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A novel photosynthetic bacterium, Rhodopseudomonas palustris P4, was isolated from an anaerobic wastewater sludge digester by virtue of its ability to utilize CO with the production of H2. P4 grew under light with CO as a sole carbon source with the doubling time of 2 h and produced H2 at 20.7 mmol –1 cell h.
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... light. After 2–3 days, fast-growing colonies were taken and stored for further studies. 16S rRNA gene (rDNA) was amplified by polymerase chain reaction (PCR) using TaKaRa PCR amplification kit (Takara Shuzo Co., Ltd. Shiga, Japan). Approximately 1.5 kb of 16S rDNA was amplified using genomic DNA of P4 as the template and two primers, 27f and 1492r (Lane 1991). Reaction conditions consisted of the preheating at 94 ◦ C for 2 min and the subsequent 30 cycles of 94 ◦ C (1 min), 55 ◦ C (1 min) and 72 ◦ C (3 min). After the 30 cycles com- pleted, final chain elongation was conducted at 72 ◦ C for 5 min. The size of the amplified product was deter- mined by agarose (1%, w/v) gel electrophoresis. The PCR product was cloned with pGEM-T Easy vector system II (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Inserts of clones were analyzed by electrophoresis after digestion with Eco RI (Promega). Total DNA and plasmid DNA were prepared as described (Ausubel et al . 1989). Agarose gel electrophoresis was performed in the TAE buffer as described (Ausubel et al. 1989). The cloned 16S rDNA was partially sequenced by a DNA sequencer (Applied Biosystems model 373A, Foster City, CA, USA) and the sequence was screened against the Ribosomal Database Project (RDP) and GenBank databases. Sequence_Match (version 2.7) (Maidak et al . 1999) and BLAST (version 2.0) (Altschul et al . 1997) were used to search the most similar sequences in the databases. Cells were grown in the liquid medium described above under the white light (1500 lux) and CO-Ar (20:80, v/v) gas mixture. The culture was adapted to a higher CO environment (60:40, v/v and/or 80:20, v/v CO-Ar mixture) before challenged to convert CO to H 2 under the dark condition. Cell growth and gas conversion were carried out in the serum bottle (150 ml) with a rubber crimp seal. Cell density was estimated by spectrophotometry (Perkin-Elmer Lambda 20, Nor- walk, CT, USA). One A 482 unit was estimated to correspond to 0.23 g dried cell mass l − 1 (designated simply as g cell l − 1 afterwards) according to a routine technique (Benson 1982). Gas concentrations were measured using the DS6200 gas chromatograph (Donam Systems Inc., Seoul, Korea) equipped with a thermal conductivity detector and a stainless steel column (1.8 m × 1/8 ) packed with Molecular Sieve 5A (Alltech, Deerfield, IL, USA). Argon was used as carrier gas. Among the several colonies formed in the agar plate, one fast-growing cell P4 was selected for further studies. Figure 1 shows the picture of the strain P4 by transmission electron microscopy. P4 had a lamella- type internal membrane structure. P4 was rod-shaped and Gram-negative. It was a facultative anaerobe and could grow in a photoautotrophic or chemo- heterotrophic manner. However, purple pigment was produced under the photoautotrophic and ...
Citations
... Most prokaryotes known to perform oxidation of CO with H 2 production are bacteria of the phylum Firmicutes [7,11,66], although a few strains belong to the Proteobacteria phylum [29,30,45,77] and one strain is from the Dictyoglomi phylum [37]. The ability to grow at the expense of hydrogenogenic CO oxidation was also shown for some hyperthermophilic archaea from the Thermococcus genus [39,40,51,65,78] and for a recombinant strain of Pyrococcus furiosus [62]. ...
