompt onset of H(2) production was due to rapid growth of Clostridium butyricum (99-100%) affiliated strains after starting continuous feed. The proportion trend of C. butyricum in FBR attached and suspended-growth phase communities coincided with H(2) and butyrate production. High glucose loading rate favoured the H(2) production by Escherichia coli (100%) affiliated strain. Decrease in H(2) production, associated with a shift from acetate-butyrate to acetate-propionate production, was due to changes in FBR attached and suspended-growth phase bacterial community compositions. During the shift, organisms, including potential propionate producers, were enriched in the communities while the proportion trend of C. butyricum decreased. We suggest that the instability of H(2) fermentation in biofilm reactors is due to enrichment and efficient adhesion of H(2) consumers on the carrier and, therefore, biofilm reactors may not favour mesophilic H(2) fermentation.
... The forward and reverse primers for PCR were GC-BacV3f and 907r, respectively. The visible bands in the polyacrylamide gel were cut by using a surgical blade, eluted in sterile water and re-amplified by PCR (primers BacV3f and 907r) before sending them to Macrogen (South Korea) for sequencing as described by Koskinen et al. [51]. The nucleotide sequences obtained were analysed using Bio-Edit [52] software version 7.2.5 and compared with the sequences in the GenBank nucleotide collection database using the BLAST software [53]. ...
... One explanation is that, in this study, the pH was set to 5.0, whereas the optimum pH for T. thermosaccharolyticum is 6.5 [60]. Furthermore, Koskinen et al. [51] showed that in a mesophilic H 2 -producing FBR, the attached microbial community is slightly different from the suspended one, which was not analysed in this study. It is thus plausible that the contribution of some H 2 producing or H 2 consuming microorganisms on the net H 2 yield was not considered. ...
... Koskinen et al. [51] studied the microbial community dynamics over time in a mesophilic (35 C) FBR reactor inoculated with digested activated sludge and concluded that the adhesion of H 2 consuming microorganisms, including homoacetogens, to the carrier material may cause an unstable H 2 production. Similarly to this study, Dinamarca and Bakke [69] reported a decrease from 1.5 to below 0.25 mol H 2 mol À1 glucose after 57 days of reactor operation at 35 C. The authors concluded that homoacetogenesis is directly correlated with the HRT and dependent on biomass density and sludge age [69]. ...
Dark fermentative biohydrogen production in a thermophilic, xylose-fed (50 mM) fluidised bed reactor (FBR) was evaluated in the temperature range 55e70 C with 5-degree increments and compared with a mesophilic FBR operated constantly at 37 �C. A significantly
higher (p ¼ 0.05) H2 yield was obtained in the thermophilic FBR, which stabilised at about 1.2 mol H2 mol-1 xylose (36% of the theoretical maximum) at 55 and 70 C, and at 0.8 mol H2 mol-1 xylose at 60 and 65 C, compared to the mesophilic FBR (0.5 mol H2 mol-1 xylose).
High-throughput sequencing of the reverse-transcribed 16S rRNA, done for the first time on biohydrogen producing reactors, indicated that Thermoanaerobacterium was the prevalent active microorganism in the thermophilic FBR, regardless of the operating temperature.
The active microbial community in the mesophilic FBR was mainly composed of Clostridium and Ruminiclostridium at 37 C. Thermophilic dark fermentation was shown to be suitable for treatment of high temperature, xylose-containing wastewaters, as it resulted in a
higher energy output compared to the mesophilic counterpart.
... DNA was extracted from defrosted pellets with PowerSoil DNA isolation kit (MO BIO Laboratories, Inc., Carlsbad, CA, USA). PCR was used to amplify partial 16S rRNA genes as described by Koskinen et al. [27] using GC-BacV3f [28] and 907r [29] primers. DGGE was performed as described by Lakaniemi et al. [30]. ...
... DGGE was performed as described by Lakaniemi et al. [30]. Separated DNA sequences were reamplified according to Koskinen et al. [27] before sequencing at Macrogen Inc. (Seoul, Korea). BioEdit software and BLAST (http://blast.ncbi.nlm.nih. ...
... The anolyte microbial communities changed slightly during the experiments. The intensity of the bands on the DGGE gel [27,37] changed at different HRTs indicating that the share of Christensenella minuta increased remarkably after the HRT decreased to 0.5 d (Fig. A1, Table 3). C. minuta is a xylose fermenting bacterium [38] and its share likely increased due to increased xylose loading rates at lower HRTs and was related to decreasing power densities and CEs. ...
Aerobic wastewater management is energy intensive and thus anaerobic processes are of interest. In this study, a microbial fuel cell was used to produce electricity from xylose which is an important constituent of lignocellulosic waste. Hydraulic retention time (HRT) was optimized for the maximum power density by gradually decreasing the HRT from 3.5 d to 0.17 d. The highest power density (430 mW/m²) was obtained at 1 d HRT. Coulombic efficiency decreased from 30% to 0.6% with HRTs of 3.5 d and 0.17 d, respectively. Microbial community analysis revealed that anode biofilm contained known exoelectrogens, including Geobacter sp. and fermentative organisms were present in both anolyte and the anode biofilm. The peak power densities were obtained at 1–1.7 d HRTs and xylose degraded almost completely even with the lowest HRT of 0.17 d, which demonstrates the efficiency of up-flow MFC for treating synthetic wastewater containing xylose.
... In AFBRs, biomass colonizes a support forming a biofilm, due to the natural development of the biofilm a portion of it remains as planktonic cells. Contact between biomass and substrate is enhanced by bed fluidization, improving mass transfer and treatment capacities [4], which in turn gives the possibility to apply high organic loading rates (OLR), low hydraulic retention times (HRT) and good mixing conditions, allowing high removal efficiencies or production rates [1,5]. Moreover, the up-flow velocities (V up ) commonly used in AFBRs drag out the H 2 produced, avoiding the inhibition by its accumulation and partial pressure [1]. ...
... In order to eliminate the hydrogen consuming methanogens different pretreatments had been tested, such as thermal, chemical or electrical treatments [12e15]. All these methods have been proved to be effective on the inhibition of methanogenic activity, but may also have an effect on the microbial physiology and the reactor performance [5,13]. ...
... It has been proposed that the prevalent fermentation pathways depend on the type of seed sludge, because their bacterial community composition is defined by their origin [16]. Comparative works relate discrepancies in hydrogen production with the use of different HRTs and pH [13], which may in fact help to select the bacterial community structure under given conditions [5]. Although other configurations and inocula sources have been tested [17], hydrogen production, performance and microbial diversity in AFBRs operated under similar conditions but seeded with pretreated biomass through two different methods have not been systematically compared. ...
The effect of two different inoculum pretreatments, thermal and cell wash-out (A1 and A2, respectively) on the performance of anaerobic fluidized bed reactors for hydrogen production was determined. The reactors were operated for 112 days under the same operational conditions using glucose as substrate at increasing organic loading rates and decreasing hydraulic retention times. Both treatments were effective avoiding methanogenesis. Reactor A2 showed better performance and stability than reactor A1 in each one of the different operational conditions. Cell wash-out treatment produced higher hydrogen volumetric production rates and yields than thermal treatment (7 L H2/L-d, 3.5 mol H2/mol hexose, respectively). DGGE analysis revealed that the microbial communities developed were affected by the inoculum treatment. Organisms from the genera Clostridium and Lactobacillus predominated in both reactors, with their relative abundances linked to hydrogen production. Resilience was observed in both reactors after a period of starvation.
... The ecological interactions can directly affect stability and/ or function. Koskinen et al. [38] monitored bacterial community dynamics inside a dark fermentation fluidized-bed bioreactor to identify the cause of the instability in hydrogen production. The authors concluded that the instability in the production was due to changes in the microbial community structure, which were caused by rapid enrichment. ...
... The results of Koskinen et al. [38] detail a community with increasing diversity, along with environmental changes. These findings bring an important question to light; what is the relationship between bacterial diversity and ecosystem stability? ...
... However, even if the community can be recovered, the system function can be highly affected, thus altering the original function. The hydrogen reactor operated by Koskinen et al. [38] did not recover hydrogen production, while the bacterial community diversity increased after the disturbance, resulting in significant changes in the bacterial community. The community showed low resilience, it recovered slowly and not to the previous structure. ...
Hydrogen has been studied as an alternative to traditional energy sources; it is a clean and renewable fuel that on combustion generates only water as a by-product. Biological production of hydrogen can occur either via photosynthesis or fermentation. The latter is technically simple and can convert substrates like organic matter present in wastewater into a renewable energy source. Microorganisms belonging to the domains Archaea and Bacteria are responsible for the conversion of various carbon sources to biogas, including hydrogen and methane. It is important to determine the microorganisms responsible for such transformations, as they are the major players of the process. Studying the bacterial diversity, population structure, and processes that modify these communities leads to a better understanding of their ecological functions and productivity. The environmental conditions within an anaerobic hydrogen reactor can exert a selective pressure on the community, thereby affecting the population structure, diversity, and heterogeneity. Combination of appropriate operational parameters and ecological factors could lead to the development of effective bioprocesses to maximize hydrogen yield. Therefore, the objective of this paper is to present a review on bacterial ecology in anaerobic hydrogen reactors and the factors that can affect bacterial diversity.
... In this sense, a simplified consortium operated under sterile conditions, constituted from hydrogen producers as dominant bacteria, showed a high hydrogen productivity of 217.7 mmole H2 L À1 d À1 , reaching a yield of 1.92 mole H2 mole glucose À1 at 40 g glucose L À1 d À1 [19]. However, as soon as the considered effluent is unsterile, these systems could be exposed to a contamination by methanogens or other hydrogen-consuming microorganisms, promoting complex and uncontrolled population dynamics in the reactor according to the operational parameters such as HRT, OLR or pH and solids retention time [20]. In the case of fixed biomass reactors, the differential ability of microorganisms to form a biofilm on a carrier can be used to select proper microorganisms according to the type of support [7,21] . ...
... and corresponded to anaerobic sludge batch growth in biofilm-based reactor operated with BESA (2- bromoethanesulfonic acid) as methanogenic inhibitor. After 50 days of continuous operation, the authors concluded that a mesophilic biofilm was not suitable for H 2 production due to the attachment of hydrogen consumers that decrease the system performances [20]. From a community point of view, our system was stable assuming that the pre-enrichment inoculum strategy generated a lower species richness within the biofilm when compared to other inocula [12,18,20,38]. ...
... After 50 days of continuous operation, the authors concluded that a mesophilic biofilm was not suitable for H 2 production due to the attachment of hydrogen consumers that decrease the system performances [20]. From a community point of view, our system was stable assuming that the pre-enrichment inoculum strategy generated a lower species richness within the biofilm when compared to other inocula [12,18,20,38]. Moreover the mixing conditions and the mode of operation in ASBBR allowed operating the reactor at low HRT with no undesirable microorganism attachment and biomass washout. ...
... They reported a HPr accumulation period (days 36-49) of 28-35 mmol L −1 dropping from 53 to 39% in the H 2 content. Koskinen et al. (2007) reported elevated concentrations of HPr in a fluidized-bed bioreactor fed with glucose, resulting in a shift from acetate/butyrate to acetate/ propionate production with a decrease and instability in H 2 production by adhesion of H 2 consumer in the biofilm of the reactor. ...
... The present experimental results indicate that the concentration of undissociated acids was insufficient to trigger a metabolic pathway shift. However, the increase of HPr concentration could suggest a change in microbial dynamic, according to Sivagurunathan et al. (2014) and Koskinen et al. (2007). The microbial community analysis can explain the depletion of hydrogen production through the presence of H 2 -consuming microorganisms and by the formation of metabolites associated with the microorganisms. ...
Food waste can be used as substrate in the dark fermentation to produce value-added products such as hydrogen, a future renewable energy supply. However, biological reactor unstable conditions might affect its potential use as green energy by low production rates. This study examined the instability of hydrogen production by dark fermentation of food waste in an anaerobic sequential biological reactor through a microbial community analysis. Hydrogen production varied significantly with a maximum of 25.74 mL H2/g VSadded to low production as 1.29–3.18 mL H2/g VSadded until the end of the experiment. Microbial community analysis showed that the unstable stage was related to the displacement of hydrogen-producing bacteria as Clostridium, Prevotella, Caloramator, and Bacteroides by a predominant abundance of Bifidobacterium, a lactic-acid bacteria. Furthermore, microbial analysis of food waste revealed the endogenous abundance of lactic-acid bacteria as Latilactobacillus (43.73%), Leuconostoc (12.1%), Lactiplantibacillus (1.84%), Lactococcus (1.37%), Lactobacillus (0.43%), Streptococcus (0.39%) and Bifidobacterium (0.19%). Thus, the inhibition of hydrogen production could be caused by the incoming of Bifidobacterium from food waste, which could compete for the substrate changing the acetic/butyric fermentation to a possible lactic acid fermentation.
... Microbial community was profiled as previously described by Haavisto et al. [38]. PowerSoil DNA isolation kit (MO BIO Laboratories, Inc., Carlsbad, CA, USA) was used for DNA extraction followed by partial 16S rRNA gene PCR amplification with GC-BacV3f [40] and 907r [41] primers as described by Koskinen et al. [42]. DNA sequences were separated with denaturing gradient gel electrophoresis (DGGE) as described by Lakaniemi et al. [43], reamplified according to Koskinen et al. [42], and sequenced at Macrogen Inc. (Seoul, Korea). ...
... PowerSoil DNA isolation kit (MO BIO Laboratories, Inc., Carlsbad, CA, USA) was used for DNA extraction followed by partial 16S rRNA gene PCR amplification with GC-BacV3f [40] and 907r [41] primers as described by Koskinen et al. [42]. DNA sequences were separated with denaturing gradient gel electrophoresis (DGGE) as described by Lakaniemi et al. [43], reamplified according to Koskinen et al. [42], and sequenced at Macrogen Inc. (Seoul, Korea). Analyzed sequence data (BioEdit software) was compared to known sequences with BLAST (https://blast.ncbi.nlm. ...
Start-up of bioelectrochemical systems (BESs) fed with brewery wastewater was compared at different adjusted anode potentials (-200 and 0 mV vs. Ag/AgCl) and external resistances (50 and 1000 Ω). Current generation stabilized faster with the external resistances (9 ± 3 and 1.70 ± 0.04 A/m3 with 50 and 1000 Ω, respectively), whilst significantly higher current densities of 76 ± 39 and 44 ± 9 A/m3 were obtained with the adjusted anode potentials of -200 and 0 mV vs. Ag/AgCl, respectively. After start-up, when operated using 47 Ω external resistance, the current densities and Coulombic efficiencies of all BESs stabilized to 9.5 ± 2.9 A/m3 and 12 ± 2%, respectively, demonstrating that the start-up protocols were not critical for long-term BES operation in microbial fuel cell mode. With adjusted anode potentials, two times more biofilm biomass (measured as protein) was formed by the end of the experiment as compared to start-up with the fixed external resistances. After start-up, the organics in the brewery wastewater, mainly sugars and alcohols, were transformed to acetate (1360 ± 250 mg/L) and propionate (610 ± 190 mg/L). Optimized start-up is required for prompt BES recovery, for example, after process disturbances. Based on the results of this study, adjustment of anode potential to -200 mV vs. Ag/AgCl is recommended for fast BES start-up.
