Spatial separation of metabolic stages in a tube anaerobic baffled reactor: reactor performance and microbial community dynamics.

Spatial separation of metabolic stages in anaerobic digesters can increase the methane content of biogas, as realized in a tube anaerobic baffled reactor. Here, we investigated the performance and microbial community dynamics of a laboratory-scale mesophilic anaerobic baffled reactor with four compartments treating an artificial substrate. Due to the activity of fermentative bacteria, organic acids mostly accumulated in the initial compartments. The methane content of the biogas increased while hydrogen levels decreased along the compartments. Microbial communities were investigated based on bacterial 16S rRNA genes, hydA genes encoding Fe-Fe-hydrogenases, and mcrA genes/transcripts encoding the methyl-CoM reductase. The metaproteome was analyzed to identify active metabolic pathways. During the reactor operation, Clostridia and Bacilli became most abundant in the first compartment. Later compartments were dominated by Sphingobacteriia, Deltaproteobacteria, Clostridia, Bacteroidia, Synergistia, Anaerolineae, Spirochaetes, vadinHA17, and W5 classes. Methanogenic communities were represented by Methanomicrobiales, Methanobacteriaceae, Methanosaeta, and Methanosarcina in the last compartments. Analysis of hydA and mcrA genes and metaproteome data confirmed the spatial separation of metabolic stages. In the first compartment, proteins of carbohydrate transport and metabolism were most abundant. Proteins assigned to coenzyme metabolism and transport as well as energy conservation dominated in the other compartments. Our study demonstrates how the spatial separation of metabolic stages by reactor design is underpinned by the adaptation of the microbial community to different niches.

Evolution of Chlamydomonas reinhardtii ferredoxins and their interactions with [FeFe]-hydrogenases.

Ferredoxins are soluble iron sulphur proteins which function as electron donors in a number of metabolic pathways in a broad range of organisms. In photosynthetic organisms, PETF, or ferredoxin 1 (FDX1), is the most studied ferredoxin due to its essential role in photosynthesis, where it transfers electrons from photosystem I to ferredoxin-NADP+ oxidoreductase. However, PETF can also transfer electrons to a large number of other proteins. One important PETF electron acceptor found in green microalgae is the biologically and biotechnologically important [FeFe]-hydrogenase HYDA, which catalyses the production of molecular hydrogen (H2) from protons and electrons. The interaction between PETF and HYDA is of considerable interest, as PETF is the primary electron donor to HYDA and electron supply is one of the main limiting factors for H2 production on a commercial scale. Although there is no three dimensional structure of the PETF-HYDA complex available, protein variants, nuclear magnetic resonance titration studies, molecular dynamics and modelling have provided considerable insight into the residues essential for forming and maintaining the interaction. In this review, we discuss the most recent findings with regard to ferredoxin-HYDA interactions and the evolution of the various Chlamydomonas reinhardtii ferredoxin isoforms. Finally, we provide an outlook on new PETF-based biotechnological approaches for improved H2 production efficiencies.

Transcription of [FeFe]-Hydrogenase Genes during H2 Production in Clostridium and Desulfovibrio spp. Isolated from a Paddy Field Soil.

Changes in the relative abundances of the transcripts of hydA gene paralogs for [FeFe]-hydrogenase in Clostridium sp. strain H2 and Desulfovibrio sp. strain A1 isolated from paddy field soil were analyzed during H2 production. Strains H2 and A1 had at least five and two phylogenetically different hydA genes, respectively. The relative abundances of their hydA transcripts differed among the paralogs and H2 production activity changed in a manner that depended on the growth phase and conditions. Increases or decreases in the relative abundances of the transcripts of two out of five hydA genes in strain H2 correlated with changes in H2 production rates, whereas those of the others remained unchanged or decreased. In strain A1, the relative abundances of the transcripts of two hydA genes differed between monoculture, sulfate-reducing, and syntrophic, methanogenic conditions. The relative abundance of the transcripts of one hydA gene, predicted to encode a cytosolic [FeFe]-hydrogenase, was higher under syntrophic, methanogenic conditions than sulfate-reducing conditions, while that of the transcripts of the other hydA gene decreased with time under both conditions. This study showed that the transcription of the hydA gene during growth with active H2 production was differently regulated among the paralogs in H2 producers isolated from paddy field soil.