The ability to grow by anaerobic CO oxidation with production of H2 from water is known for some thermophilic bacteria, most of which belong to Firmicutes, as well as for a few hyperthermophilic Euryarchaeota isolated from deep-sea hydrothermal habitats. A hyperthermophilic, neutrophilic, anaerobic filamentous archaeon strain 1505=VKM B-3180=KCTC 15798 was isolated from a terrestrial hot spring in Kamchatka (Russia) in the presence of 30% CO in the gas phase. Strain 1505 could grow lithotrophically using carbon monoxide as the energy source with the production of hydrogen according to the equation CO+H2O→CO2+H2; mixotrophically on CO plus glucose; and organotrophically on peptone, yeast extract, glucose, sucrose, or Avicel. The genome of strain 1505 was sequenced and assembled into a single chromosome. Based on 16S rRNA gene sequence analysis and in silico genome-genome hybridization, this organism was shown to be closely related to the Thermofilum adornatum species. In the genome of Thermofilum sp. strain 1505, a gene cluster (TCARB_0867-TCARB_0879) was found that included genes of anaerobic (Ni,Fe-containing) carbon monoxide dehydrogenase and genes of energy-converting hydrogenase ([Ni,Fe]-CODH-ECH gene cluster). Compared to the [Ni,Fe]-CODH-ECH gene clusters occurring in the sequenced genomes of other H2-producing carboxydotrophs, the [Ni,Fe]-CODH-ECH gene cluster of Thermofilum sp. strain 1505 presented a novel type of gene organization. The results of the study provided the first evidence of anaerobic CO oxidation coupled with H2 production performed by a crenarchaeon, as well as the first documented case of lithotrophic growth of a Thermofilaceae representative.
... The length and diameter of each Rhodopseudomonas sp. cell was taken to be 2 mm and 0.8 mm respectively, as apparent from the transmission electron microscope images provided in a recent literature (Jung, Jung, Kim, Ahn, & Park, 1999). ...
Flat plate photobioreactors (FPPBRs) using bacterial biofilm have gained much recent attention due to operational ease, improved light conversion efficiency and reduction of process cost, particularly in hydrogen production. In this study, two comprehensive mathematical models, one explaining the dynamics of a batch type FPPBR used for the development of biofilm and the other a deterministic model (both temporal and spatial) to predict the performance of a continuous FPPBR using Rhodopseudomonas sp. have been developed for both circular and rectangular configurations. The system equations have been solved using MATLAB 2013. From batch studies, the maximum specific growth rate and half saturation constant for the microorganism have been determined to be 0.07 h−1 and 1.946 g l−1 respectively. An “Instantaneous attachment and proliferation” mechanism has been proposed to explain the behaviour of biofilm right from the early stage of attachment to the reversal from attached to planktonic state. The flow patterns of substrate medium through the biofilm have been generated using COMSOL Multiphysics software. From the perspective of the hydrogen yield, the models predict that the FPPBR geometry plays a crucial role by demonstrating the superior performance of the circular reactor in comparison to the rectangular counterpart. It is expected that the mathematical models developed here will help in the design, scale-up and control of FPPBRs to be used particularly for hydrogen production using suitable microorganisms.
... Few mesophiles have been identified as anaerobic hydrogenotrophic carboxydotrophs. These mesophiles are all phototrophic Proteobacteria and include the Betaproteobacterium Rubrivivax gelatinosus (Uffen 1976), the Gammaproteobacterium Citrobacter amalonaticus Y19 (Jung et al. 1999b), and the Alphaproteobacteria Rhodospirillum rubrum (Ensign and Luddens 1991;Kerby, Ludden and Roberts 1995) and Rhodopseudomonas palustris (Jung et al. 1999a). Despite aerobic carboxydotrophy being widespread among the Proteobacteria (Tolli, Sievert and Taylor 2006;King and Weber 2007), the aforementioned mesophiles are the sole Proteobacteria reported to grow anaerobically with CO. ...