... The samples were stored at -20°C and microbial communities were analyzed from defrosted samples as described by Haavisto et al. [30]. DNA was extracted with a PowerSoil DNA isolation kit (MO BIO Laboratories, Inc., Carlsbad, CA, USA) and partial 16SrRNA genes were amplified with PCR using GC-BacV3f [36] and 907r [37] primers as described by Koskinen et al. [38]. After separating DNA sequences with denaturing gradient gel electrophoresis (DGGE) according to Lakaniemi et al. [39], the sequences were reamplified according to Koskinen et al. [38] and sequenced at Macrogen Inc. (Seoul, Korea). ...
... DNA was extracted with a PowerSoil DNA isolation kit (MO BIO Laboratories, Inc., Carlsbad, CA, USA) and partial 16SrRNA genes were amplified with PCR using GC-BacV3f [36] and 907r [37] primers as described by Koskinen et al. [38]. After separating DNA sequences with denaturing gradient gel electrophoresis (DGGE) according to Lakaniemi et al. [39], the sequences were reamplified according to Koskinen et al. [38] and sequenced at Macrogen Inc. (Seoul, Korea). Sequence data were analyzed with BioEdit software and compared to known sequences by using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). ...
Starting up a microbial fuel cell (MFC) requires often a long-term culture enrichment period, which is a challenge after process upsets. The purpose of this study was to develop low cost storage for microbial fuel cell enrichment culture to enable prompt process recovery after upsets. Anolyte of an operating xylose-fed MFC was stored at different temperatures and for different time periods. Storing the anolyte for one week or one month at +4 °C did not significantly affect power production, but lag time for power production was increased from 2 days to 3 or 5 days, respectively. One month storing at -20 °C increased the lag time to 7 days. The average power density in these MFCs varied between 1.2 and 1.7 W/m³. The share of dead cells (measured by live/dead staining) increased with storing time. After six-month storage the power production was insignificant. However, xylose removal remained similar in all cultures (99-100%) whilst volatile fatty acids production varied. The results indicate that fermentative organisms tolerated the long storage better than the exoelectrogens. As storing at +4 °C is less energy intensive compared to freezing, anolyte storage at +4 °C for maximum of one month is recommended as start-up seed for MFC after process failure to enable efficient process recovery.
... Authors have sampled different environments, such as anaerobic sludge (Koskinen et al., 2007;Davila-Vazquez et al., 2009;Baghchehsaraee et al., 2010;Yossan et al., 2012;Sivagurunathan et al., 2013), compost (Akutsu et al., 2008;Huang et al., 2010;Nissilä et al., 2011aNissilä et al., , 2011b, and wastewater treatment plants (Akutsu et al., 2009;Baghchehsaraee et al., 2010;Chaganti et al., 2012;Lay et al., 2012;Sivagurunathan et al., 2013) with the aim of selecting an active microbial community able to produce H 2 . Since in these consortia non-H 2 -producing bacteria, H 2 -producing bacteria, and H 2 -consuming bacteria coexist, the literature is full of methods and strategies for enriching the inocula with H 2 -producing bacteria (Baghchehsaraee et al., 2008;Ren et al., 2008a;Adav et al., 2009;Hafez et al., 2009;Kim et al., 2009;O-Thong et al., 2009;Ohnishi et al., 2010;Nasr et al., 2011;Nissilä et al., 2011aNissilä et al., , 2011bRossi et al., 2011;Li et al., 2012b;Ning et al., 2012;Jeong et al., 2013;Wan et al., 2013). ...
... These species, in conjunction with clostridia, may be a more robust system for H 2 production at a large scale. Molecular approaches have also given insights into the microbial interactions that led to the instability of H 2 production, detecting changes in the microbial structure toward Lactobacillus (Jo et al., 2007), non-H 2 -producing acidogens (Koskinen et al., 2007;D.-H. Kim et al., 2008), and H 2 -consuming bacteria, such as Desulfovibrio desulfuricans (Koskinen et al., 2006). ...
... When comparing the settling and non-settling fractions in an ASBR converting starch to hydrogen, high propionate concentrations were detected only in the settleable sludge, which had the lowest specific H 2 activity; it was attributed to the propionate producers Selenomonas sp. which were favored by high settling times (Arooj et al. 2007). In a fluidized bed bioreactor fed with glucose, the shift from acetatebutyrate to acetate-propionate production (resulting in reduced H 2 production) was explained by community structure changes rather than metabolic changes, through the enrichment of propionate producers (identified as Schwartzia succinivorans) due to their efficient adhesion on the carrier forming a biofilm, regardless of the HRT changes (Koskinen, Kaksonen and Puhakka 2007). In a CSTR treating real industrial wastewater from a beverage plant, propionic acid accumulation was attributed to Selenomonas lacticifex and Bifidobacterium catenulatum, which could be successfully eliminated by a temperature increase from 37 • C to 45 • C leading to improved hydrogen production performance (Sivagurunathan, Sen and Lin 2014). ...
... One of the most studied SRB, Desulfovibrio spp., was detected in wastewater treatment plant sludge used as hydrogen production inoculum (Chaganti, Lalman and Heath 2012) and has [NiFe]-hydrogenase genes encoding uptake hydrogen enzymes (Wawer and Muyzer 1995). During H 2 fermentation from glucose in a fluidized bed bioreactor, the SRB Desulfovibrio desulfuricans was detected in the attached and suspended growth phase communities (Koskinen, Kaksonen and Puhakka 2007), but the authors suggested that it was not involved in H 2 consumption or production in that case, because (i) the reactor did not contain sulfate for sulfate reduction activity and (ii) the substrates and products of other fermenting activities in D. desulfuricans (choline and pyruvate fermentation) do not include hydrogen. In kitchen waste fermenter, Desulfovibrio sp. was detected and suspected to be involved in lactate degradation (Li et al. 2011) which involves hydrogen production (McInerney and Bryant 1981). ...
One of the most important biotechnological challenges is to develop environment friendly technologies to produce new sources of energy. Microbial production of biohydrogen through dark fermentation, by conversion of residual biomass, is an attractive solution for short-term development of bioH2 producing processes. Efficient biohydrogen production relies on complex mixed communities working in tight interaction. Species composition and functional traits are of crucial importance to maintain the ecosystem service. The analysis of microbial community revealed a wide phylogenetic diversity that contributes in different—and still mostly unclear—ways to hydrogen production. Bridging this gap of knowledge between microbial ecology features and ecosystem functionality is essential to optimize the bioprocess and develop strategies toward a maximization of the efficiency and stability of substrate conversion. The aim of this review is to provide a comprehensive overview of the most up-to-date biodata available and discuss the main microbial community features of biohydrogen engineered ecosystems, with a special emphasis on the crucial role of interactions and the relationships between species composition and ecosystem service. The elucidation of intricate relationships between community structure and ecosystem function would make possible to drive ecosystems toward an improved functionality on the basis of microbial ecology principles.
... Several research groups have shown that the composition of microbial communities in the biofilm has a strong influence on H 2 production performances (Ren et al., 2008;Wong et al., 2014;Li and Fang, 2007;Koskinen et al., 2007). Most of the studies dealt with biofilm reactors inoculated with seed sludge from wastewater treatment plants and anaerobic digesters, thus resulting in a large variability of microbial communities (Ren et al., 2008;Wong et al., 2014). ...
... On the one hand, mixed bacterial communities may provide a large variety of metabolic pathways for the degradation of complex substrate (Li and Fang, 2007). On the other hand, the variability in the microbial composition may lead to unstable H 2 production (Koskinen et al., 2007). Therefore, the most important challenge for future research is to improve operation and design of the reactor in order to obtain a stable and efficient H 2 production. ...
Dark fermentation systems often show low H2 yields and unstable H2 production, as the result of the variability of microbial dynamics and metabolic pathways. Recent batch investigations have demonstrated that an artificial consortium of two anaerobic bacteria, Clostridium acetobutylicum and Desulfovibrio vulgaris Hildenborough, may redirect metabolic fluxes and improve H2 yields. This study aimed at evaluating the scale-up from batch to continuous H2 production in an up-flow anaerobic packed-bed reactor (APBR) continuously fed with a glucose-medium. The effects of various parameters, including void hydraulic retention time (HRTv), pH, and alkalinity, on H2 production performances and metabolic pathways were investigated. The results demonstrated that a stable H2 production was reached after 3-4 days of operation. H2 production rates increased significantly with decreasing HRTv from 4 to 2 h. Instead, H2 yields remained almost stable despite the change in HRTv, indicating that the decrease in HRTv did not affect the global metabolism.
... High H 2 partial pressures and low temperatures make unfavorable the catabolic reactions that give rise to H 2 production, thus, directing metabolic activity to ethanol or lactate production [15e17]. On the other hand, stability, meaning the capability of bioreactors to operate for long periods, has proved to be an obstacle [13,18,19], except in a few cases [20]. Because significant diversity is lost after pretreatments of inocula and due to culture conditions, some attributes related to their ecological structure might also be lost. ...
... Because Bacillaceae and Enterobacteriaceae members are facultative anaerobes, they are thought to enhance H 2 production by consuming O 2 in mixed cultures [27,69], although they also produce H 2 via pyruvate formate-lyase (with a maximum H 2 yield of just 2 mol of H 2 per mol of glucose) [70], acting as substrate competitors decreasing H 2 production. Additionally, it might be possible that hydrogen consumers or lactate producers take advantage of these conditions and outgrow hydrogen producers as previously reported [18]. ...
Research on hydrogen production by dark fermentation has improved the management and performance of bioreactors, although yield and long-term stability challenges still exist. To understand the causes of these challenges we propose to investigate the ecological properties of microbial consortia residing in hydrogen-producing bioreactors. In this study, we analyzed scientific literature (until 2016) on hydrogen fermentation and investigated the relationships between bioreactor operational conditions and microbial composition, as well as co-occurrences of microbial groups through multivariate and network analyses, respectively. The results of the analyses highlight ecological aspects that may need to be considered when aiming for increased performance and stability of bioreactors, such as: (i) the relationship between key parameters and their influence on microbial diversity (e.g. positive relationship between richness with H2 yield and higher diversity of certain inocula), (ii) the positive relationship of the presence of specific microbial families and genera (often overlooked) with increased H2 yield (e.g. Tissierellaceae, Oxalobacteraceae and Tepidimicrobium), (iii) the importance of specific groups and their interactions (e.g. potential cooperative interactions between H2 producers, biofilm producers and facultative anaerobic bacteria) and (iv) the importance of such interactions on the systemic properties of the consortia (e.g. increased network robustness with higher diversity). Beyond the ecological implications of our analyses, we also discuss the limitations of current methods for characterizing microbial communities and the potential for the application of modern methodologies such as high throughput sequencing and proteomics to re-evaluate the diversity and functional information thus far published in helping to disentangle the ecological phenomena that occur within hydrogenogenic consortia.
... However, HP has an inhibitory effect on H 2 production rate due to its low biodegradability (Cohen et al., 1982). Thus, the production of HP negatively affect the HPB activity (Koskinen et al., 2007). The accumulation of HP might be attributed to overloading (Kennedy and Van den Berg, 1982) or due to shifting in the dominant species of acidogenic populations (Inanc et al., 1996). ...
Large amounts of swine manure (SM) and livestock waste production in South Korea, leading to enormous environmental pollution and requiring extensive treatment before discharge. To address these issues, the upstream dark fermentation (DF) technique provides a sustainable solution to recover biohydrogen (bio-H2) using solubilized organics; however, the process parameters need to be optimized to boost the yield. The modulation of process variables, including input chemical oxygen demand (COD) doses (2, 6.4, 10, and 12 g/L), pH (5, 5.5, and 6), hydraulic retention time (HRT: 4.5 days, 2 days, and 1 day), and cosubstrate (SM: food waste (FW) = 8:2), was studied to improve the performance of DF. Mixing FW and SM maintains the feedstock pH in the range of 5.8–6.21 in DF, boosting hydrogen (H2) production to 0.5 L, which is considerably higher than that in DF fed with individual substrates. Similarly, a higher H2 generation and hydrogen yield (HY) of 1.32 L/L/day and 275.57 mL/gVSadded, respectively, were obtained for HRT of 1 day compared to HRT of 2 days (H2 production of 0.830 L/L/day and HY of 159.05 mL/gVSadded) and 4.5 days (H2 production of 0.403 L/L/day and HY of 80.6 mL/gVSadded) in 5 L DF. The produced H2 had a high purity of 83.21%. Thus, DF with operational variables of HRT of 1 day and pH of 5.5–6.2 with cosubstrate feeding was found to be effective for boosting the bio-H2 yield and treatment efficiency. Such optimized process parameters guide the operation of scalable DF to keep the stability and equilibrium of the process and maximize the HY.
... Clostridium-Enterobacter a été observée dans plusieurs études (Koskinen et al., 2007;Maintinguer et al., 2008). Comme relevé par l'étude de Tolvanen et al. (2010), quand à la fois des anaérobes stricts et facultatifs coexistent en culture mixte, Enterobacter (présent après 400 jours sans réensemencement dans notre étude) contribue probablement à la production d'hydrogène, bien que n'étant pas le producteur majoritaire. ...
Cette étude porte sur l’intensification de la production d’hydrogène par fermentation obscure en bioréacteur membranaire liquide/gaz (BRM L/G) à fibres creuses et la valorisation de coproduits agricoles et agroalimentaires. A partir d’une solution modèle, sans réensemencement bactérien, des productions d’hydrogène stables (2,6±0,2 LH2/Lréacteur/j et 1,0±0,1 molH2/molglucose) ont été obtenues pendant plus d’un an, en favorisant l’émergence répétée de bactéries productrices d’hydrogène (Clostridium, Enterobacter), ayant colonisé le module membranaire. L’extraction efficace de l’hydrogène via la lumière des fibres creuses a été montrée et un optimum de concentration en glucides a été atteint (14 g/L). En bioréacteur semibatch, des biomasses variées ont généré par fermentation endogène (0,7-55 LH2/kgbiomasse), caractérisées par une identité métabolique et microbiologique et parfois l’inhibition de bactéries productrices d’hydrogène. Enfin, la mise en œuvre de la fermentation obscure endogène en BRM L/G utilisant des biomasses diversifiées a été démontrée, avec des performances de production élevées (4,1 LH2/L/j, 86,8 mLH2/gDCO).
... Acid Production: Acidification activity of the organisms isolated was measured by change in pH during time (Ayad et al., 2004). Strains were investigated for acid production using MRS broth as described by Koskinen et al., (2007). ...
