Showing papers by "Alfons J. M. Stams published in 2013"

Microbial Community Analysis of a Methane-Producing Biocathode in a Bioelectrochemical System

Abstract: A methane-producing biocathode that converts CO2 into methane was studied electrochemically and microbiologically. The biocathode produced methane at a maximum rate of 5.1 L CH4/m2 projected cathode per day (1.6 A/m2) at −0.7 V versus NHE cathode potential and 3.0 L CH4/m2 projected cathode per day (0.9 A/m2) at −0.6 V versus NHE cathode potential. The microbial community at the biocathode was dominated by three phylotypes of Archaea and six phylotypes of bacteria. The Archaeal phylotypes were most closely related to Methanobacterium palustre and Methanobacterium aarhusense. Besides methanogenic Archaea, bacteria seemed to be associated with methane production, producing hydrogen as an intermediate. Biomass density varied greatly with part of the carbon electrode covered with a dense biofilm, while only clusters of cells were found on other parts. Based on our results, we discuss how inoculum enrichment and changing operational conditions may help to increase biomass density and to select for microorganisms that produce methane.

Activity and viability of methanogens in anaerobic digestion of unsaturated and saturated long-chain fatty acids.

Abstract: Lipids can be anaerobically digested to methane, but methanogens are often considered to be highly sensitive to the long-chain fatty acids (LCFA) deriving from lipids hydrolysis. In this study, the effect of unsaturated (oleate [C18:1]) and saturated (stearate [C18:0] and palmitate [C16:0]) LCFA toward methanogenic archaea was studied in batch enrichments and in pure cultures. Overall, oleate had a more stringent effect on methanogens than saturated LCFA, and the degree of tolerance to LCFA was different among distinct species of methanogens. Methanobacterium formicicum was able to grow in both oleate- and palmitate-degrading enrichments (OM and PM cultures, respectively), whereas Methanospirillum hungatei only survived in a PM culture. The two acetoclastic methanogens tested, Methanosarcina mazei and Methanosaeta concilii, could be detected in both enrichment cultures, with better survival in PM cultures than in OM cultures. Viability tests using live/dead staining further confirmed that exponential growth-phase cultures of M. hungatei are more sensitive to oleate than are M. formicicum cultures; exposure to 0.5 mM oleate damaged 99% ± 1% of the cell membranes of M. hungatei and 53% ± 10% of the cell membranes of M. formicicum. In terms of methanogenic activity, M. hungatei was inhibited for 50% by 0.3, 0.4, and 1 mM oleate, stearate, and palmitate, respectively. M. formicicum was more resilient, since 1 mM oleate and >4 mM stearate or palmitate was needed to cause 50% inhibition on methanogenic activity.

Enrichment and isolation of acidophilic sulfate‐reducing bacteria from Tinto River sediments

Abstract: Although some acidophilic and alkaliphilic species have been described recently, most of the known sulfate-reducing bacteria (SRB) grow optimally at neutral pH. In this study, sulfate reduction was studied with sediment samples from the extremely acidic Tinto River basin. Stable enrichments of SRB were obtained at pH 4 with glycerol, methanol and hydrogen; at pH 4.5 with lactate and at pH 5.5 with succinate as substrates. Inhibition of sulfate reduction by organic acids below their pKa was observed. Cloning and sequencing of 16S rRNA gene showed that fermentative bacteria (Paludibacter spp., Oscillibacter spp.) and SRB (Thermodesulfobium spp., Desulfosporosinus spp., Desulfitobacterium spp., Desulfotomaculum spp.) were co-enriched. By repeated serial dilutions and streaking on agar plates, four strains of SRB belonging to the Firmicutes phylum were obtained. Two of them show 96% 16S rRNA gene sequence similarity with Desulfosporosinus acidophilus, and a third one with Desulfosporosinus orientis. Another isolate has just 93% rRNA gene sequence similarity with the Desulfosporosinus/Desulfitobacterium cluster and might represent a novel species within a novel genus. One of the Desulfosporosinus strains was further investigated showing maximum growth at pH 5.5, and a pH-dependent inhibitory effect of organic acids and sulfide.