Carbon monoxide (CO)-metabolism and phenotypic and phylogenetic characterization of a novel anaerobic, mesophilic and hydrogenogenic carboxydotroph are reported. Strain SVCO-16 was isolated from anaerobic sludge and grows autotrophically and mixotrophically with CO. The genes cooS and cooF, coding for a CO dehydrogenase complex, and genes similar to hycE2, encoding a CO-induced hydrogenase, were present in its genome. The isolate produces H2 and CO2 from CO, and acetate and formate from organic substrates. Based on the 16S rRNA sequence, it is an Alphaproteobacterium most closely related to the genus Pleomorphomonas (98.9%-99.2% sequence identity). Comparison with other previously characterized Pleomorphomonas showed that P. diazotrophica and P. oryzae do not metabolize CO, and P. diazotrophica does not grow anaerobically with organic substrates. Average nucleotide identity values between strain SVCO-16 and P. diazotrophica, P. oryzae or P. koreensis were 86.66 ± 0.21%. These values are below the boundary to define species (95%-96%). Digital DNA-DNA hybridization estimates between strain SVCO-16 and reference strains were also below the 70% threshold for species delineation: 29.1%-34.5%. Based on the differences in CO metabolism, genome analyses and cellular fatty acid composition, the isolate should be classified into the genus Pleomorphomonas as a representative of a novel species, Pleomorphomonas carboxyditropha. The type strain of Pleomorphomonas carboxyditropha is SVCO-16T (strain deposit numbers, DSM 106132T and TSD-119T).
... Although CO is a well-known toxic ingredient to most organisms, acting as a respiratory inhibitor, several microorganisms from various taxonomic groups can tolerate and even convert CO to value-added chemicals, such as acetate and ethanol, via a microbial CO conversion process, called gas fermentation (Chang et al., 1999;Geelhoed et al., 2016;Sipma et al., 2006). Pure culture-based CO conversion was examined using Citrobacter sp., Clostridium sp., Rhodospirillum sp., and Eubacterium limosum KIST612 (Diender et al., 2015;Jung et al., 1999aJung et al., , 2002Park et al., 2017;Younesi et al., 2005). Genetic modification was attempted to improve the low conversion rate and yield of wild type strains. ...
... R. gelatinosus and R. palustris exhibit a similar hydrogenogenic CO metabolism as R. rubrum. However, in contrast to R. rubrum, these bacteria were able to perform the water-gas shift reaction and grow on CO as a sole carbon source, but merely in presence of light (Jung et al., 1999;Maness et al., 2005). Growth was significantly slowed down for R. gelatinosus in the dark, which was not assessed for R. palustris. ...
Carbon monoxide can act as a substrate for different modes of fermentative anaerobic metabolism. The trait of utilizing CO is spread among a diverse group of microorganisms, including members of bacteria as well as archaea. Over the last decade this metabolism has gained interest due to the potential of converting CO-rich gas, such as synthesis gas, into bio-based products. Three main types of fermentative CO metabolism can be distinguished: hydrogenogenesis, methanogenesis, and acetogenesis, generating hydrogen, methane and acetate, respectively. Here, we review the current knowledge on these three variants of microbial CO metabolism with an emphasis on the potential enzymatic routes and bio-energetics involved.
... Non-sulfur purple bacteria are predominant in this group, including Rubrivivax gelatinosus and Rhodospirillum rubrum, which require light for optimal cell growth. Although Rhodopseudomonas palustris P4 is capable of hydrogenogenic CO conversion in the dark, this strain does not grow under such a condition (Jung et al., 1999a). The non-phototrophic Citrobacter strain Y19 also converts CO to H 2 , but this strain only grows slowly under anaerobic conditions, and an aerobic growth phase is required to generate sufficient biomass before the anaerobic CO conversion phase (Jung et al., 1999b). ...
Among four basic mechanisms for biological hydrogen (H2) production, dark fermentation has been considered to show the highest hydrogen evolution rate (HER). H2 production from one-carbon (C1) compounds such as formate and carbon monoxide (CO) is promising because formate is an efficient H2 carrier, and the utilization of CO-containing syngas or industrial waste gas may render the industrial biohydrogen production process cost-effective. A variety of microbes with the formate hydrogen lyase (FHL) system have been identified from phylogenetically diverse groups of archaea and bacteria, and numerous efforts have been undertaken to improve the HER for formate through strain optimization and bioprocess development. CO-dependent H2 production has been investigated to enhance the H2 productivity of various carboxydotrophs via an increase in CO gas-liquid mass transfer rates and the construction of genetically modified strains. Hydrogenogenic CO-conversion has been applied to syngas and by-product gas of the steel-mill process, and this low-cost feedstock has shown to be promising in the production of biomass and H2. Here, we focus on recent advances in the isolation of novel phylogenetic groups utilizing formate or CO, the remarkable genetic engineering that enhances H2 productivity, and the practical implementation of H2 production from C1 substrates.