Lianes have gained notoriety as species that suppress and kill desired forest tree species among foresters and silviculturists; a situation that has contributed to their wanton destruction. However, lianes play important ecological roles by contributing significantly to carbon sequestration and forest regeneration. The composition, diversity and structural characteristics of lianes in the Subri River Forest Reserve in Ghana were studied on a 5 ha patch of the forest divided in 50 random 0.1 ha circular subplots each. Eleven species of lianes ≥ 3.2 cm diameter at breast height (dbh, 1.3 m above ground) belonging to 10 taxonomic families were identified. The abundance and diversity of the lianes were low. Relative diversity among the families was low with all but one family being represented by single species. The density, basal area and ecological importance of the species, as determined by the importance value index were low. The results of the study have shown that liane species are rare and less diverse coupled with low structural attributes The need to preserve the lianes in the Subri Forest Reserve is therefore very crucial, for forest biodiversity conservation, which will in turn contribute to climate change mitigation.
... The performance of microbial communities is often affected by factors such as pH, temperature, substrate and inoculum pretreatment (Cisneros-Pérez et al., 2015;Penteado et al., 2013). Changes in environmental conditions during dark fermentative H2 production cause change in the population dynamics, which in turn can lead to instability of H2 producing systems (Bakonyi et al., 2014;Koskinen et al., 2007). Previous studies investigating the effects of temperature on fermentative H2 production have focused on comparing batch and reactor performances at different fixed operating temperatures (Dessì et al., 2018;Zhang and Shen, 2006). ...
The aim of this thesis was to enhance thermophilic dark fermentative hydrogen production by using microbial strategies (bioaugmentation and synthetic co-cultures) and by increasing the understanding on the microbial community dynamics especially during stress conditions such as fluctuating temperatures and elevated substrate concentrations. To study the effects of sudden short-term temperature fluctuations, batch cultures initially incubated at 55°C (control) were subjected to downward (from 55°C to 35°C or 45°C) or upward (from 55°C to 65°C or 75°C) temperature shifts for 48 hours after which they were incubated again at 55°C for two consecutive batch cycles. The results showed that sudden, temporal upward and downward temperature fluctuations had a direct impact on the hydrogen yield as well as the microbial community structure. Cultures exposed to downward temperature fluctuation recovered more rapidly enabling almost similar hydrogen yield (92-96%) as the control culture kept at 55 °C. On the contrary, upward temperature shifts from 55 to 65 or 75 °C had more significant negative effect on dark fermentative hydrogen production as the yield remained significantly lower (54-79%) for the exposed cultures compared to the control culture. To improve the stability of hydrogen production during temperature fluctuations and to speed up the recovery, mixed microbial consortium undergoing a period of either downward or upward temperature fluctuation was augmented with a synthetic mix culture containing well-known hydrogen producers. The addition of new species into the native consortium significantly improved hydrogen production both during and after the fluctuations. However, when the bioaugmentation was applied during the temperature fluctuation, hydrogen production was enhanced. This study also investigated the dynamics between pure cultures and co-cultures of highly specialized hydrogen producers, Caldicellulosiruptor saccharolyticus and Thermotoga neapolitana. The highest hydrogen yield (2.8 ± 0.1 mol H2 mol-1 glucose) was obtained with a synthetic co-culture which resulted in a 3.3 or 12% increase in hydrogen yield when compared to pure cultures of C. saccharolyticus or T. neapolitana, respectively. Furthermore, quantitative polymerase chain reaction (qPCR) based method for monitoring the growth and contribution of T. neapolitana in synthetic co-cultures was developed. With this method, it was verified that T. neapolitana was an active member of the synthetic co-culture. The effect of different feed glucose concentrations (from 5.6 to 111.0 mmol L-1) on hydrogen production was investigated with and without augmenting the culture with T. neapolitana. Compared to the control (without T. neapolitana), bioaugmentated culture resulted in higher hydrogen yields in almost all the concentrations studied even though hydrogen yield decreased the feed glucose concentration was increased. The presence of T. neapolitana also had a significant impact on the metabolite distribution when compared to the control.In summary, this study showed that thermophilic dark fermentative hydrogen production can be enhanced by using synthetic co-cultures or bioaugmentation. The highest hydrogen yield in this study was obtained with the synthetic co-culture, although it should be considered that the incubation conditions differed from those used for the mixed cultures in this study. The use of molecular methods such as qPCR and high-throughput sequencing also helped to understand the role of certain species in the microbial consortia and improved the understanding of the microbial community dynamics during stress conditions
... Within the Firmicutes phylum (94.0%), 64.7% belonged to Clostridiaceae, 15.8% belonged to Enterococcaceae and 13.3% belonged to Sporolactobacillaceae; only 5.6% belonged to the Gammaproteobacteria phylum and more precisely to the Enterobacteriaceae family. Affiliation to sequences of main OTUs ( Koskinen et al., 2007). It is also consistent with the butyrate production during Pinot Gris grape must deposits endogenous fermentation. ...
The aim of this work was to assess the performances of wine byproduct biomass for hydrogen production by dark fermentation. Grape must deposits from two grape varieties (Pinot Gris and Chardonnay) were considered, either with external microbial inoculum or without. We show that grape must residues contain endogenous microflora, well adapted to their environment, which can degrade sugars (initially contained in the biomass) to hydrogen without any nutrient addition. Indeed, hydrogen production during endogenous fermentation is as efficient as with an external heat-treated inoculum (2.5 ± 0.4 LH2.L⁻¹reactor and 1.61 ± 0.41 molH2.mol⁻¹consumed hexose, respectively) with a lower energy cost. Hydrogen-producing bacteria were selected from the endogenous microflora during semi-batch bioreactor operation, as shown by T-RFLP profiles and 16S rRNA sequencing, with Clostridium spp. (butyricum, beijerinckii, diolis, roseum) identified as the major phylotype. Such hydrogen production efficiency opens new perspectives for innovating in the valorization of winery by-products.
... Besides pH 6, which seems to be the favorable condition for PA producing bacteria, a reason for this could be that the thermal pretreatment inhibited the bacteria present in the dog food which can be considered to be the main competitors for the inoculum bacteria (Hu et al., 2014;Wang & Yin, 2017). At the same time, it might have selected for spore-producing microorganisms that include hydrogen producing bacteria which stimulate the inoculum bacteria to produce PA (Kim et al., 2008;Koskinen et al., 2007;Ren et al., 2007;Vavilin et al., 1995). This might also explain the higher amount of butyric acid produced from thermally treated food in some tests. ...
In this study, lab-scale batch fermentation tests were carried out at mesophilic temperature (30 °C) to examine the influence of inoculum type, pH-value, and thermal pretreatment of substrate on propionic acid production from dog food. The selected inocula comprised a mixed bacterial culture, milk, and soft goat cheese. The batch tests were performed at pH 4, pH 6, and pH 8 for both, untreated and thermally pretreated food. Results show that the production of PA and volatile fatty acids (VFAs) in general were significantly dependent on the chosen inoculum and adjusted pH value. The maximum PA production rates and yields were determined for the cheese inoculum at pH 6 using untreated and pretreated dog food. PA concentration reached 10 g L-1 and 26.5 g L-1, respectively. Our findings show that by selecting optimal process parameters, an efficient PA production from model food waste can be achieved.
... Cependant, les réacteurs les plus utilisés sont les réacteurs infiniment mélangés Li and Fang, 2007). En effet, les réacteurs à biomasse fixée présentent d'éventuels problèmes de colmatage (Zhang and Shen, 2006) et imposent des temps de séjours importants aux bactéries fixées, ce qui favorise les consommatrices d'hydrogène Koskinen et al., 2007). De plus, Ueno et al. (1996) ont montré que l'homoacétogénèse était favorisée par les longs temps de séjour, ce qui a un impact négatif sur la production d'hydrogène. ...
Soutenue le 24 octobre Diplôme : Dr. d'Universite
... Alors que les procédés photosynthétiques présentent des rendements de conversion plus élevés, les bioprocédés utilisant la voie fermentaire fournissent un panel métabolique plus large qui leur permet de s'adapter à un plus grand nombre de substrats organiques. Néanmoins, la complexité des communautés microbiennes utilisées engendre une instabilité des procédés mis en oeuvre (Koskinen et al., 2007) et le manque de connaissances des interactions microbiennes conduit le plus souvent à une optimisation en « boite noire » de ces bioprocédés. Dans le milieu naturel, la production de biohydrogène par voie fermentaire correspond à une étape intermédiaire et très spécifique de la digestion anaérobie de la matière organique qui comporte de nombreuses voies possibles de dégradation. ...
Colloque « Les biotechnologies pour relever le défi du carbone renouvelable » Toulouse 18/04/2013
... The formation of 1 mol of 1,3-PD is accompanied by the consumption of 1 mol of H 2 , and higher HYs are associated with the generation of acetic and butyric acid; however, these phenomena did not occur in the tests previously analyzed. Additionally, according to Koskinen et al. [42], the microbial community present in the biofilm may become enriched with propionate producers over time since they have better adhesion properties than H 2 producers. This peculiarity causes a decrease in hydrogen production and favors propionic acid production. ...
The individual and interactive effects of the influent glycerol, Gin (2.9–17.1 g. L⁻¹), and hydraulic retention time, HRT (0.76–9.24 h), were evaluated to optimize the production of hydrogen (H2 content and yield), 1,3-propanediol (1,3-PD) and propionic acid (HPr) in a mesophilic (30 °C) anaerobic fluidized bed reactor (AFBR). The maximum H2 content of 89.6% was obtained under the optimum conditions of 12.6 g. L⁻¹ and 4.58 h, while the maximum H2 yield of 0.31 mol H2.kg COD applied⁻¹ was obtained under the conditions of 18.0 g. L⁻¹ and 7.72 h. For both responses, Gin was the more significant individual variable; however, the interactive effects between the Gin and HRT variables also suggest their significance in the process. The influence of the organic loading rate (OLR) on the production of 1,3-PD and HPr was also investigated in the reactor. The maximum yield of 1,3-PD was 0.87 g g glycerolconsumed⁻¹ and was obtained under the conditions of 10 g. L⁻¹ and 9.24 h, which is equivalent to an OLR of 23.62 kg m.⁻³.day⁻¹. In contrast, HPr was produced in the highest yield of 0.57 g g glycerolconsumed⁻¹ under the conditions of 15 g. L⁻¹ and 2 h, which is equivalent to an OLR of 160.60 kg m.⁻³.day⁻¹. The different specific conditions determined to favor each product enhance the mixed fermentation and the existence of competing metabolic pathways in the AFBR.
... The expanded clay (Barros et al. 2010; N. C. S. Amorim et al. 2014;Ferreira et al. 2019) has the advantage of being a cheap and abrasion-insensitive material and has been used in AFBRs to treat synthetic and industrial effluents. On the other hand, activated carbon (Zhang et al. 2007) and celite (Koskinen, Kaksonen, and Puhakka 2007) are more expensive materials and require care in regard to abrasion because the turbulence in the system can easily reduce the size of these support materials (Barros et al. 2010). In addition, polymeric materials such as polystyrene (Barros et al. 2010;Barros and Silva 2012;Rosa et al. 2014a), ethylene vinyl acetate copolymer (Lin et al. 2009), silicone gel (Lin, Wu, and Chang 2006), polymeric waste such as ground tire (Barros et al. 2011;Silva et al. 2019) and polyethylene terephthalate (PET) (Barros et al. 2011;Barros and Silva 2012), were also efficient in the adhesion of the H2-producing microbial biomass in high rate reactors. ...
The Dark Fermentation of organic substrates is a low-cost process mediated by microorganisms which is an economically feasible method for hydrogen production. In addition to the possibility of reusing wastewater as a substrate, the use of Dark Fermentation for hydrogen production leads to positive energy gain by generating a renewable, carbon-free and a product with a high energy (120 kJ g-1). Several reactor configurations used for this purpose were reported in the literature. Among these configurations, the use of high rate reactors is attributed to the greater stability of Dark Fermentation leading to higher hydrogen production rates. In high-rate reactors, the microorganisms responsible for hydrogen production have a longer retention time than the substrate. An example of high rate reactor successfully used for fermentative hydrogen production is the three-phase Fluidized Bed Reactor, in which, the microorganisms are immobilized on the surface of a support material. The high superficial velocity of the liquid in the Fluidized Bed Reactor provides high mass transfer and, consequently, high rates of the biochemical reactions. These characteristics associated with biomass immobilization lead to the application of lower hydraulic retention times in the reactor, thus, enabling the design of lower reactor volumes. Therefore, this chapter will present the main results reported in the literature on the application of the Fluidized Bed Reactor in the fermentative production of hydrogen using different synthetic and complex substrates such as glucose, sucrose, xylose, sugarcane vinasse, cheese whey, cassava wastewater and glycerin. In addition, will be indicat the influences of the main operational parameters as support material, temperature, organic loading rate, hydraulic retention time and pH on hydrogen production in the Fluidized Bed Reactor.
... The low proportions of formic, acetic and propionic acid did not interfere in the bioH 2 production. The low concentrations of propionic acid suggest that the system was not under organic overloading conditions [61,62] highlighting that propionic acid accumulation leads to a drop in bioH 2 rates, which is not desired in acidogenic systems [63]. In addition, the literature indicates the favoring of propionic fermentation only at higher pH values (>6.0) [34,64], different from this study. ...
By-products from sugarcane mills have a considerable energy potential, and therefore have been studied aiming to generate biogas emphasising biohydrogen (bioH2). Sugarcane molasses, a byproduct from sugar production, are rich in carbohydrates, thus easily biodegraded by anaerobic microorganisms. This study evaluated the production of bioH2 in unfavorable pH (3.80) using molasses as a feedstock in an anaerobic structured bed reactor (AnSTBR-A) under thermophilic conditions (55 °C). The AnSTBR-A operated with an organic loading rate (OLR) of 60 g L⁻¹ d⁻¹ was able to produce bioH2 under long-term operation (392 days). The hydrogen yield (HY) was 1.18 mol H2 mol total carbohydrates⁻¹. The results highlighted HY variation concomitant with metabolite concentrations. The main role to bioH2 production in AnSTBR-A was acetate + lactate → butyric + bioH2, with a predominance of the organism belonging to the Thermoanaerobacterium genus.
... Microbial communities are not static and have their dynamics, in some processes this dynamic was associated with a stable process as demonstrated for methanogenic reactors [29]. However, in H 2 -producing reactors, changes in the community composition could be a cause of instability leading to the fluctuation of H 2 production [30][31][32][33]. Biodiversity is a community-level attribute that is generally associated with productivity in soil ecosystems [34] and also in methanogenic process [35]. ...