Archaeal (Per)Chlorate Reduction at High Temperature: An Interplay of Biotic and Abiotic Reactions

Abstract: Perchlorate and chlorate anions [(per)chlorate] exist in the environment from natural and anthropogenic sources, where they can serve as electron acceptors for bacteria. We performed growth experiments combined with genomic and proteomic analyses of the hyperthermophile Archaeoglobus fulgidus that show (per)chlorate reduction also extends into the archaeal domain of life. The (per)chlorate reduction pathway in A. fulgidus relies on molybdo-enzymes that have similarity with bacterial enzymes; however, chlorite is not enzymatically split into chloride and oxygen. Evidence suggests that it is eliminated by an interplay of abiotic and biotic redox reactions involving sulfur compounds. Biological (per)chlorate reduction by ancient archaea at high temperature may have prevented accumulation of perchlorate in early terrestrial environments and consequently given rise to oxidizing conditions on Earth before the rise of oxygenic photosynthesis.

Genome analysis of Desulfotomaculum kuznetsovii strain 17T reveals a physiological similarity with Pelotomaculum thermopropionicum strain SIT.

Abstract: Desulfotomaculum kuznetsovii is a moderately thermophilic member of the polyphyletic spore-forming genus Desulfotomaculum in the family Peptococcaceae. This species is of interest because it originates from deep subsurface thermal mineral water at a depth of about 3,000 m. D. kuznetsovii is a rather versatile bacterium as it can grow with a large variety of organic substrates, including short-chain and long-chain fatty acids, which are degraded completely to carbon dioxide coupled to the reduction of sulfate. It can grow methylotrophically with methanol and sulfate and autotrophically with H2 + CO2 and sulfate. For growth it does not require any vitamins. Here, we describe the features of D. kuznetsovii together with the genome sequence and annotation. The chromosome has 3,601,386 bp organized in one contig. A total of 3,567 candidate protein-encoding genes and 58 RNA genes were identified. Genes of the acetyl-CoA pathway, possibly involved in heterotrophic growth with acetate and methanol, and in CO2 fixation during autotrophic growth are present. Genomic comparison revealed that D. kuznetsovii shows a high similarity with Pelotomaculum thermopropionicum. Genes involved in propionate metabolism of these two strains show a strong similarity. However, main differences are found in genes involved in the electron acceptor metabolism.

Genome Analysis and Physiological Comparison of Alicycliphilus denitrificans Strains BC and K601T

Abstract: The genomes of the Betaproteobacteria Alicycliphilus denitrificans strains BC and K601T have been sequenced to get insight into the physiology of the two strains. Strain BC degrades benzene with chlorate as electron acceptor. The cyclohexanol-degrading denitrifying strain K601T is not able to use chlorate as electron acceptor, while strain BC cannot degrade cyclohexanol. The 16S rRNA sequences of strains BC and K601T are identical and the fatty acid methyl ester patterns of the strains are similar. Basic Local Alignment Search Tool (BLAST) analysis of predicted open reading frames of both strains showed most hits with Acidovorax sp. JS42, a bacterium that degrades nitro-aromatics. The genomes include strain-specific plasmids (pAlide201 in strain K601T and pAlide01 and pAlide02 in strain BC). Key genes of chlorate reduction in strain BC were located on a 120 kb megaplasmid (pAlide01), which was absent in strain K601T. Genes involved in cyclohexanol degradation were only found in strain K601T. Benzene and toluene are degraded via oxygenase-mediated pathways in both strains. Genes involved in the meta-cleavage pathway of catechol are present in the genomes of both strains. Strain BC also contains all genes of the ortho-cleavage pathway. The large number of mono- and dioxygenase genes in the genomes suggests that the two strains have a broader substrate range than known thus far.