... In fact, in the hydrogen production stage, CO was consumed to obtain energy via WGS reaction [21]. Jung et al. [22] have reported the application of pure CO during the hydrogen production stage and revealed that the bacterium R. palustris P4 managed to convert 60 % of the CO during a comparatively shorter period of time (40 h) [22], than the 68 % conversion of initial CO concentration of 60 % during 72 h achieved in present study. It could be concluded that the high initial CO concentration (60 %), used during the growth stage in this investigation (compare to 20 % CO concentration used in their study), led to prolonged time period required to convert CO to H 2 . ...
... In fact, in the hydrogen production stage, CO was consumed to obtain energy via WGS reaction [21]. Jung et al. [22] have reported the application of pure CO during the hydrogen production stage and revealed that the bacterium R. palustris P4 managed to convert 60 % of the CO during a comparatively shorter period of time (40 h) [22], than the 68 % conversion of initial CO concentration of 60 % during 72 h achieved in present study. It could be concluded that the high initial CO concentration (60 %), used during the growth stage in this investigation (compare to 20 % CO concentration used in their study), led to prolonged time period required to convert CO to H 2 . ...
... Among the bacteria described, the maximum hydrogen production achieved (0.67 mmol/l h) was through R. rubrum-catalyzed WGS reaction using COrich syngas as gas substrate, with 88 % hydrogen production yield [24]. Moreover, in two different studies conducted by a group of Korean scholars [22,25,26] isolation of two hydrogenogenic bacteria, i.e., Citrobacter sp. Y19 and R. palustris P4 was reported. ...
Biohydrogen production through water–gas
shift (WGS) reaction by a biocatalyst was conducted in
batch fermentation. The isolated photosynthetic bacterium
Rhodopseudomonas palustris PT was able to utilize carbon
monoxide and simultaneously produce hydrogen. Light
exposure was provided as an indispensable requirement for
the first stage of bacterial growth, but throughout the
hydrogen production stage, the energy requirement was
met through the WGS reaction. At ambient pressure and
temperature, the effect of various sodium acetate concentrations
in presence of CO-rich syngas on cell growth,
carbon monoxide consumption, and biohydrogen production
was also investigated. Maximal efficiency of hydrogen
production in response to carbon monoxide consumption
was recorded at 86 % and the highest concentration of
hydrogen at 33.5 mmol/l was achieved with sodium acetate
concentration of 1.5 g/l. The obtained results proved that
the local isolate; R. palustris PT, was able to utilize COrich
syngas and generate biohydrogen via WGS reaction
... In fact, in the hydrogen production stage, CO was consumed to obtain energy via WGS reaction [21]. Jung et al. [22] have reported the application of pure CO during the hydrogen production stage and revealed that the bacterium R. palustris P4 managed to convert 60 % of the CO during a comparatively shorter period of time (40 h) [22], than the 68 % conversion of initial CO concentration of 60 % during 72 h achieved in present study. It could be concluded that the high initial CO concentration (60 %), used during the growth stage in this investigation (compare to 20 % CO concentration used in their study), led to prolonged time period required to convert CO to H 2 . ...
... In fact, in the hydrogen production stage, CO was consumed to obtain energy via WGS reaction [21]. Jung et al. [22] have reported the application of pure CO during the hydrogen production stage and revealed that the bacterium R. palustris P4 managed to convert 60 % of the CO during a comparatively shorter period of time (40 h) [22], than the 68 % conversion of initial CO concentration of 60 % during 72 h achieved in present study. It could be concluded that the high initial CO concentration (60 %), used during the growth stage in this investigation (compare to 20 % CO concentration used in their study), led to prolonged time period required to convert CO to H 2 . ...
... Among the bacteria described, the maximum hydrogen production achieved (0.67 mmol/l h) was through R. rubrum-catalyzed WGS reaction using COrich syngas as gas substrate, with 88 % hydrogen production yield [24]. Moreover, in two different studies conducted by a group of Korean scholars [22,25,26] isolation of two hydrogenogenic bacteria, i.e., Citrobacter sp. Y19 and R. palustris P4 was reported. ...