H2 production by dark fermentation using mixed cultures has been studied intensively during the last two decades, and its feasibility has been demonstrated. Different substrates, operational conditions, and reactor technologies have been widely studied and there is a general agreement that the use of non-sterile fermentable substrates is required to make the process feasible for scaling up. Nonetheless, stability problems during long term operation may hinder its application at large scale. This work, written by members of the Latin American Biohydrogen Network, analyse and discuss instability causes and possible solutions in the H2 production by dark fermentation. It is concluded that instability is mostly linked to the biotic aspects of the process (i.e., changes in the microbial community composition, presence of organisms that consume hydrogen and compete for the substrate, and accumulation of fermentation products); regardless of the reactor configuration. However, some problems like excessive growth of microorganisms and methanogens presence were mostly reported in fixed bed reactors and granular sludge reactors. The novelty of this work relies on the comprehensive revision of the main causes behind the unstable and low hydrogen production and how these causes are linked to the technology used. The strategies to overcome the problems as well as the potential implications are also analysed.
... The cellular metabolism of the fermentative microorganisms is easily affected by the physical and physicochemical conditions of the medium, and sudden changes in these conditions can lead to variations in the metabolic pathways, resulting in the production of unwanted products ( Koskinen et al., 2007). ...
An assessment of the biodegradability of the biogas digester when fed with co-substrates at different at different mixtures.
... Dominant bands were eluted in sterile water overnight. The re-amplification of bands for sequencing was conducted as described by Koskinen et al. (2007). The sequence data from this research have been compared with the data in the NCBI's Sequence Read Archive database (http://www.ncbi.nlm.nih.gov/blast). ...
The alternative methods should be used to treat sewage sludge and municipal solid wastes which are mostly landfilled in Turkey. Therefore co-composting of these waste streams is a suitable disposal method yielding a useful product. The aim of this study was to investigate the microbial community during the field scale co-composting of sewage sludge and organic municipal solid wastes with addition of bulking agents. Aerated static pile of approximately 26 m3 was used for composting process during 56 days. Investigations of diversity dynamics depending on the temperature were determined by denaturing gradient gel electrophoresis and sequencing of bacterial 16SrDNA-PCR products. The variations of physicochemical parameters and biodegradability during the process were also monitored. The results showed that most of microbial group’s role in composting process was temperature dependent and composting was designated by its characteristic thermal profile.
... Dominant bands were eluted in sterile water overnight. The re-amplification of bands for sequencing was conducted as described by Koskinen et al. (2007). The sequence data from this research have been compared with the data in the NCBI's Sequence Read Archive database (http://www.ncbi.nlm.nih.gov/blast). ...
The alternative methods should be used to treat sewage sludge and municipal solid wastes which are mostly landfilled in Turkey. Therefore co-composting of these waste streams is a suitable disposal method yielding a useful product. The aim of this study was to investigate the microbial community during the field scale co-composting of sewage sludge and organic municipal solid wastes with addition of bulking agents. Aerated static pile of approximately 26 m3 was used for composting process during 56 days. Investigations of diversity dynamics depending on the temperature were determined by denaturing gradient gel electrophoresis and sequencing of bacterial 16SrDNA-PCR products. The variations of physicochemical parameters and biodegradability during the process were also monitored. The results showed that most of microbial group's role in composting process was temperature dependent and composting was designated by its characteristic thermal profile.
... Support materials such as sand, coal, clay, and synthetic polymers are generally used in AFBR in wastewater treatment, and for better hydrogen production, activated carbon, celite, and expanded clay were generally employed. Studies show that better H 2 production mainly attributed to high microbial population, higher OLRs, volumetric H 2 production rates (HPR), and proper supporting material [9,22,[25][26][27]. Biogas generated from AFBR contains hydrogen, carbon dioxide, and free from methane. ...
... Moreover, there was a low lactate concentration and absolutely zero propionate in the produced OAs. Previous studies have reported that the propionate-type dark fermentation is a hydrogen-consuming pathway that produces mainly propionate, acetate and some valerate, without significant gas production (Cabrol et al., 2017;Koskinen et al., 2007) meanwhile, as already explained before, the presence of lactic acid as a metabolite during dark fermentation has been associated with lower hydrogen production. ...
In this study a two-steps bioprocess approach aimed at biohydrogen production via dark-fermentation, and polyhydroxyalkanoates-PHA production by mixed microbial cultures, was proposed to valorise two dairy-waste streams coming from cheese whey deproteinization (i.e. Ricotta cheese production and ultrafiltration). During the first step, the increase of OLR was tested, resulting in higher daily H 2 volume (3.47 and 5.07 NL H 2 d À1 for second cheese whey-SCW and concentrated cheese whey permeate-CCWP) and organic acids production (14.6 and 12.6 g L À1 d À1 for SCW and CCWP) for both the substrates, keeping good conversion of sugars into H 2 (1.37 and 1.93 mol H 2 mol À1 sugars for SCW and CCWP). During the second step, the organic acids were used for PHA production reaching high conversion yields for both the fermented streams (as average 0.74 ± 0.14 mg COD PHA mg À1 COD OA-in), with a maximum polymer content of 62 ± 4.5 and 55.1 ± 1.3% (g PHA g À1 VSS) for fermented SCW and fermented CCWP respectively. For the results reported, this study could be taken into consideration for larger scale application.
... Previous FBR publications often focus on specific aspects, such as process modelling (Shen et al., 2012) and or use in specific applications such as low-temperature bioremediation of polychlorophenols (Puhakka and Melin, 1998), biological iron oxidation (Nurmi et al., 2009), anaerobic sulfate treatment (Kaksonen et al., 2006) and acid mine drainage treatment using sulfate reduction based processes Papirio et al., 2013) as well as bio-hydrogen production (Koskinen et al., 2007). Some papers review the use of FBR technology in particular fields, such as wastewater treatment (Bello et al., 2017;Nelson et al., 2017;Burghate and Ingole, 2013). ...
Fluidized bed reactors (FBR) are characterized by two-phase mixture of fluid and solid, in which the bed of solid particles is fluidized by means of downward or upward recirculation stream. FBRs are widely used for multiple environmental engineering solutions, such as wastewater treatment, as well as some industrial applications. FBR offers many benefits such as compact reactor size due to short hydraulic retention time, long biomass retention on the carrier, high conversion rates due to fully mixed conditions and consequently high mass transfer rates, no channelling of flow, dilution of influent concentrations due to recycle flow, suitability for enrichment of microbes with low Km values. The disadvantages of FBRs include reactor size limitations due to the height-to-diameter ratio, high-energy requirements due to high recycle ratios, and long start-up period for biofilm formation. This paper critically reviews key studies on biomass enrichment via immobilisation of low growth yield microorganisms, high-rates via fully mixed conditions, technical developments in FBRs and ways of overcoming toxic effects via solution recycling. This technology has many potential new uses as well as hydrodynamic characteristics which enable high-rate environmental engineering and industrial applications.
... Therefore, based on the results obtained in this batch experiment (Figure 1), dark fermentation of TMP wastewater at 37 and 65 °C appears favourable if suspended biomass bioreactors are used, as homoacetogenic bacteria would be flushed out ( Figure 1). However, bioreactors retaining high active biomass content, such as FBRs or upflow anaerobic sludge bioreactors (UASBs), would enable higher organic loadings and conversion rates than suspended biomass bioreactors (Koskinen et al., 2006 Figure 1) with a thermophilic inoculum previously stored at 4 °C for one week. ...
This study evaluates the use of non-pretreated thermo-mechanical pulping (TMP) wastewater as a potential substrate for hydrogen production by dark fermentation. Batch incubations were conducted in a temperature gradient incubator at temperatures ranging from 37 to 80 °C, using an inoculum from a thermophilic, xylose-fed, hydrogen-producing fluidised bed reactor. The aim was to assess the short-term response of the microbial communities to the different temperatures with respect to both hydrogen yield and composition of the active microbial community. High throughput sequencing (MiSeq) of the reversely transcribed 16S rRNA showed that Thermoanaerobacterium sp. dominated the active microbial community at 70 °C, resulting in the highest hydrogen yield of 3.6 (±0.1) mmol H2 g⁻¹ CODtot supplied. Lower hydrogen yields were obtained at the temperature range from 37 to 65 °C, likely due to consumption of the produced hydrogen by homoacetogenesis. No hydrogen production was detected at temperatures above 70 °C. Thermomechanical pulping wastewaters are released at high temperatures (50–80 °C), and thus dark fermentation at 70 °C could be sustained using the heat produced by the pulp and paper plant itself without any requirement for external heating.
... 8% polyacrylamide gel (acrylamide/bisacrylamide gel stock solution 37.5:1) was prepared with a denaturing gradient from 40% to 60% (100% denaturing solution contains 7 M of urea and 40% formamide). Gel were run at 60 C in 1xTAE with 130 V for 8 h, DGGE bands were cut directly from the gel and incubated in 40 ml of deionized water overnight at 4 C [22]. At the end of the incubation period, 2 ml of the solution was used as a template in a re-amplification reaction using specific target primers (338 Fwithout GC clamp and 518R). ...
Trace elements are one of the important parameters for dark fermentative H2 production because they work as co-factors in H2 formation biochemistry. Lack or excess of trace element and its concentrations could be an important reason for the low yield of H2 production. In this study, the effects of 11 different trace elements (Fe, Ni, Zn, Co, Cu, Mn, Al, B, Se, Mo and W) were tested at two levels in terms of biohydrogen production from Fruit and Vegetable Wastes (FVW) with Biochemical Hydrogen Potential (BHP) Tests using Plackett-Burman statistical design. 1.1–2.8 times enhancement of biohydrogen production was determined with its addition. The most effective trace elements were found as Zn and Ni. In order to reveal the resident microbial flora, Denaturing Gradient Gel Electrophoresis (DGGE) analysis was carried out on all BHP effluent samples. Results of DGGE analysis, four microbial sequences evaluated as Clostridium sp., Clostridium baratii, Uncultured bacterium, Uncultured Streptococcus sp., and their similarity rates were 99%, 100%, 89%, 98%, respectively.
... The experiment also revealed the presence of possible hydrogen consumers related to the class Bacteroidetes and other strains affiliated with Lachnospiraceae and Veillonellaceae. 64 The presence of hydrogen consumers is a good indication of the reduction in hydrogen production at the later stage of fermentation. ...
Microbial degradation of straw, the main by-product of agricultural production, has proved to be the most economical and effective means of producing hydrogen. Mixed cultures provide stable combinations to process complex materials, thereby supporting more efficient decomposition and hydrogenation of biomass than pure bacterial species. Cellulose, the main component of straw, is degraded by microorganisms found in the rumen fluid of cows and converted to hydrogen gas, ethanol, and other fuels. This study investigated hydrogen production and microbial community structures during cellulose degradation by rumen microorganisms at pH values in the range of 5.5-7.5. The highest degradation efficiency was 81% at pH 6.5 with no methane and with the maximum hydrogen yield of 178.16 mL L⁻¹ (culture medium) (8.42 mmol H2 g⁻¹ Avicel). The yield value is higher than that associated with most mesophilic bacteria subjected to serial inoculation. The microbial diversity of rumen liquid enrichments at different pH values was analyzed using 454 pyrosequencing techniques. The dominant bacteria changed from Bacteroides, Enterococcus, and Enterobacter to Oscillibacter. This study thus examined the effect of pH on the efficiency of cellulose utilization in rumen bacteria growth. The results provide information about the mechanisms of metabolization at different pH values in diverse microflora.
... -1 (Table 3). Variations in the concentration of the metabolites suggest that the system presented changes among the fermentative pathways that were established in accordance with the operational conditions imposed to the reactor or even by the growth of different microbial communities (KOSKINEN et al., 2007;GUO et al., 2008). ...
Fixed-bed reactors have been considered promise alternatives for hydrogen production due to their simple construction and increase in the biomass retention. The purpose of this study was to investigate the biological production of hydrogen in anaerobic fixed-bed reactors with cassava starch wastewater used as substrate. Different support materials and arrangements of fixed-bed were used to evaluate the biological production of hydrogen in anaerobic continuous fixed-bed reactors, with cassava starch wastewater as substrate - recycled low-density polyethylene scraps, in packed bed (R1), recycled low-density polyethylene cylinders, in ordained bed (R3) and bamboo stems, in vertical arrangement (R2 and R4). In R1 the initial pH was adjusted for 6.0, with hydraulic retention time (HRT) of 4 h and organic loading rate (OLR) of 9.5 g.L−1.d−1. In R2 the initial pH was maintained in 4.5, with HRT of 4 h and OLR of 9.5 g.L−1.d−1. R3 and R4 were operated with initial pH of 4.5, HRT of 4 h and OLR of 13.5 g.L−1.d−1. The volumetric hydrogen production (VHPR) was favored by lower OLR applied, evenin different pH ranges (6.0 and 4.5). VHPR values of 229 mL H2.L−1.d−1and 248 mL H2.L−1.d−1 were obtained in R1 and R2, respectively. Both in the bamboo stems bed as in the polyethylene cylinders bed, the increase of OLR and the reduction of the initial pH resulted in a diminishing of VHPR to 175 mL H2.d−1.L−1 (R3) and 145 m LH2 .d−1.L−1 (R4). Higher concentrations of butanol (821.32 and 1,529.22 mg.L−1) and ethanol (915.41 and 924.41 mg.L−1) were verified in the reactors with bamboo stems. In R4, the increase of OLR and the reduction of the initial pH contributed to the increase of butanol concentration in 1.8 times, diminishing the VHPR in 41.68%, yield in 63.95% and H2 in 37.6%, and indicating that the effect of pH is more pronounced with the increase of OLR, leading to the solventogenesis.
... Unsurprisingly, the observed enrichment represented by the most abundant OTUs, consisted of species known to produce hydrogen and VFA products in both mono-and mixedculture bioreactors. More specifically, all bioreactors were enriched for Clostridium pasteurianum, Clostridium acetobutyricum, Escherichia coli, as well as other OTUs classified within the Clostridium and Enterobacter genera (Fig 4A) [1,18,[30][31][32]. All enriched community members, with the exception of the C. pasteurianum, were present at very low abundances in at least two out of three replicates at cycle zero. ...
The sustainable recovery of resources from wastewater streams can provide many social and environmental benefits. A common strategy to recover valuable resources from wastewater is to harness the products of fermentation by complex microbial communities. In these fermentation bioreactors high microbial community diversity within the inoculum source is commonly assumed as sufficient for the selection of a functional microbial community. However, variability of the product profile obtained from these bioreactors is a persistent challenge in this field. In an attempt to address this variability, the impact of inoculum on the microbial community structure and function within the bioreactor was evaluated using controlled laboratory experiments. In the course of this work, sequential batch reactors were inoculated with three complex microbial inocula and the chemical and microbial compositions were monitored by HPLC and 16S rRNA amplicon analysis, respectively. Microbial community dynamics and chemical profiles were found to be distinct to initial inoculate and highly reproducible. Additionally we found that the generation of a complex volatile fatty acid profile was not specific to the diversity of the initial microbial inoculum. Our results suggest that the composition of the original inoculum predictably contributes to bioreactor community structure and function.