Compartmentalisation of [FeFe]-hydrogenase maturation in Chlamydomonas reinhardtii.

Molecular hydrogen (H2 ) can be produced in green microalgae by [FeFe]-hydrogenases as a direct product of photosynthesis. The Chlamydomonas reinhardtii hydrogenase HYDA1 contains a catalytic site comprising a classic [4Fe4S] cluster linked to a unique 2Fe sub-cluster. From in vitro studies it appears that the [4Fe4S] cluster is incorporated first by the housekeeping FeS cluster assembly machinery, followed by the 2Fe sub-cluster, whose biosynthesis requires the specific maturases HYDEF and HYDG. To investigate the maturation process in vivo, we expressed HYDA1 from the C. reinhardtii chloroplast and nuclear genomes (with and without a chloroplast transit peptide) in a hydrogenase-deficient mutant strain, and examined the cellular enzymatic hydrogenase activity, as well as in vivo H2 production. The transformants expressing HYDA1 from the chloroplast genome displayed levels of H2 production comparable to the wild type, as did the transformants expressing full-length HYDA1 from the nuclear genome. In contrast, cells equipped with cytoplasm-targeted HYDA1 produced inactive enzyme, which could only be activated in vitro after reconstitution of the [4Fe4S] cluster. This indicates that the HYDA1 FeS cluster can only be built by the chloroplastic FeS cluster assembly machinery. Further, the expression of a bacterial hydrogenase gene, CPI, from the C. reinhardtii chloroplast genome resulted in H2 -producing strains, demonstrating that a hydrogenase with a very different structure can fulfil the role of HYDA1 in vivo and that overexpression of foreign hydrogenases in C. reinhardtii is possible. All chloroplast transformants were stable and no toxic effects were seen from HYDA1 or CPI expression.

Improvement of hydrogen yield of ethanol-producing Escherichia coli recombinants in acidic conditions

Background: An effective single culture with high glycerol consumption and hydrogen and ethanol coproduction yield is still in demand. A locally isolated glycerol-consuming Escherichia coli SS1 was found to produce lower hydrogen levels under optimized ethanol production conditions. Molecular approach was proposed to improve the hydrogen yield of E. coli SS1 while maintaining the ethanol yield, particularly in acidic conditions. Therefore, the effect of an additional copy of the native hydrogenase gene hycE and recombinant clostridial hydrogenase gene hydA on hydrogen production by E. coli SS1 at low pH was investigated. Results: Recombinant E. coli with an additional copy of hycE or clostridial hydA was used for fermentation using 10 g/L (108.7 mmol/L) of glycerol with an initial pH of 5.8. The recombinant E. coli with hycE and recombinant E. coli with hydA showed 41% and 20% higher hydrogen yield than wild-type SS1 (0.46 ± 0.01 mol/mol glycerol), respectively. The ethanol yield of recombinant E. coli with hycE (0.50 ± 0.02 mol/mol glycerol) was approximately 30% lower than that of wild-type SS1, whereas the ethanol yield of recombinant E. coli with hydA (0.68 ± 0.09 mol/mol glycerol) was comparable to that of wild-type SS1. Conclusions: Insertion of either hycE or hydA can improve the hydrogen yield with an initial pH of 5.8. The recombinant E. coli with hydA could retain ethanol yield despite high hydrogen production, suggesting that clostridial hydA has an advantage over the hycE gene in hydrogen and ethanol coproduction under acidic conditions. This study could serve as a useful guidance for the future development of an effective strain coproducing hydrogen and ethanol.

Towards improved butanol production through targeted genetic modification of Clostridium pasteurianum.