Anaerobic Degradation of Lindane and Other HCH Isomers

Abstract: Lindane (γ-HCH) is a pesticide that has mainly been used in agriculture. Lindane and the other HCH isomers are highly chlorinated hydrocarbons. The presence of a large number of electron withdrawing chlorine groups makes some of the HCH isomers rather recalcitrant in oxic environments. Especially β-HCH is poorly degraded by aerobic bacteria. The chlorine groups make HCH isomers more accessible for an initial reductive attack, a common mechanism in anoxic environments. Among the HCH isomers, γ-HCH is degraded most easily while β-HCH is most persistent. Little is known about the diversity of the microorganisms involved in anaerobic HCH degradation. Thus far, species within the genera Clostridium and Bacillus, two Desulfovibrio species, and one species each of Desulfococcus, Desulfobacter, Citrobacter and Dehalobacter have been found to metabolize lindane and other HCH isomers. Benzene and monochlorobenzene are the end products of anaerobic degradation, while in some studies pentachlorocyclohexane, tetrachlorocyclohexene, chlorobenzenes and chlorophenols have been detected as intermediates. Enzymes and coding genes involved in the reductive dechlorination of HCH isomers are largely unknown. Recently, a metagenomic analysis has indicated the presence of numerous putative reductive dehalogenase genes in the genome of β-HCH degrading Dehalobacter sp. High-throughput omics techniques can help to explore the key players and enzymes involved in the reductive dehalogenation of lindane and other HCH isomers.

Metabolic response of Alicycliphilus denitrificans strain BC toward electron acceptor variation.

Abstract: Alicycliphilus denitrificans is a versatile, ubiquitous, facultative anaerobic bacterium. Alicycliphilus denitrificans strain BC can use chlorate, nitrate, and oxygen as electron acceptor for growth. Cells display a prolonged lag-phase when transferred from nitrate to chlorate and vice versa. Furthermore, cells adapted to aerobic growth do not easily use nitrate or chlorate as electron acceptor. We further investigated these responses of strain BC by differential proteomics, transcript analysis, and enzyme activity assays. In nitrate-adapted cells transferred to chlorate and vice versa, appropriate electron acceptor reduction pathways need to be activated. In oxygen-adapted cells, adaptation to the use of chlorate or nitrate is likely difficult due to the poorly active nitrate reduction pathway and low active chlorate reduction pathway. We deduce that the Nar-type nitrate reductase of strain BC also reduces chlorate, which may result in toxic levels of chlorite if cells are transferred to chlorate. Furthermore, the activities of nitrate reductase and nitrite reductase appear to be not balanced when oxygen-adapted cells are shifted to nitrate as electron acceptor, leading to the production of a toxic amount of nitrite. These data suggest that strain BC encounters metabolic challenges in environments with fluctuations in the availability of electron acceptors. All MS data have been deposited in the ProteomeXchange with identifier PXD000258.

Biomethanation Potential of Biological and Other Wastes

Abstract: Anaerobic technology has been traditionally applied for the treatment of carbon rich wastewater and organic residues. Anaerobic processes can be fully integrated in the biobased economy concept for resource recovery. After a brief introduction about applications of anaerobic processes to industrial wastewater treatment, agriculture feedstock and organic fraction of municipal solid waste, the position of anaerobic processes in biorefinery concepts is presented. Integration of anaerobic digestion with these processes can help in the maximisation of the economic value of the biomass used, while reducing the waste streams produced and mitigating greenhouse gases emissions. Besides the integration of biogas in the existing full-scale bioethanol and biodiesel production processes, the potential applications of biogas in the second generation lignocellulosic, algae and syngas-based biorefinery platforms are discussed.

Effect of long-chain fatty acids (LCFA) on the prevalence and viability of hydrogenotrophic methanogens

Abstract: Anaerobic degradation of long-chain fatty acids (LCFA) is essential for efficient biogas production from complex lipid-containing wastewaters. Methanogens play a key role in this process, but the general idea is that LCFA exert a toxic effect towards these microorganisms that impairs good methane recovery. In this work, the effect of saturated (palmitate, C16:0) and unsaturated (oleate, C18:1) LCFA towards hydrogenotrophic methanogens was studied in batch enrichments and in pure cultures. Methanospirillum hungatei and Methanobacterium formicicum were added to oleateand palmitate-degrading enrichments, cultures OM and PM, and their prevalence was subsequently monitored by PCR-DGGE. M. formicicum grew in both OM and PM cultures, while M. hungatei only prevailed in PM culture. Viability tests using live/dead staining further confirmed that M. hungatei is more sensitive to oleate than M. formicicum. The percentage of damaged cells, caused by the exposure to 0.5 mM of oleate, was higher in M. hungatei cultures (99 ± 1 %) than in M. formicicum cultures (53 ± 10 %). These results suggest that oleate is more inhibitory to methanogens than palmitate, although methane production was not completely inhibited with either LCFA.