... Acetic, propionic, butyric, and lactic acids, as well as acetone, methanol, ethanol, and butanol, were the main soluble microbial products detected in this work ( Table 2). The ethanolic fermentation has been linked to a low production of H 2 in different studies (Guo et al., 2008;Koskinen et al., 2007Koskinen et al., , 2008Ren et al., 1997). In our case, one of the reasons for the low yield of H 2 might be the high production of ethanol, which generally showed concentrations that accounted for ca. ...
This research assessed the viability to use disposable diapers as a substrate for the production of biohydrogen, a valuable clean-energy source. The important content of cellulose of disposable diapers indicates that this waste could be an attractive substrate for biofuel production. Two incubation temperatures (35 °C and 55 °C) and three diaper conditioning methods (whole diapers with faeces, urine, and plastics, WD; diapers without plastic components, with urine and faeces, DWP; diapers with urine but without faeces and plastic, MSD) were tested in batch bioreactors. The bioreactors were operated in the solid substrate anaerobic hydrogenogenic fermentation with intermittent venting mode (SSAHF-IV). The batch reactors were loaded with the substrate at ca. 25% of total solids and 10% w/w inoculum. The average cumulative bioH2 production followed the order WD > MSD > DWP. The bio-H2 production using MSD was unexpectedly higher than DWP; the presence of plastics in the first was expected to be associated to lower degradability and H2 yield. BioH2 production at 55 °C was superior to that of 35 °C, probably owing to a more rapid microbial metabolism in the thermophilic regime. The results of this work showed low yields in the production of H2 at both temperatures compared with those reported in the literature for municipal and agricultural organic waste. The studied process could improve the ability to dispose of this residue with H2 generation as the value-added product. Research is ongoing to increase the yield of biohydrogen production from waste disposable diapers.
... The 16S rRNA genes from the extracted DNA were amplified by polymerase chain reaction (PCR) as described by Ahoranta et al.[25]. Denaturing gradient gel electrophoresis (DGGE) was performed with a denaturing gradient from 30 to 70% and the gel was prepared and run (100 V for 22.5 h) as described by Koskinen et al.[26]. DNA purification and sequencing was performed by Macrogen (Seoul, Korea). ...
This study investigated the impacts of thiosulfate (S2O32−) as well as chemically synthesized and biogenic elemental sulfur (S0) on the rates of sulfur-based denitrification in batch bioassays. The use of S2O32− resulted in the highest denitrification rate (52.5 mg N-NO3−/L d), whereas up to 10 times slower nitrate (NO3−) removal was observed with S0. Biogenic S0 was tested for the first time as electron donor for chemolithotrophic denitrification, resulting in 1.7-fold faster NO3− removal than that achieved with chemically synthesized S0. The effects of increasing concentrations of S2O32− and chemically synthesized S0 on denitrification were evaluated testing three different sulfur-to-nitrogen (S/N) ratios (1.8, 3.5 and 5.1) on a pure culture of _Thiobacillus denitrificans_ and a mixotrophic enrichment dominated by _Thiobacillus thioparus_. S2O32− concentrations exceeding 2.2 g/L inhibited the activity of _T. denitrificans_, whereas a stimulatory effect was observed on mixotrophic denitrification. The increase in S0 concentration slightly enhanced denitrification by both microbial cultures due to the low solubility of chemically synthesized S0. The temperature dependence of thiosulfate-driven denitrification by _T. denitrificans_ was investigated to further optimize the process and modeled by the Arrhenius equation with an apparent activation energy _Ea_ of 76.6 kJ/mol and a temperature coefficient _Q10_ of 3.0.
... The low and unstable hydrogen production was found to be associated with high propionate production during the initial start-up period (Fig. 3). Formation of propionate (HPr) lowers hydrogen production as it consumes the produced hydrogen for its formation [36,37]. During the steady-state conditions at various HRTs, butyrate, and acetate were major metabolites with low propionate concentra-tion. ...
... However, there are also several issues associated with their use. As complex communities, their composition can vary over time, with changes in process parameters and from reactor to reactor, as was shown by molecular (16sRNA) studies [25][26][27][28]. A possible way to overcome this issue might be to construct 'designer' consortia [29] with the goal of creating a community of diverse members, each contributing a unique and essential metabolic capacity. ...
... The washout pressure due to the low HRT applied to CSTR could explain the lower diverse community compared to biomass fixed reactors with higher solid retention prompt by the bacteria adhesion capability. In this sense, Koskinen et al. (2007) have previously determined that biofilm-based reactors could enrich hydrogen-consuming microorganisms at mesophilic conditions due to their adhesion capability. In order to explain the differences inside sample B, the influence of the time of enrichment and the packing material should be analyzed. ...
To provide new insight into the dark fermentation process, a multi-lateral study was performed to study the microbiology of 20 different lab-scale bioreactors operated in four different countries (Brazil, Chile, Mexico, and Uruguay). Samples (29) were collected from bioreactors with different configurations, operation conditions, and performances. The microbial communities were analyzed using 16S rRNA genes 454 pyrosequencing. The results showed notably uneven communities with a high predominance of a particular genus. The phylum Firmicutes predominated in most of the samples, but the phyla Thermotogae or Proteobacteria dominated in a few samples. Genera from three physiological groups were detected: high-yield hydrogen producers (Clostridium, Kosmotoga, Enterobacter), fermenters with low-hydrogen yield (mostly from Veillonelaceae), and competitors (Lactobacillus). Inocula, reactor configurations, and substrates influence the microbial communities. This is the first joint effort that evaluates hydrogen-producing reactors and operational conditions from different countries and contributes to understand the dark fermentation process.
The present study investigated the effect of the initial proportions of carbohydrates, proteins and lipids within the substrate on the resulting biohydrogen productivity by dark fermentation. Organic matter removal and the related metabolic by-products generated during the process were also assessed. The results obtained showed that initial substrate composition in terms of carbohydrates, proteins and lipids has a significant effect on maximal potential hydrogen production (Hmax), hydrogen production rate (Rmax), hydrogen yield (YH2) and metabolites distribution. Tests with proteins and lipids as unique substrate did not produce H2. A simplex-centroid design (SCD) and compositional data analysis of the substrate was used to determine the best condition to convert the substrate into H2. Hmax, Rmax and YH2 were significantly increased using an initial proportion of 56% carbohydrates (15 g/L), 22% proteins (6 g/L), and 22% lipids (6 g/L), which was concomitant with the generation of acetic and butyric acids. Protein and lipid proportions higher than 29% and lower than 12% led to decreased Hmax, Rmax and YH2 values with a consequent accumulation of propionic acid.
A liquid/gas membrane bioreactor (L/G MBR) was developed to intensify the dark fermentation process. A hollow fiber membrane module was used to combine biohydrogen production, in situ liquid-gas separation and hydrogen producing bacteria retention in a single unit. The L/G MBR was seeded once and did not require further microbial input, as consistent average hydrogen yield of 0.97 ± 0.09 mol-H2/added mol-glucose and hydrogen production rate of 106.5 ± 10.6 mL-H2/L-medium/h were reached over a year. Different biogas extraction strategies showed that efficient in situ H2 extraction is possible without sweeping gas in the lumen of the fibers, thus facilitating H2 purification in an industrial setting. Modelling predicted an optimal hydrogen yield of 1.2 mol/mol-glucose added for a glucose concentration in the feed of 13.1 g/L, close to experimental hydraulic retention time of 8-10 h with an organic loading rate of 1.4 g-glucose/L-medium/h. No washout of hydrogen-producing bacteria was observed at low HRT (2 h), suggesting the possibility of further hydrogen production rate enhancement using an optimized organic loading rate. Acetate and butyrate were the main metabolites identified. Clostridium and Enterobacter dominated in the liquid outlet. The relative abundance of Clostridium pasteurianum increased with glucose concentration in the bioreactor, as opposed to Clostridium beijerinckii which was more abundant at low glucose concentration. The original hollow fiber L/G MBR configuration enabled the testing and selection of fermentation strategies that greatly simplified the implementation of the dark fermentation process by addressing its key operational bottlenecks. Indeed, the L/G membrane surface served as a support and reservoir for the hydrogen producing bacteria across a wide range of HRT conditions.
This study conducted the utilization of vegetable residues by an enriched microflora inoculum to produce biohydrogen via anaerobic batch reactor. Dark fermentation processes were carried out with 3 kinds of vegetable residue substrates including broccoli (Brassica oleracea var. italica.), onion (Alium cepa Linn.), and sweet potato (Ipomoea batatas (L.) Lam). Vegetable wastes were pretreated into 2 forms, i.e. mashed and powdered vegetable, prior to the fermentation. The substrate used for the biohydrogen production were vegetable residues and inoculum at the vegetable residues/inoculum ratio of 1:1 (based on TS). The digestion processes were performed under 120 rpm speed of shaking bottle in the incubator with control temperature of 35๐C. In this work, the maximum hydrogen production was achieved by anaerobic digestion at mashed onion with bioreactor inoculum that produced total hydrogen of 424.1 mL H2 with hydrogen yield and hydrogen concentration of 151.67 mL H2/g VSadded and 43.54%, respectively. In addition, the hydrogen production continues took only 7 days for the vegetables blended with the bioreactor inoculum. Finally, it was found that the high potential of degradation of vegetable wastes an enriched microflora in dark fermentation also showed alternative solution to eliminate agricultural wastes to produce green energy.
Dark fermentation and microbial fuel cells (MFCs) are two emerging technologies for biological conversion of the chemical energy of organic compounds into hydrogen (H2) and electricity, respectively. Due to kinetic and thermodynamic advantages, high temperature can be the key for increasing both dark fermentative H2 production and electricity production in MFCs. Therefore, this thesis focuses on delineating how temperature influences biological production of H2 and electricity from organic carbon-containing wastewaters. Two heat-treated inocula (fresh and digested activated sludge) were compared, for H2 production from xylose at 37, 55 and 70 °C. At both 37 and 55 °C, a higher H2 yield was achieved by the fresh than digested activated sludge, whereas a very low H2 yield was obtained by both inocula at 70 °C. Then, four different inoculum pretreatments (acidic, alkaline, heat and freezing shocks) were evaluated for creating an efficient mesophilic (37 °C) or thermophilic (55 °C) H2 producing community. Acidic and alkaline shocks selected known H2 producing microorganisms belonging to Clostridiaceae at the expenses of lactate producing bacteria, resulting in the highest H2 yield at 37 and 55 °C, respectively. Although a heat shock resulted in a low H2 yield in a single batch, H2 production by the heat-treated fresh activated sludge was shown to increase in the experiment with four consecutive batch cycles.Heat-treated fresh activated sludge was selected as inoculum for continuous H2 production from a xylose-containing synthetic wastewater in a mesophilic (37 °C) and a thermophilic (55-70 °C, increased stepwise) fluidized bed reactor (FBR). A higher H2 yield was obtained in the thermophilic than in the mesophilic FBR. Furthermore, H2 production at 70 °C, which failed in the earlier batch study, was successful in the FBR, with a stable yield of 1.2 mol H2 mol-1 xyloseadded. Operation temperature of 70 °C was also found optimal for H2 production from thermomechanical pulping (TMP) wastewater in a temperature gradient incubator assay.A RNA approach was used to study the structure and role of the anode-attached, membrane-attached and planktonic microbial communities in a mesophilic (37 °C) and a thermophilic (55 °C) two-chamber, xylose-fed MFC. An anode attached community dominated by Geobacteraceae sustained electricity production at 37 °C, whereas the establishment of methanogenic and H2 oxidizing microorganisms resulted in a low electricity production at 55 °C. However, the development of a thermophilic exoelectrogenic community can be promoted by applying a start-up strategy which includes imposing a negative potential to the anode and chemical inhibition of methanogens. A mesophilic exoelectrogenic community was also shown to produce electricity from TMP wastewater in an upflow MFC operated at 37 °C. In conclusion, a higher and more stable H2 yield can be achieved in thermophilic rather than mesophilic dark fermentation. Dark fermentation at 70 °C is particularly suitable for treatment of TMP wastewater as it is released at high temperature (50-80 °C) and could be treated on site. TMP wastewater can be also used as substrate for electricity production in mesophilic MFCs. Electricity production in thermophilic MFCs is feasible, but enrichment of thermophilic exoelectrogenic microorganisms may require a long start-up period
Glycerol, the main residue of biodiesel production, can be used to produce organic acids and energy through anaerobic digestion. This study aimed to assess microbial structure, diversity, productivity, and stability and the influence of these parameters on the performance of an anaerobic reactor. The experimental setup consisted of an upflow anaerobic sludge blanket (UASB) reactor fed residual glycerol and nutrients. The organic loading rate (OLR) was gradually increased through five stages, and sludge samples were collected at each, followed by DNA extraction and PCR denaturing gradient gel electrophoresis (PCR-DGGE). The resulting bands were excised, amplified, and purified. The results showed increased bacterial diversity and richness from the inoculum (Rr 38.72 and H 2.32) and along stages I and II, reaching the highest populational parameters (Rr 194.06 and H 3.32). The following stages promote decreases in richness and diversity, achieving the lowest populational parameters on this study (Rr 11.53 and H 2.04). Biogas production increased along with functional organization due to the specialization of the bacterial community and a decrease in the methanogenic population, both promoted by the increase in OLR.
Background
The versatility of microbial metabolic pathways enables their utilization in vast number of applications. However, the electron and carbon recovery rates, essentially constrained by limitations of cell energetics, are often too low in terms of process feasibility. Cocultivation of divergent microbial species in a single process broadens the metabolic landscape, and thus, the possibilities for more complete carbon and energy utilization.
Results
In this study, we integrated the metabolisms of two bacteria, an obligate anaerobe Clostridium butyricum and an obligate aerobe Acinetobacter baylyi ADP1. In the process, a glucose-negative mutant of A. baylyi ADP1 first deoxidized the culture allowing C. butyricum to grow and produce hydrogen from glucose. In the next phase, ADP1 produced long chain alkyl esters (wax esters) utilizing the by-products of C. butyricum, namely acetate and butyrate. The coculture produced 24.5 ± 0.8 mmol/l hydrogen (1.7 ± 0.1 mol/mol glucose) and 28 mg/l wax esters (10.8 mg/g glucose).
Conclusions
The cocultivation of strictly anaerobic and aerobic bacteria allowed the production of both hydrogen gas and long-chain alkyl esters in a simple one-pot batch process. The study demonstrates the potential of ‘metabolic pairing’ using designed microbial consortia for more optimal electron and carbon recovery.
Electronic supplementary material
The online version of this article (10.1186/s13068-018-1186-9) contains supplementary material, which is available to authorized users.