Biohydrogen production through water-gas shift (WGS) reaction by a biocatalyst was conducted in batch fermentation. The isolated photosynthetic bacterium Rhodopseudomonas palustris PT was able to utilize carbon monoxide and simultaneously produce hydrogen. Light exposure was provided as an indispensable requirement for the first stage of bacterial growth, but throughout the hydrogen production stage, the energy requirement was met through the WGS reaction. At ambient pressure and temperature, the effect of various sodium acetate concentrations in presence of CO-rich syngas on cell growth, carbon monoxide consumption, and biohydrogen production was also investigated. Maximal efficiency of hydrogen production in response to carbon monoxide consumption was recorded at 86 % and the highest concentration of hydrogen at 33.5 mmol/l was achieved with sodium acetate concentration of 1.5 g/l. The obtained results proved that the local isolate; R. palustris PT, was able to utilize CO-rich syngas and generate biohydrogen via WGS reaction.
... The methods that have shown the most potential are photo-fermentation and methanogenesis. However, the application of photofermentation is limited due to low conversion efficiencies, the requirement of light and expensive reactors [5][6][7]. Methanogenisis also has problems like slow biodegradation kinetics, the requirement of gas treatment and the need for post-treatment [8,9]. Electricity generation through biomethanation process from the organic wastes is a two stage process whereas that of MFC is a single step process [8,10]. ...
A major limitation associated with fermenta-tive hydrogen production is the low substrate conversion efficiency. This limitation can be overcome by integrating the process with a microbial fuel cell (MFC) which converts the residual energy of the substrate to electricity. Studies were carried out to check the feasibility of this integration. Biohydrogen was produced from the fermen-tation of cane molasses in both batch and continuous modes. A maximum yield of about 8.23 mol H 2 /kg COD removed was observed in the batch process compared to 11.6 mol H 2 /kg COD removed in the continuous process. The spent fermentation media was then used as a substrate in an MFC for electricity generation. The MFC parameters such as the initial anolyte pH, the substrate concentration and the effect of pre-treatment were studied and optimized to maximize coulombic efficiency. Reductions in COD and total carbohydrates were about 85% and 88% respectively. A power output of 3.02 W/m 3 was obtained with an anolyte pH of 7.5 using alkali pre-treated spent media. The results show that integrating a MFC with dark fermentation is a promising way to utilize the substrate energy.
... Predominant within this group are nonsulfur purple bacteria, including Rubrivivax gelatinosus and Rhodospirillum rubrum, which require light for optimal cell growth. Although Rhodopseudomonas palustris P4 is capable of hydrogenogenic CO conversion in the dark, it does not grow under this condition (4). Nonphototrophic Citrobacter strain Y19 also converts CO to H 2 , but it only grows slowly under anaerobic conditions and an aerobic growth phase is required to generate sufficient biomass before the anaerobic CO conversion phase (5). ...
Hydrogenogenic CO oxidation (CO + H2O → CO2 + H2) has the potential for H2 production as a clean renewable fuel. Thermococcus onnurineus NA1, which grows on CO and produces H2, has a unique gene cluster encoding the carbon monoxide dehydrogenase (CODH) and the hydrogenase. The gene cluster was identified
as essential for carboxydotrophic hydrogenogenic metabolism by gene disruption and transcriptional analysis. To develop a
strain producing high levels of H2, the gene cluster was placed under the control of a strong promoter. The resulting mutant, MC01, showed 30-fold-higher transcription
of the mRNA encoding CODH, hydrogenase, and Na+/H+ antiporter and a 1.8-fold-higher specific activity for CO-dependent H2 production than did the wild-type strain. The H2 production potential of the MC01 mutant in a bioreactor culture was 3.8-fold higher than that of the wild-type strain. The
H2 production rate of the engineered strain was severalfold higher than those of any other CO-dependent H2-producing prokaryotes studied to date. The engineered strain also possessed high activity for the bioconversion of industrial
waste gases created as a by-product during steel production. This work represents the first demonstration of H2 production from steel mill waste gas using a carboxydotrophic hydrogenogenic microbe.