or Methanobacterium thermoformicicum with Methanobacterium thermoautotrophicum Propionate-Oxidizing Bacteria in Syntrophy Enrichment of Thermophilic

Abstract: Above 60 and below 45°C, no growth occurred in cultureswith strain AH. No propionate oxidation occurred whenbacteriawereculturedwithMethanospirillumhungatiiDSM864at 37°C, showingthat the enriched bacteriawereclearlydifferent from previously described mesophilic propionateoxidizers (7, 9, 21).DISCUSSIONResults obtained in this study show clearly that thestability ofmethanogens against starvation is the determin-ing factor for a successful enrichment of thermophilic,

Enrichment and microbial characterization of syngas converting anaerobic cultures

Abstract: Bioconversion of recalcitrant biomass/waste into bulk chemicals or biofuels is often not feasible. By gasification of these materials, syngas (mainly composed of CO2, CO and H2) is generated and can be used for the production of high value compounds by thermochemical or biotechnological processes. Here, three thermophilic cultures enriched with syngas mixtures or pure CO (T-Syn, T-Syn-CO and T-CO) were studied. Stable enriched cultures obtained by subsequent transfers for over a year, convert syngas/CO to mainly acetate and hydrogen (CO partial pressure up to 0.88 bar). 16S rRNA based techniques (PCR-DGGE) showed that predominant microorganisms in the cultures belonged to Desulfotomaculum, Caloribacterium, Thermincola and Thermoanaerobacter genera. Moreover, from the syngasand CO-degrading cultures, a novel Thermoanaerobacter sp. (strain PCO) and a novel Moorella sp. (strain E3-O) were isolated.

Addition of electron acceptors stimulates methanogenesis from lipids by anaerobic sludge

Abstract: A.P. Guedes*, A.J. Cavaleiro*, S.A. Silva*, M.M. Alves*, A.J.M. Stams** and D.Z. Sousa* * IBB-Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, University of Minho, Portugal (E-mail: anaguedes@ceb.uminho.pt; acavaleiro@deb.uminho.pt; sergiosilva@ceb.uminho.pt; madalena.alves@deb.uminho.pt; dianasousa@deb.uminho.pt) ** Laboratory of Microbiology, Wageningen University, The Netherlands (E-mail: fons.stams@wur.nl)

Non-syntrophic reactions in anaerobic unsaturated LCFA conversion by methanogenic sludges

Abstract: Lipids are energy-rich compounds. This energy can be conserved as biogas in anaerobic bioreactors but the process is frequently hindered by long-chain fatty acids (LCFA) accumulation. LCFA catabolism is thought to occur via beta-oxidation, performed by anaerobic bacteria that live in obligatory syntrophy with H2-consuming methanogens, but the initial steps of unsaturated LCFA biodegradation are still unclear. In this work we hypothesize that these initial steps do not depend on interspecies H2 transfer. To test this, six anaerobic bioreactors were continuously fed with saturated or unsaturated C16and C18-LCFA, and operated in the presence or absence of bromoethanesulfonate, a selective inhibitor of methanogens. Intermediates of LCFA degradation including longand medium-chain fatty acids, volatile fatty acids and methane were monitored. Bacterial community composition was analysed in the different bioreactors by denaturing gradient gel electrophoresis of 16S rRNA reverse transcriptase-PCR products. In the presence or absence of the inhibitor of methanogenesis, palmitate (C16:0) accumulated during the degradation of oleate (C18:1), accounting for more than 50% of total accumulated LCFA. Palmitoleate (C16:1) feeding resulted in the build-up of myristate (C14:0) and palmitate (C16:0). Accumulation of saturated intermediary-LCFA was two to four times higher in bioreactors in which methanogenesis was inhibited compared to methanogenic bioreactors. Beta-oxidation of saturated intermediates only occurred in methanogenic bioreactors. No catabolic activity was observed in the bioreactor fed with saturated LCFA when methanogenesis was inhibited. These results show that the first steps of unsaturated LCFA degradation are not obligatorily syntrophic, and suggest that beta-oxidation is the limiting step in the overall conversion of LCFA to methane.