Hydrogen is the element of greatest abundance in the universe; however, its production from renewable resources remains a major challenge. Biohydrogen produced from biorenewables is a promising alternative for a sustainable energy source. Biohydrogen is a renewable biofuel produced from biorenewable feedstocks by chemical, thermochemical, biological, biochemical, and biophotolytical methods. As a sustainable energy supply with minimal or zero use of hydrocarbons, hydrogen is a promising alternative to fossil fuel. It is a clean and environmentally friendly fuel, which produces water instead of greenhouse gases when combusted.
Anaerobic ammonium oxidation (anammox) has been studied extensively while no widely accepted optimum values for nitrite (both a substance and inhibitor) has been determined. In the current paper, nitrite spiking (abruptly increasing nitrite concentration in reactor over 20 mg NO−2-NL−1) effect on anammox process was studied on three systems: a moving bed biofilm reactor (MBBR), a sequencing batch reactor (SBR) and an upflow anaerobic sludge blanket (UASB). The inhibition thresholds and concentrations causing 50% of biomass activity decrease (IC50) were determined in batch tests. The results showed spiked biomass to be less susceptible to nitrite inhibition. Though the values of inhibition threshold and IC50 concentrations were similar for non-spiked biomass (81 and 98 mg NO−2-NL−1, respectively, for SBR), nitrite spiking increased IC50 considerably (83 and 240 mg NO−2-NL−1, respectively, for UASB). As the highest total nitrogen removal rate was also measured at the aforementioned thresholds, there is basis to suggest stronger limitational effect of nitrite on anammox process than previously reported. The quantitative polymerase chain reaction analysis showed similar number of anammox 16S rRNA copies in all reactors, with the lowest quantity in SBR and the highest in MBBR (3.98 × 108 and 1.04 × 109 copies g−1 TSS, respectively).
Most of the fermentative hydrogen production studies based on mixed cultures have shown enrichment of the microbial community by means of a heat treatment. This heat treatment enrichment strategy selects for Clostridum spp., an efficient hydrogen producer; however, other bacteria that may contribute to the systems performance could be excluded. Another enrichment strategy based on high dilution rates selects different taxonomic groups, which may affect hydrogen production and the system stability. In this work, two enrichment strategies were evaluated, heat shock and cell wash-out, for hydrogen production and the system stability in continuous stirred reactors. The enriched communities were then inoculated in packed bed reactors and operated up to 70 days. Both strategies selected hydrogen producing bacteria, mainly Clostridium spp. The highest hydrogen production rate (6.01 L H2/L-d), molar yield (1.29 mol H2/mol glucoseconsumed), and stability were achieved by the wash-out procedure; this high performance was attributed to facultative bacteria like Lactobacillus and Lactococcus. Furthermore, there was a transition within the community (along the operation time in the reactor with cell wash-out inoculum) and a selection for methanogenic activity (due to the long solids retention time).
In this study, an adaptive laboratory evolution strategy was originally developed to enhance fermentative hydrogen production by directionally regulating the metabolic heterogeneity in anaerobic mixed culture. The results indicated that the co-introduction of 4-methylpyrazole and oxamate could redistribute the metabolic flux to butyrate-type hydrogen fermentation. Subsequently, a synergistic evolutionary pressure, combining exogenous butyrate stress with 4-methylpyrazole and oxamate, was employed to evolve hydrogen-producing mixed culture with continuous fermentation system. The metabolic engineering strategy could directionally regulate the metabolic heterogeneity through efficiently shaping powerful butyrate-type hydrogen-producing community, by which evolved culture acquired a significantly improved hydrogen yield and productivity. Furthermore, compared with original culture, evolved culture possessed much higher activities of pyruvate-ferredoxin oxidoreductase and hydrogenase but a much lower ferredoxin-NAD+ oxidoreductase activity, and these enzymatic evolutionary mechanisms were crucially important for the enhanced hydrogen fermentation.
Denaturing gradient gel electrophoresis (DGGE) of 16S rDNA profiles were objectively digitized using an image analyzer; the individual microbial species in a community can thus be precisely quantified. The similarity between various microbial communities was compared to the digitized DGGE profiles using the cluster analyses technique. The microbial community in a biofilm was considerably different from that in suspended sludge obtained from the same system.
This paper reviews information from continuous laboratory studies of fermentative hydrogen production useful when considering practical applications of the technology. Data from reactors operating with pure cultures and mixed microflora enriched from natural sources are considered. Inocula have been derived from heat-treated anaerobically digested sludge, activated sludge, aerobic compost and soil, and non-heat-treated aerobically composted activated sludge. Most studies are on soluble defined substrates, and there are few reports of continuous operation on complex substrates with mixed microflora to produce H2. Methanogenesis which consumes H2 may be prevented by operation at short hydraulic retention times (around 8– on simple substrates) and/or pH below 6. Although the reactor technology for anaerobic digestion and biohydrogen production from complex substrates may be similar, there are important microbiological differences, including the need to manage spore germination and oxygen toxicity on start-up and control sporulation in adverse circumstances during reactor operation.
The Ribosomal RNA Operon Copy Number Database (rrndb) is an Internet-accessible database containing annotated information
on rRNA operon copy number among prokaryotes. Gene redundancy is uncommon in prokaryotic genomes, yet the rRNA genes can vary
from one to as many as 15 copies. Despite the widespread use of 16S rRNA gene sequences for identification of prokaryotes,
information on the number and sequence of individual rRNA genes in a genome is not readily accessible. In an attempt to understand
the evolutionary implications of rRNA operon redundancy, we have created a phylogenetically arranged report on rRNA gene copy
number for a diverse collection of prokaryotic microorganisms. Each entry (organism) in the rrndb contains detailed information
linked directly to external websites including the Ribosomal Database Project, GenBank, PubMed and several culture collections.
Data contained in the rrndb will be valuable to researchers investigating microbial ecology and evolution using 16S rRNA gene
sequences. The rrndb web site is directly accessible on the WWW at http://rrndb.cme.msu.edu.
Suh, Byungse (University of Kansas, Lawrence), and J. M. Akagi. Pyruvate-carbon dioxide exchange reaction of Desulfovibrio desulfuricans. J. Bacteriol.
91
2281–2285. 1966.—The pyruvate-CO2 exchange reaction, catalyzed by Desulfovibrio desulfuricans, required the presence of phosphate and coenzyme A. However, the requirement for phosphate disappeared when the concentration of coenzyme A was increased to a level of 3.8 × 10⁻³m. Passing crude extracts through a diethylaminoethyl-cellulose column and an Amberlite CG-50 ion-exchange column, to remove ferredoxin and cytochrome c3, resulted in a marked decrease in exchange activity; full activity was restored by the addition of ferredoxin or cytochrome c3. Fe⁺⁺ or Co⁺⁺ stimulated the exchange of CO2 into pyruvate.
A methanogenic bacterium, commonly seen in digested sludge and referred to as the "fat rod" or Methanobacterium soehngenii, has been enriched to a monoculture and is characterized. Cells are gramnegative, non-motile and appear as straight rods with flat ends. They form filaments which can grow to great lengths. The structure of the outer cell envelop is similar to Methanospirillum hungatii. The organism grows on a mineral salt medium with acetate as the only organic component. Acetate is the energy source, and methane is formed exclusively from the methyl group. Acetate and carbon dioxide act as sole carbon source and are assimilated in a molar ratio of about 1.9:1. The reducing equivalents necessary to build biomass from these two precursors are obtained from the total oxidation of some acetate. Hydrogen is not used for methane formation and is not needed for growth. Formate is cleaved into hydrogen and carbon dioxide. Coenzyme M was found to be present at levels of 0.35 nmol per mg of dry cells and F420 amounted to 0.55 microgram per mg protein. The mean generation time was 9 days at 33 degrees C.
We describe a new molecular approach to analyzing the genetic diversity of complex microbial populations. This technique is based on the separation of polymerase chain reaction-amplified fragments of genes coding for 16S rRNA, all the same length, by denaturing gradient gel electrophoresis (DGGE). DGGE analysis of different microbial communities demonstrated the presence of up to 10 distinguishable bands in the separation pattern, which were most likely derived from as many different species constituting these populations, and thereby generated a DGGE profile of the populations. We showed that it is possible to identify constituents which represent only 1% of the total population. With an oligonucleotide probe specific for the V3 region of 16S rRNA of sulfate-reducing bacteria, particular DNA fragments from some of the microbial populations could be identified by hybridization analysis. Analysis of the genomic DNA from a bacterial biofilm grown under aerobic conditions suggests that sulfate-reducing bacteria, despite their anaerobicity, were present in this environment. The results we obtained demonstrate that this technique will contribute to our understanding of the genetic diversity of uncharacterized microbial populations.
Strictly anaerobic, gram-positive, nonsporing, thin rod-shaped organisms whose cells were 0.2 to 0.6 by 3 to 6 microns were isolated from a Hoechst Biohochreaktor (strain FaeT [T = type strain]) and from the biofilm population of a fixed-film reactor treating sour whey (strain FT). Strain FT was vigorously motile during early logarithmic growth by means of peritrichously inserted flagella, while strain FaeT was seldom motile and usually possessed no flagella. During the stationary growth phase both strains formed spheroplasts. The temperature optimum was close to 37 degrees C (temperature range for growth, > or = 17 to < 45 degrees C) and the pH optimum was 7.0 to 7.4 (pH range, 6.5 to 8.0) for both strains. The two organisms grew chemoorganotrophically on a number of mono- and disaccharides, including glucose and xylose; yeast extract was required for growth. The principal fermentation products from glucose included lactate, acetate, ethanol, formate, and CO2. Hydrogen was not generated. The G + C contents of the DNAs of strains FaeT and FT were 55 and 54.5 mol%, respectively. The cell wall architecture was typical of gram-positive bacteria; the cells had an extraordinarily thin type A3 alpha' peptidoglycan layer containing muramic acid. Analysis of 16S ribosomal DNA sequences of the two new isolates demonstrated that they represent members of a new genus of bacteria in Clostridium cluster IV of the domain Bacteria and that the misclassified organism Fusobacterium prausnitzii and Clostridium leptum are among their closest relatives. The names Anaerofilum pentosovorans gen. nov., sp. nov. (type strain, strain Fae [= DSM 7168]) and Anaerofilum agile sp. nov. (type strain, strain F [= DSM 4272]) are proposed.
Characteristics of continuous hydrogen production and fatty acid formation by an active hydrogen-producing anaerobic bacterium, Clostridium butyricum strain SC-E1, was examined under vacuum and non-vacuum culture systems. The continuous cultures were performed using 1040 ml anaerobic glass bottles containing 600 ml of medium including glucose and polypeptone at a concentration of 0.5 or 1.0% as substrate, and were conducted at pH 6.7, hydraulic retention time (HRT) 8h, and 30°C on a reciprocal shaker. The non-vacuum cultures at 16 days of incubation showed 2.0 to 2.3 mol-H2/mol-glucose and 1.4 to 2.0 mol-H2/mol-glucose of hydrogen productivity at 0.5 and 1.0% of substrate concentration, respectively. The vacuum cultures conducted at 0.28 atm gave 1.8 to 2.3 mol-H2/mol-glucose and 1.3 to 2.2 mol-H2/mol-glucose of hydrogen productivity at 0.5 and 1.0% of substrate concentration, respectively. The fatty acid production from the vacuum cultures exhibited approximately the same yield of fatty acids as those of the non-vacuum cultures. It was concluded that the maximal hydrogen production potential by anaerobic bacteria is 1.3 to 2.2 mol-H2/mol-glucose, which is less than 50% of theoretical. In addition, the total hydrogen production rate by a two-stage bioreactor consisting of a 1-litre anaerobic fermenter (HRT 10h) and a 4-litre photobioreactor (HRT 36h) feeding at 2.4-litre of 1.0% glucose per day was estimated at 1.4 to 5.6 mol-H2/mol-glucose, which is 12 to 47% theoretical.
Previous studies suggested three distinctive DNA homology groups among the bifidobacteria; these were provisionally named 'catenulatum', 'dentium', and 'angulatum'. 184 strains isolated from sewage, in addition to many of the strains from the previous study, were investigated and their DNA homology relationships were assessed using 23 reference systems. Strains in the catenulatum group were found not to differ significantly from those of B. adolescentis Reuter in their main physiological characters, such as sugars fermented and temperature, pH, and oxygen relationships; however, their DNA reciprocal homology is only some 50%, their guanine plus cytosine values were 54.7 ± 0.2 and 59.4 ± 0.4 mol %, respectively, and there were some morphological differences between them. The DNA of the dentium group has only about 45% homology with the DNA of B. adolescentis and is even less related to other members of the genus. The dentium strains can also be distinguished from other bifidobacteria by means of their sugar fermentations. The DNA of the angulatum group has little or no homology with that of any other bifidobacteria; the angulatum group also has a distinctive pattern of sugar fermentation and a unique morphology, resembling that of the coryneform bacteria. The three groups are named and described as new species of the genus Bifidobacterium: B. catenulatum, B. dentium, and B. angulatum. The type strains of these species are B669 (= ATCC 27539), B764 (=ATCC 27534), and B677 (= ATCC 27535), respectively. DNA-DNA homology relationships are basic to currently proposed species concepts, and data are presented confirming the reliability of critical experimental parameters influencing filter bound DNA and thus the final relative homology values (e.g., temperature and time of incubation and annealing of DNA in the presence of homologous and heterologous competitive or nonspecific DNA, and the replicability of homology values using different homologous DNA preparations with single DNA competitor and reference DNA).
The fermentation of glucose by Clostridium butyricum strains NCTC 7423, IFO 3315t1, 3847, 3858 and IAM 19001 was investigated in batch cultures at the defined pH values of 5.5, 6, 7, 7.5 or 8. Acetate, butyrate, lactate, formate, CO2 and H2 formations were determined quantitatively. The pH range for growth and fermentation is strain-linked and varies between 5.5–6 (for strain IFO 3847) and 5.5–8 (for strain NCTC 7423). The higher the pH of the fermentation medium, the more the H2 production differs from the one, theoretically expected according to the acetate and butyrate production. For at least one strain (NCTC 7423) formate then serves as an additional electron sink. With increasing incubation pH the acetate/butyrate ratio increases above 1 for strains NCTC 7423, IFO 3315t1, 3858 and IAM 19001.
No change in the fermentation pattern occurred with increasing acetate or butyrate concentrations up to 200 mM, except for the maximum velocity of gas production, which slowed down gradually with added butyrate. Between 400 and 500 mM acetate or butyrate added the glucose consumption was completely or almost completely inhibited.