Declining fossil fuel reserves, coupled with environmental concerns over their continued extraction and exploitation have led to strenuous efforts to identify renewable routes to energy and fuels. One attractive option is to convert glycerol, a by-product of the biodiesel industry, into n-butanol, an industrially important chemical and potential liquid transportation fuel, using Clostridium pasteurianum. Under certain growth conditions this Clostridium species has been shown to predominantly produce n-butanol, together with ethanol and 1,3-propanediol, when grown on glycerol. Further increases in the yields of n-butanol produced by C. pasteurianum could be accomplished through rational metabolic engineering of the strain. Accordingly, in the current report we have developed and exemplified a robust tool kit for the metabolic engineering of C. pasteurianum and used the system to make the first reported in-frame deletion mutants of pivotal genes involved in solvent production, namely hydA (hydrogenase), rex (Redox response regulator) and dhaBCE (glycerol dehydratase). We were, for the first time in C. pasteurianum, able to eliminate 1,3-propanediol synthesis and demonstrate its production was essential for growth on glycerol as a carbon source. Inactivation of both rex and hydA resulted in increased n-butanol titres, representing the first steps towards improving the utilisation of C. pasteurianum as a chassis for the industrial production of this important chemical.

Metagenomic and PCR-Based Diversity Surveys of [FeFe]-Hydrogenases Combined with Isolation of Alkaliphilic Hydrogen-Producing Bacteria from the Serpentinite-Hosted Prony Hydrothermal Field, New Caledonia.

High amounts of hydrogen are emitted in the serpentinite-hosted hydrothermal field of the Prony Bay (PHF, New Caledonia), where high-pH (~11), low-temperature (< 40°C), and low-salinity fluids are discharged in both intertidal and shallow submarine environments. In this study, we investigated the diversity and distribution of potentially hydrogen-producing bacteria in Prony hyperalkaline springs by using metagenomic analyses and different PCR-amplified DNA sequencing methods. The retrieved sequences of hydA genes, encoding the catalytic subunit of [FeFe]-hydrogenases and, used as a molecular marker of hydrogen-producing bacteria, were mainly related to those of Firmicutes and clustered into two distinct groups depending on sampling locations. Intertidal samples were dominated by new hydA sequences related to uncultured Firmicutes retrieved from paddy soils, while submarine samples were dominated by diverse hydA sequences affiliated with anaerobic and/or thermophilic submarine Firmicutes pertaining to the orders Thermoanaerobacterales or Clostridiales. The novelty and diversity of these [FeFe]-hydrogenases may reflect the unique environmental conditions prevailing in the PHF (i.e., high-pH, low-salt, mesothermic fluids). In addition, novel alkaliphilic hydrogen-producing Firmicutes (Clostridiales and Bacillales) were successfully isolated from both intertidal and submarine PHF chimney samples. Both molecular and cultivation-based data demonstrated the ability of Firmicutes originating from serpentinite-hosted environments to produce hydrogen by fermentation, potentially contributing to the molecular hydrogen balance in situ.

Enhanced butanol production by increasing NADH and ATP levels in Clostridium beijerinckii NCIMB 8052 by insertional inactivation of Cbei_4110.

Clostridium beijerinckii is identified as a promising Clostridium strain for industrialization of acetone and butanol (AB) fermentation. It has been reported that high reducing power levels are associated with high butanol yield. In this study, we regulated reducing power by blocking NAD(P)H consumption in C. beijerinckii NCIMB 8052. Gene Cbei_4110, encoding NADH-quinone oxidoreductase (nuoG), is a subunit of the electron transport chain complex I. After inactivation of gene Cbei_4110, the generated mutant strain exhibited a remarkable increase in glucose utilization ratio and enhanced butanol production to 9.5 g/L in P2 medium containing 30 g/L of glucose. NAD(P)H and ATP levels were also increased by one to two times and three to five times, respectively. Furthermore, a comparative transcriptome analysis was carried out in order to determine the mechanism involved in the enhanced activity of the Cbei_4110-inactivated mutant strain. This strategy may be extended for making industrial bio-butanol more economically attractive.

Simultaneous production of butanol and acetoin by metabolically engineered Clostridium acetobutylicum.