Abstract Hydrogen gas is recognized as a promising energy resource in the future. Microbial hydrogen fermentation would be an attractive process for hydrogen recovery. In particular, hydrogen production using fermentative bacteria has some advantages such as a high rate of hydrogen,production without light. In this study, the hydrogen production from organic wastes was investigated using batch experiments. Bean curd manufacturing waste, rice bran and wheat bran were used as the organic wastes. The effects of solid concentration on the hydrogen production potential and the characteristics of substrate decomposition,were investigated. The percentages of hydrogen in the produced gas were between 54‐78%, 43‐68% and 42‐72% for bean curd manufacturing waste, rice bran and wheat bran, respectively. The hydrogen production potentials of bean curd manufacturing waste, rice bran and wheat bran were 14‐21, 31‐61 and 10‐43 ml.g VS, of hexose for bean curd manufacturing waste, rice bran and wheat bran, respectively. The carbohydrate was rapidly consumed just after inoculation. On the other hand, soluble protein was hardly degraded for each substrate, indicating that carbohydrate was the main source of the hydrogen production. Keywords,Bean curd manufacturing waste; hydrogen fermentation; organic wastes; rice bran; soluble carbohydrate; wheat bran
Characteristics of continuous hydrogen production and fatty acid fonnation by an active hydrogen-producing anaerobic bacterium, Clostridium butyricum strain SC-E1, was examined under vacuum and non-vacuum culture systems. The continuous cultures were performed using 1040 ml anaerobic glass bottles containing 600 ml of medium including glucose and polypeptone at a concentration of 0.5 or 1.0% as substrate, and were conducted at pH 6.7, hydraulic retention time (HRT) 8h, and 30°C on a reciprocal shaker. The non-vacuum cultures at 16 days of incubation showed 2.0 to 2.3 mol-H2/mol-glucose and 1.4 to 2.0 mol-H2/mol-glucose of hydrogen productivity at 0.5 and 1.0% of substrate concentration, respectively. The vacuum cultures conducted at 0.28 atm gave 1.8 to 2.3 mol-H2/mol-glucose and 1.3 to 2.2 mol-H2/mol-glucose of hydrogen productivity at 0.5 and 1.0% of substrate concentration, respectively. The fatty acid production from the vacuum cultures exhibited approximately the same yield of fatty acids as those of the non-vacuum cultures. It was concluded that the maximal hydrogen production potential by anaerobic bacteria is 1.3 to 2.2 mol-H2/mol-glucose, which is less than 50% of theoretical. In addition, the total hydrogen production rate by a two-stage bioreactor consisting of a 1-litre anaerobic fennenter (HRT 10h) and a 4-litre photobioreactor (HRT 36h) feeding at 2.4-litre of 1.0% glucose per day was estimated at 1.4 to 5.6 mol-H2/mol-glucose, which is 12 to 47% theoretical.
In ammonium-limitation (4.55 mM NH4+) at a dilution rate (D)=0.081 h−1,Clostridium butyricum produced 2 mol H2 per mol glucose consumed at pH 5.0, but at a low fermentation rate. At higher pH, important amounts of extracellular protein were produced. Phosphatelimitation (0.5 mM PO4−3) at D=0.061 h−1 and pH 7.0 were the best conditions tested for hydrogen gas production (2.22 mol H2 per mol glucose consumed) at a high fermentation rate. Steady-state growth at lower pH and with 0.1 mM PO4−3 resulted in proportional higher glucose incorporation into biomass and lower H2 production.
C. pasteurianum in NH4+ limitation showed higher fermentation rates thanC. butyricum and a stabilized H2 production around 2.08 (±0.06) mol per mol glucose consumed at various defined pH conditions, although the acetate/butyrate ratio increased to 1 at pH 7.0. The latter was also observed in phosphate-limitation, but here H2 production was maximal (1.90 mol. per mol glucose consumed) at the lowest pH (5.5) tested.
An overview of the sulfate-reduction process is given in Chapter 24. Most types of dissimilatory sulfate-reducing bacteria that have been isolated from nature and described so far are mesophilic, nonsporeforming anaerobes. They are members of the delta subdivision of the proteobacteria. The earliest known representative of this category is Desulfovibrio (Beijerinck, 1895). Further investigations have revealed a great morphological and nutritional diversity within this group. Various cell types have been described including cocci; oval or long straight rods; more or less curved rods or spirilla; cell packets; cells with gas vesicles; and gliding, multicellular filaments (Figs. 7–9). Electron donors used for sulfate reduction include H2, alcohols, fatty acids, other monocarboxylic acids, dicarboxylic acids, some amino acids, a few sugars, phenyl-substituted acids, and some other aromatic compounds (Table 2). Even long-chain alkanes can be anaerobically oxidized by a particular type of sulfate-reducing bacterium (Aeckersberg et al., 1991). The utilization of polysaccharides or polypeptides, such as has been observed with the extremely thermophilic sulfate-reducing archaebacterium Archaeoglobus (Stetter, 1988; Stetter et al., 1987), has not been reported for mesophilic sulfate reducers.
Techniques are presented for measuring the biodegradability (Biochemical Methane Potential—BMP) and toxicity (Anaerobic Toxicity Assay—ATA) of material subjected to anaerobic treatment. These relatively simple bioassays can be conducted in most research laboratories without the need for sophisticated equipment. BMP is a measure of substrate biodegradability determined by monitoring cumulative methane production from a sample which is anaerobically incubated in a chemically defined medium. The ATA measures the adverse effect of a compound on the rate of the total gas production from an easily-utilized, methanogenic substrate. These techniques are demonstrated by an analysis of the BMP and ATA of processed samples of peat.
The hydrogen productivity of a chemostat-type anaerobic reactor operating without pH control on a glucose-mineral salts feed at ambient temperatures (15–34°C) was examined. The reactor, with an inoculum from sewage sludge, operated continuously for 330 days at solids retention times (SRTs) varied manually in response to the progress of the fermentation. SRTs of 2.5, 2.0, 1.5, 1.0, 0.5 and 0.25 days were used at the temperatures of 15–18°C, 16–19°C, 24–28°C, 25–29°C, 28–32°C and 30–34°C, respectively. The sewage sludge microflora (dominated by Clostridium species) produced hydrogen during anaerobic acidogenic conversion of glucose at ambient temperatures. At the shortest tested SRT of 0.25 days, the organic loading rate was 416mmol-glucose/l/day, the hydrogen productivity peaked with each mole of glucose in the temperature-uncontrolled fermenter converted into 1.42mol of H2; each gram of biomass produced 0.214mol of H2 per day. The experimental results show that without temperature control the anaerobic sewage sludge microflora could be acclimated to produce hydrogen. Moreover, we prove the stable performance characteristics of a hydrogen-producing fermenter using anaerobic sewage sludge microflora.
The growth of a wide variety of eubacteria, three nonmethanogenic archaebacteria, and a yeast was not significantly inhibited by 25 mM bromoethanesulfonate, whereas three methanogenic bacteria were completely inhibited by this compound. We conclude that bromoethanesulfonate is a specific and effective inhibitor of methanogenic bacteria, even when used at this high concentration.
Experiments on hydrogen production using chemostat-type anaerobic digesters were conducted. The results indicate that the anaerobic acidogenic conversion of glucose can produce hydrogen. The hydrogenic activity of acclimated anaerobic sewage sludge is high at a short solids retention time (SRT) and low pH. At pH 5.7, SRT 0.25 days and an organic loading rate of 416 mmol-glucose dm−3 day−1, each mole of glucose in the mesophilic acidogenic reactor can produce 1.7 mol of hydrogen; each gram of biomass produces 0.456 mole of hydrogen per day. Moreover, the hydrogen productivity of the sludge is comparable to that of an enrichment culture.© 1999 Society of Chemical Industry
Citrus exocortis viroid (CEV) is widespread in citrus production areas where trifoliate orange [Poncirus trifoliata (L.) Raf.] is used as rootstock. Citrus reticulata Blanco cv. Red tangerine, a different rootstock, is tolerant to CEV. Embryogenic protoplasts of C. reticulata cv. Red tangerine were electrically fused with mesophyll protoplasts from P. trifoliata, and five embryoids were regenerated after 40 days of culture. The embryoids were cut into several pieces and subcultured on shoot induction medium. After 5 months and several subcultures, shoots initially regenerated. The plants grew vigorously with well-developed root systems and exhibited the trifoliate leaf character of P. trifoliata. Chromosome counts on four randomly selected root tips revealed them to be tetraploids (2n=4x=36). RAPD analysis of four randomly selected plants verified their hybridity. This hybridity was further confirmed by AFLP analysis using four primer pairs, from which a total of 65 specific bands were detected. Cytoplasmic genome analysis using universal primers revealed that their chloroplast DNA banding pattern was identical to that of trifoliate orange, while the banding pattern of mitochondrial DNA was identical to that of Red tangerine. The potential of this somatic hybrid as a means to control tree size and provide multi-resistance is discussed.
Clostridium butyricum was grown in a glucose-limited chemostat culture at a dilution rate of 0.1 h–1 at pH 6.0. With 0.9% w/v input glucose in the medium the cells were found to grow in suspension and glucose was fermented completely to acetate and butyrate. An increase in the input concentration of glucose resulted in increased concentrations of end-products, but not all extra glucose was consumed. It could be demonstrated that this was due to a lowering of the maximal growth rate by elevated levels of butyric acid. However, prolonged growth in the presence of high glucose concentrations led to an increase in biomass. This was caused by the selection of a variant that was less sensitive to butyrate. This variant was able to form aggregates in an anaerobic gas-lift reactor at high dilution rates. Inoculation of these aggregates in a conventional chemostat culture with high glucose input resulted in an aggregated culture that remained stable for at least 6 months, and in which all glucose was consumed. Whether the organisms grew in suspension or in aggregates was found to be determined by the concentration of butyrate. The isolation of aggregate-forming variants from chemostat cultures leads to a very simple and new type of immobilization technique.
A mixed continuous culture of Clostridium butyricum and Enterobacter aerogenes removed O2 in a reactor and produced H2 from starch with yield of more than 2 mol H2/mol glucose without any reducing agents in the medium. Co-immobilized cells of the bacteria on porous glass beads evolved H2 from starch at 1.3 l/l.h, with H2 yield of 2.6 mol H2/ mol glucose at dilution rate of 1.0 h–1 in a continuous culture.
The effect of substrate concentration on hydrogen production was investigated using a continuous-flow stirred-tank reactor (CSTR). Sucrose was used as a model substrate. The CSTR was started at a sucrose concentration of 30 g COD/L and exhibited stable H2 production for 271 days at inlet sucrose concentrations of 10–60 g COD/L. Hydrogen production depended on the substrate concentration such that the highest values of 1.09 mol H2/mol hexoseadded, 1.22 mol H2/mol hexoseconsumed, 7.65 L H2/L/d, and 3.80 L H2/g VSS/d were recorded at a sucrose concentration of 30 g COD/L. All bacterial species detected by polymerase chain reaction-denaturing gradient gel electrophoresis analysis were H2-producing Clostridium spp. At inlet sucrose concentrations below 20 g COD/L, the H2 yield per hexoseconsumed decreased along with a significant decrease in the n-butyrate/acetate ratio. At the same range of sucrose concentrations, Clostridium scatologenes (an H2-consuming acetogen) was found in the sludge. At inlet sucrose concentrations over 35 g COD/L, substrate overload occurred and caused a decrease in the carbohydrate degradation efficiency and H2 yield per hexoseadded.
Continuous production of hydrogen from sugary wastewater by anaerobic microflora in chemostat culture was examined as a function of hydraulic retention time (HRT) in the reactor. The measured volumes of the evolved gas at each HRT were almost constant (Avg. 3590 ml/l-feed) and the composition of the gas was approximately 64% hydrogen, 36% carbon dioxide, and less than 0.13% methane. Steady states on evolution of gas were observed for 190 d at HRTs from 0.5 to 3 d giving hydrogen production rates from 198 to 34 mmol/l/d. Significant amounts of acetate and butyrate were formed as by-products. A maximum production yield of hydrogen of 14 mmol/g carbohydrate removed was obtained at an HRT of 0.5 d. The maximum removal efficiency of carbohydrates was approximately 97% at an HRT of 3 d. The patterns of fermentation by anaerobic microflora changed with HRT, i.e., acid formation decreased with decreasing HRT.
Continuous production of hydrogen from the anaerobic acidogenesis of a high-strength rice winery wastewater by a mixed bacterial flora was demonstrated. The experiment was conducted in a 3.0-l upflow reactor to investigate individual effects of hydraulic retention time (HRT) (2–), chemical oxygen demand (COD) concentration in wastewater (14– COD/l), pH (4.5–6.0) and temperature (20–55°C) on bio-hydrogen production from the wastewater. The biogas produced under all test conditions was composed of mostly hydrogen (53–61%) and carbon dioxide (37–45%), but contained no detectable methane. Specific hydrogen production rate increased with wastewater concentration and temperature, but with a decrease in HRT. An optimum hydrogen production rate of was achieved at an HRT of , COD of , pH 5.5 and 55°C. The hydrogen yield was in the range of 1.37–. In addition to acetate, propionate and butyrate, ethanol was also present in the effluent as an aqueous product. The distribution of these compounds in the effluent was more sensitive to wastewater concentration, pH and temperature, but was less sensitive to HRT. This upflow reactor was shown to be a promising biosystem for hydrogen production from high-strength wastewaters by mixed anaerobic cultures.
Temperature effects on H2 production performance of a novel carrier-induced granular sludge bed (CIGSB) reactor were investigated. Using sucrose-based synthetic wastewater as the feed, the CIGSB system was operated at 30– to identify the optimal working temperature. It was found that H2 production was the most efficient at , especially when it was operated at a low hydraulic retention time (HRT) of 0.5 h. The overall maximal hydrogen production rate and yield were 7.66 l/h/l and 3.88 mol H2/mol sucrose, respectively, both of them occurred at . The biomass content tended to decrease as the temperature was increased, suggesting that granular sludge formation may be inhibited at high temperatures. However, increasing temperature gave better specific H2 production rate, signifying that the average cellular activity for H2 production may be enhanced as the temperature was increased. The H2 yield and gas phase H2 content did not vary considerably regardless of changes in temperature and HRT. This reflects that the CIGSB was a relatively stable H2-producing system. The major soluble products from hydrogen fermentation were butyric acid and acetic acid, accounting for 46±3% and 28±2% of total soluble microbial products (SMP), respectively. Thus, the dominant H2 producers in the mixed culture belonged to acidogenic bacteria that underwent butyrate-type fermentation.
The effects of lactic acid bacteria (LAB) on hydrogen fermentation of organic waste were investigated. For this three hydrogen producing strains of Clostridium were cultured with two lactic acid bacteria, i.e. Lactobacillus paracasei and Enterococcus durans, which were isolated from the wastes generated in the bean curd manufacturing. The decrease or cessation of hydrogen production by Clostridium was caused by the addition of LAB. The supernatants of L. paracasei and E. durans suspensions also inhibited hydrogen production by Clostridium. This inhibition was partially destroyed in the presence of trypsin, which is a protease inactivating a bacteriocin. These results suggest that the inhibitory effect of lactic acid bacteria on hydrogen production was caused by bacteriocins excreted from LAB which have a deleterious effect on other bacteria. To suppress any effect by LAB, heat treatment of this waste was investigated as a possible pretreatment step. The inhibition of hydrogen production was reduced by heat treatment for at temperatures ranging from 50°C to 90°C. This means that a temperature of 50°C is already adequate to prevent growth of LAB.