Biobutanol is a potential fuel substitute and has been receiving increased attention in recent years. However, the economics of biobutanol production have been hampered by a number of bottlenecks such as high cost of raw material and low yield of solvent. Co-production of value-added products is a possible way to improve the economics of biobutanol production. Here, we present metabolic engineering strategies to substitute the major by-product acetone for a value-added product acetoin during butanol fermentation. By overexpressing the α-acetolactate decarboxylase gene alsD in Clostridium acetobutylicum B3, the acetoin yield was markedly increased while acetone formation was reduced. Subsequent disruption of adc gene effectively abolished acetone formation and further increased acetoin yield. After optimization of fermentation conditions, the alsD-overexpressing adc mutant generated butanol (13.8g/L), acetoin (4.3g/L), and ethanol (3.9g/L), but no acetone. Thus, acetone was completely substituted for acetoin, and both mass yield and product value were improved. This study provides valuable insights into the regulation of acetoin synthesis and should be highly useful for the development of acetoin-derived products like 2,3-butanediol and 2-butanol in C. acetobutylicum.

Total solid content drives hydrogen production through microbial selection during thermophilic fermentation.

In this study, the effect of total solid content (TS) on thermophilic hydrogen production from wheat straw was investigated. Six TS contents ranging from wet to dry conditions (10-34%TS) were tested in batch tests. A decrease of H2 yields was observed and three statistical groups were distinguished according to the TS content: wet conditions (10% and 14%TS) with 15.3 ± 1.6 NmlH2 gTS(-1), intermediate conditions (19%TS) with 6.4 ± 1.0 NmlH2 gTS(-1) and dry conditions (25-34%TS) with 3.4 ± 0.8 NmlH2 gTS(-1). Such a decrease in biohydrogen yields was related to a metabolic shift with an accumulation of lactic acid under dry conditions. Concomitantly, a microbial population shift was observed with a dominance of species related to the class Clostridia under wet conditions, and a co-dominance of members of Bacilli, Clostridia classes and Bacteroidetes phylum under dry conditions.

Knockdown of PsbO leads to induction of HydA and production of photobiological H2 in the green alga Chlorella sp. DT.

Green algae are able to convert solar energy to H2 via the photosynthetic electron transport pathway under certain conditions. Algal hydrogenase (HydA, encoded by HYDA) is in charge of catalyzing the reaction: 2H(+)+2e(-)↔H2 but usually inhibited by O2, a byproduct of photosynthesis. The aim of this study was to knockdown PsbO (encoded by psbO), a subunit concerned with O2 evolution, so that it would lead to HydA induction. The alga, Chlorella sp. DT, was then transformed with short interference RNA antisense-psbO (siRNA-psbO) fragments. The algal mutants were selected by checking for the existence of siRNA-psbO fragments in their genomes and the low amount of PsbO proteins. The HYDA transcription and the HydA expression were observed in the PsbO-knockdown mutants. Under semi-aerobic condition, PsbO-knockdown mutants could photobiologically produce H2 which increased by as much as 10-fold in comparison to the wild type.

Molecular basis of [FeFe]-hydrogenase function: an insight into the complex interplay between protein and catalytic cofactor.

The precise electrochemical features of metal cofactors that convey the functions of redox enzymes are essentially determined by the specific interaction pattern between cofactor and enclosing protein environment. However, while biophysical techniques allow a detailed understanding of the features characterizing the cofactor itself, knowledge about the contribution of the protein part is much harder to obtain. [FeFe]-hydrogenases are an interesting class of enzymes that catalyze both, H2 oxidation and the reduction of protons to molecular hydrogen with significant efficiency. The active site of these proteins consists of an unusual prosthetic group (H-cluster) with six iron and six sulfur atoms. While H-cluster architecture and catalytic states during the different steps of H2 turnover have been thoroughly investigated during the last 20 years, possible functional contributions from the polypeptide framework were only assumed according to the level of conservancy and X-ray structure analyses. Due to the recent development of simpler and more efficient expression systems the role of single amino acids can now be experimentally investigated. This article summarizes, compares and categorizes the results of recent investigations based on site directed and random mutagenesis according to their informative value about structure function relationships in [FeFe]-hydrogenases. This article is part of a Special Issue entitled: Metals in Bioenergetics and Biomimetics Systems.