Organic municipal solid waste (OFMSW) and two seed microorganisms, namely heat-pretreated digested sludge and hydrogen-producing bacteria enriched from soybean-meal silo, were varied according to a full factorial central composite experimental design with the aim of assessing the feasibility of hydrogen production from OFMSW. A simple model developed from the Gompertz equation was suitable for estimating the hydrogen production potential and rate. Through response surface methodology, empirical equations for specific hydrogen production potential and rate were fitted and plotted as contour diagrams in order to facilitate examination of experimental results. The contour plots showed that high hydrogen production potentials of 140 and 180 ml H2·g TVS−1 occurred when the pretreated digested sludge and the hydrogen-producing bacteria consumed OFMSW, respectively. A high hydrogenic activity for the pretreated digested sludge (45 ml·g VSS−1·h−1) was obtained at a high food-to-microorganism (F/M) ratio; however, that for the hydrogen-producing bacteria (36 ml·g VSS−1·h−1) was found at a low F/M ratio. The experimental results showed that the hydrogen composition of the biogas was greater than 60% except for initial incubation and no significant methane was found throughout this study. Further experiments confirmed that the results of this study were highly reliable and the OFMSW had a considerable potential on biological hydrogen production. Metabolic responses confirmed that characteristics of the heat-pretreated digested sludge converting the OFMSW into hydrogen were similar to that of anaerobic spore-forming bacteria of the genus Clostridium.
Hydrogen may be produced by a number of processes, including electrolysis of water, thermocatalytic reformation of hydrogen-rich organic compounds, and biological processes. Currently, hydrogen is produced, almost exclusively, by electrolysis of water or by steam reformation of methane. Biological production of hydrogen (Biohydrogen) technologies provide a wide range of approaches to generate hydrogen, including direct biophotolysis, indirect biophotolysis, photo-fermentations, and dark-fermentation. The practical application of these technologies to every day energy problems, however, is unclear. In this paper, hydrogen production rates of various biohydrogen systems are compared by first standardizing the units of hydrogen production and then by calculating the size of biohydrogen systems that would be required to power proton exchange membrane (PEM) fuel cells of various sizes.
The food processing industry produces highly concentrated, carbohydrate-rich wastewaters, but their potential for biological hydrogen production has not been extensively studied. Wastewaters were obtained from four different food-processing industries that had chemical oxygen demands of 9 g/L (apple processing), 21 g/L (potato processing), and 0.6 and 20 g/L (confectioners A and B). Biogas produced from all four food processing wastewaters consistently contained 60% hydrogen, with the balance as carbon dioxide. Chemical oxygen demand (COD) removals as a result of hydrogen gas production were generally in the range of 5–11%. Overall hydrogen gas conversions were 0.7–0.9 L-H2/L-wastewater for the apple wastewater, 0.1 L/L for Confectioner-A, 0.4–2.0 L/L for Confectioner B, and 2.1–2.8 L/L for the potato wastewater. When nutrients were added to samples, there was a good correlation between hydrogen production and COD removal, with an average of -COD. However, hydrogen production could not be correlated to COD removal in the absence of nutrients or in more extensive in-plant tests at the potato processing facility. Gas produced by a domestic wastewater sample (concentrated 25×) contained only 23±8% hydrogen, resulting in an estimated maximum production of only 0.01 L/L for the original, non-diluted wastewater. Based on an observed hydrogen production yield from the effluent of the potato processing plant of 1.0 L-H2/L, and annual flows at the potato processing plant, it was estimated that if hydrogen gas was produced at this site it could be worth as much as $65,000/year.
An investigation on anaerobic hydrogen production was conducted in fixed-bed bioreactors containing hydrogen-producing bacteria originated from domestic sewage sludge. Three porous materials, loofah sponge (LS), expanded clay (EC) and activated carbon (AC), were used as the support matrix to allow retention of the hydrogen-producing bacteria within the fixed-bed bioreactors. The carriers were assessed for their effectiveness in biofilm formation and hydrogen production in batch and continuous modes. It was found that LS was inefficient for biomass immobilization, while EC and AC exhibited better biomass yields. The fixed-bed reactors packed with EC or AC (denote as EC or AC reactors) were thus used for continuous hydrogen fermentation at a hydraulic retention time (HRT) of 0.5–. Sucrose was utilized as the major carbon source. With a sucrose concentration of ca. COD/l in the feed, the EC reactor () was able to produce H2 at an optimal rate of at . In contrast, the AC reactor ( in volume) exhibited a better hydrogen production rate of , which occurred at . When the AC reactor was scaled up to , the hydrogen production rate was nearly 0.53– for HRT=1–, but after a short thermal treatment (75°C, ) the rate rose to ca. at . The biogas produced with EC and AC reactors typically contained 25–35% of H2 and the rest was mainly CO2, while production of methane was negligible (less than 0.1%). During the efficient hydrogen production stage, the major soluble metabolite was butyric acid, followed by propionic acid, acetic acid, and ethanol.
Palm oil mill effluent (POME) sludge, sludge compost from Malaysia and CREST compost from Philippines were collected for the study. The capability of these microflora to produce hydrogen was examined with artificial wastewater containing 1% glucose, 0.2% yeast extract and 0.018% magnesium chloride hexahydrate under anaerobic fermentation in a batch culture. The microflora in POME sludge, sludge compost and CREST compost were found to produce significant amounts of hydrogen. The maximum production yield of hydrogen per decomposed glucose was -glucose at a conversion rate of at 50°C obtained by sludge compost. All fermentations were carried out without pH control. It was also found that the addition of nitrogen source in the medium caused a change in hydrogen produced. There was no methane gas in the evolved gas.
It has been found, in this study, that a new ethanol-type fermentation can be obtained in a continuous flow, high-rate acidogenic reactor receiving molasses as the feed. The operating pH must be maintained at about 4.5 to avoid onset of propionic fermentation. The acidogenic reactor had a VSS level of 20 g/L and its organic loading was as high as 80 to 90 kg COD/m(3) d. The operating ORP was around -250 mV. The ethanol-type fermentation was characterized by a simultaneous production of acetic acid and ethanol, while the yield of propionic was minimal even at a high organic loading rate of 80 to 90 kg COD/m(3) d, and also, the hydrogen partial pressure was as high as 50 kPa. Thus, this study has shown that the production of propionic acid is not always related to high hydrogen partial pressure. When the operating pH was increased to 5.5, the yield of propionic acid became significant.
The technique of percolation was applied as an improvement over the usual technique of maceration for extraction of carbohydrates from small amounts of plant material. Soluble carbohydrates were extracted by percolation with 80% ethanol and starch was extracted by percolation with 35% perchloric acid. The course of percolation was studied and the technique was demonstrated to give reproducible results. The anthrone method was applied for starch determination. The influence by cellulose on the determination of starch, as well as, the influence of perchloric acid on the anthrone reaction was investigated. An analytical procedure based on the obtained results is given.
Archaea (archaebacteria) are a phenotypically diverse group of microorganisms that share a common evolutionary history. There are four general phenotypic groups of archaea: the methanogens, the extreme halophiles, the sulfate-reducing archaea, and the extreme thermophiles. In the marine environment, archaeal habitats are generally limited to shallow or deep-sea anaerobic sediments (free-living and endosymbiotic methanogens), hot springs or deep-sea hydrothermal vents (methanogens, sulfate reducers, and extreme thermophiles), and highly saline land-locked seas (halophiles). This report provides evidence for the widespread occurrence of unusual archaea in oxygenated coastal surface waters of North America. Quantitative estimates indicated that up to 2% of the total ribosomal RNA extracted from coastal bacterioplankton assemblages was archaeal. Archaeal small-subunit ribosomal RNA-encoding DNAs (rDNAs) were cloned from mixed bacterioplankton populations collected at geographically distant sampling sites. Phylogenetic and nucleotide signature analyses of these cloned rDNAs revealed the presence of two lineages of archaea, each sharing the diagnostic signatures and structural features previously established for the domain Archaea. Both of these lineages were found in bacterioplankton populations collected off the east and west coasts of North America. The abundance and distribution of these archaea in oxic coastal surface waters suggests that these microorganisms represent undescribed physiological types of archaea, which reside and compete with aerobic, mesophilic eubacteria in marine coastal environments.
The influence of a number of environmental parameters on the fermentation of glucose, and on the energetics of growth of Clostridium butyricum in chemostat culture, have been studied. With cultures that were continuously sparged with nitrogen gas, glucose was fermented primarily to acetate and butyrate with a fixed stoichiometry. Thus, irrespective of the growth rate, input glucose concentration specific nutrient limitation and, within limits, the culture pH value, the acetate/butyrate molar ratio in the culture extracellular fluids was uniformly 0.74±0.07. Thus, the efficiency with which ATP was generated from glucose catabolism also was constant at 3.27±0.02 mol ATP/mol glucose fermented. However, the rate of glucose fermentation at a fixed growth rate, and hence the rate of ATP generation, varied markedly under some conditions leading to changes in the Y
glucose and Y
ATP values. In general, glucose-sufficient cultures expressed lower yield values than a correponding glucose-limited culture, and this was particularly marked with a potassium-limited culture. However, with a glucose-limited culture increasing the input glucose concentration above 40g glucose·l-1 also led to a significant decrease in the yield values that could be partially reversed by increasing the sparging rate of the nitrogen gas. Finally glucose-limited cultures immediately expressed an increased rate of glucose fermentation when relieved of their growth limitation. Since the rate of cell synthesis did not increase instantaneously, again the yield values with respect to glucose consumed and ATP generated transiently decreased.
Two conditions were found to effect a change in the fermentation pattern with a lowering of the acetate/butyrate molar ratio. First, a significant decrease in this ratio was observed when a glucose-limited culture was not sparged with nitrogen gas; and second, a substantial (and progressive) decrease was observed to follow addition of increasing amounts of mannitol to a glucose-limited culture. In both cases, however, there was no apparent change in the Y
ATP value.
These results are discussed with respect to two imponder-ables, namely the mechanism(s) by which C. butyricum might partially or totally dissociate catabolism from anabolism, and how it might dispose of the excess reductant [as NAD(P)H] that attends both the formation of acetate from glucose and the fermentation of mannitol. With regards to the latter, evidence is presented that supports the conclusion that the ferredoxin-mediated oxidation of NAD(P)H, generating H2, is neither coupled to, nor driven by, an energy-yielding reaction.
Acidaminococcus gen. n. and the type species Acidaminococcus fermentans sp. n. were described. Amino acids, of which glutamic acid is the most important, could serve as the sole energy source for growth. Acetic and butyric acids and CO(2) were produced; propionic acid and hydrogen were not produced. Amino acid media supporting growth and the amino acid and vitamin requirements were described. Glucose was frequently not fermented or was weakly catabolized. Derivative products from glucose autoclaved in media, but not glucose itself, stimulated or were required for growth in amino acid media. A wide range of polyols and carbohydrates were not attacked. Lactate, fumarate, malate, succinate, citrate, and pyruvate were not used as energy sources for growth. Pyruvate completely suppressed growth. Cytochrome oxidase and benzidine reactions were negative; catalase, indole, acetyl methyl carbinol, and H(2)S were not produced; nitrate and sulfonthalein indicators were not reduced; ammonia was produced; gelatin liquefaction was negative or slow and partial; vancomycin (7.5 mug/ml) was resisted. Acidaminococcus was different from Veillonella in morphology, serology, nutrition, utilization of substrates, and accumulation of products in media supporting growth; Acidaminococcus resembled Peptococcus in utilization of glutamic acid and accumulation of similar products, but the two genera differed in morphology, gram reaction, serology, guanine plus cytosine content of deoxyribonucleic acid, and nutrition.
Two hundred and ninety strains of 29 species of bifidobacteria from human and animal origin were surveyed for their ability to ferment complex carbohydrates. The substrates fermented by the largest number of species were D-galactosamine, D-glucosamine, amylose and amylopectin. Many of the species isolated from animal habitats showed reduced fermentation activity. Bifidobacterium dentium strains fermented gum guar and gum locust bean; porcine gastric mucin was fermented only by B. bifidum, B. infantis was the only species to ferment D-glucuronic acid; strains of B. longum fermented arabinogalactan and the gums arabic, ghatti and tragacanth; alpha-L-fucose was fermented by strains of B. breve, B. infantis and B. pseudocatenulatum. A key to the differentiation of Bifidobacterium species of human origin is provided.
The potential for biological transformation of 23 xenobiotic compounds by microorganisms in municipal solid waste (MSW) samples from a laboratory scale landfill reactor was studied. In addition the influence of these xenobiotic compounds on methanogenesis was investigated. All R11, 1,1 dichloroethylene, 2,4,6 trichlorophenol, dimethyl phthalate, phenol, benzoate and phthalic acid added were completely transformed during the period of incubation ( > 100 days). Parts of the initially added perchloroethylene, trichloroethylene, R12, R114, diethyl phthalate, dibutyl phthalate and benzylbutyl phthalate were transformed. Methanogenesis from acetate was completely inhibited in the presence of 2,5 dichlorophenol, whereas 2,4,6 trichlorophenol and R11 showed an initial inhibition, whenafter methane formation recovered. No transformation or effect on the anaerobic microflora occurred for R13, R22, R114, 3 chlorobenzoate, 2,4,6 trichlorobenzoate, bis(2 ethyl)hexyl phthalate, diisodecyl phthalate and dinonyl phthalate. The results indicate a limited potential for degradation, of the compounds tested, by microorganisms developing in a methanogenic landfill environment as compared with other anaerobic habitats such as sewage digestor sludge and sediments.
Acidaminococcus fermentans is able to ferment glutamate to ammonia, CO2, acetate, butyrate, and H2. The molecular hydrogen (approximately 10 kPa; E' = -385 mV) stems from NADH generated in the 3-hydroxybutyryl-CoA dehydrogenase reaction (E degrees ' = -240 mV) of the hydroxyglutarate pathway. In contrast to growing cells, which require at least 5 mM Na+, a Na+-dependence of the H2-formation was observed with washed cells. Whereas the optimal glutamate fermentation rate was achieved already at 1 mM Na+, H2 formation commenced only at > 10 mM Na+ and reached maximum rates at 100 mM Na+. The acetate/butyrate ratio thereby increased from 2.0 at 1 mM Na+ to 3.0 at 100 mM Na+. A hydrogenase and an NADH dehydrogenase, both of which were detected in membrane fractions, are components of a model in which electrons, generated by NADH oxidation inside of the cytoplasmic membrane, reduce protons outside of the cytoplasmic membrane. The entire process can be driven by decarboxylation of glutaconyl-CoA, which consumes the protons released by NADH oxidation inside the cell. Hydrogen production commences exactly at those Na+ concentrations at which the electrogenic H+/Na+-antiporter glutaconyl-CoA decarboxylase is converted into a Na+/Na+ exchanger.