J. I. Alves

Effect of sulfate on methanogenic communities that degrade unsaturated and saturated long-chain fatty acids (LCFA)

Anaerobic bacteria involved in the degradation of long-chain fatty acids (LCFA), in the presence of sulfate as electron acceptor, were studied by combined cultivation-dependent and molecular techniques. The bacterial diversity in four mesophilic sulfate-reducing enrichment cultures, growing on oleate (C(18:1), unsaturated LCFA) or palmitate (C(16:0), saturated LCFA), was studied by denaturing gradient gel electrophoresis (DGGE) profiling of polymerase chain reaction (PCR)-amplified 16S rRNA gene fragments. These enrichment cultures were started using methanogenic inocula in order to assess the competition between methanogenic communities and sulfate-reducing bacteria. Phylogenetic affiliation of rRNA gene sequences corresponding to predominant DGGE bands demonstrated that members of the Syntrophomonadaceae, together with sulfate reducers mainly belonging to the Desulfovibrionales and Syntrophobacteraceae groups, were present in the sulfate-reducing enrichment cultures. Subculturing of LCFA-degrading methanogenic cultures in the presence of sulfate resulted in the inhibition of methanogenesis and, after several transfers, archaea could no longer be detected by real-time PCR. Competition for hydrogen and acetate was therefore won by sulfate reducers, but acetogenic syntrophic bacteria were the only known LCFA-degrading organisms present after subculturing with sulfate. Principal component analysis of the DGGE profiles from methanogenic and sulfate-reducing oleate- and palmitate-enrichment cultures showed a greater influence of the substrate than the presence or absence of sulfate, indicating that the bacterial communities degrading LCFA in the absence/presence of sulfate are rather stable.

Comparative Analysis of Carbon Monoxide Tolerance among Thermoanaerobacter Species

An anaerobic thermophilic strain (strain PCO) was isolated from a syngas-converting enrichment culture. Syngas components cannot be used by strain PCO, but the new strain is very tolerant to carbon monoxide (pCO = 1.7 × 105 Pa, 100% CO). 16S rRNA gene analysis and DNA-DNA hybridization revealed that strain PCO is a strain of Thermoanaerobacter thermohydrosulfuricus. The physiology of strain PCO and other Thermoanaerobacter species was compared, focusing on their tolerance to carbon monoxide. T. thermohydrosulfuricus, T. brockii subsp. finnii, T. pseudethanolicus, and T. wiegelii were exposed to increased CO concentrations in the headspace, while growth, glucose consumption and product formation were monitored. Remarkably, glucose conversion rates by Thermoanaerobacter species were not affected by CO. All the tested strains fermented glucose to mainly lactate, ethanol, acetate, and hydrogen, but final product concentrations differed. In the presence of CO, ethanol production was generally less affected, but H2 production decreased with increasing CO partial pressure. This study highlights the CO resistance of Thermoanaerobacter species.

Anaerobic microbial LCFA degradation in bioreactors

This paper reviews recent results obtained on long-chain fatty acids (LCFA) anaerobic degradation. Two LCFA were used as model substrates: oleate, a mono-unsaturated LCFA, and palmitate, a saturated LCFA, both abundant in LCFA-rich wastewaters. 16S rRNA gene analysis of sludge samples submitted to continuous oleate- and palmitate-feeding followed by batch degradation of the accumulated LCFA demonstrated that bacterial communities were dominated by members of the Clostridiaceae and Syntrophomonadaceae families. Archaeal populations were mainly comprised of hydrogen-consuming microorganisms belonging to the genus Methanobacterium, and acetate-utilizers from the genera Methanosaeta and Methanosarcina. Enrichment cultures growing on oleate and palmitate, in the absence or presence of sulfate, gave more insight into the major players involved in the degradation of unsaturated and saturated LCFA. Syntrophomonas-related species were identified as predominant microorganisms in all the enrichment cultures. Microorganisms clustering within the family Syntrophobacteraceae were identified in the methanogenic and sulfate-reducing enrichments growing on palmitate. Distinct bacterial consortia were developed in oleate and palmitate enrichments, and observed differences might be related to the different degrees of saturation of these two LCFA. A new obligately syntrophic bacterium, Syntrophomonas zehnderi, was isolated from an oleate-degrading culture and its presence in oleate-degrading sludges detected by 16S rRNA gene cloning and sequencing.

Moorella stamsii sp. nov., a new anaerobic thermophilic hydrogenogenic carboxydotroph isolated from digester sludge.

A novel anaerobic, thermophilic, carbon monoxide-utilizing bacterium, strain E3-O(T), was isolated from anaerobic sludge from a municipal solid waste digester. Cells were straight rods, 0.6-1 µm in diameter and 2-3 µm in length and grew as single cells or in pairs. Cells formed round terminal endospores. The temperature range for growth was 50-70 °C, with an optimum at 65 °C. The pH range for growth was 5.7-8.0, with an optimum at 7.5. Strain E3-O(T) had the ability to ferment various sugars, such as fructose, galactose, glucose, mannose, raffinose, ribose, sucrose and xylose, producing mainly H2 and acetate. In addition, the isolate was able to grow with CO as the sole carbon and energy source. CO oxidation was coupled to H2 and CO2 formation. The G+C content of the genomic DNA was 54.6 mol%. Based on 16S rRNA gene sequence analysis, this bacterium is most closely related to Moorella glycerini (97 % sequence identity). Based on the physiological features and phylogenetic analysis, it is proposed that strain E3-O(T) should be classified in the genus Moorella as a representative of a novel species, Moorella stamsii. The type strain of Moorella stamsii is E3-O(T) ( = DSM 26271(T) = CGMCC 1.5181(T)).

Strategies to suppress hydrogen‐consuming microorganisms affect macro and micro scale structure and microbiology of granular sludge

Treatment of anaerobic granules with heat and two chemical treatments, contacting with 2‐bromoethanesulfonate (BES) and with BES + Chloroform, were applied to suppress hydrogen‐consuming microorganisms. Three mesophilic expanded granular sludge bed (EGSB) reactors—RHeat, RBES, and RBES + Chlo—were inoculated with the treated sludges and fed with synthetic sugar‐based wastewater (5 gCOD L−1, HRT 20–12 h). Morphological integrity of granules and bacterial communities were assessed by quantitative image analysis and 16S rRNA gene based techniques, respectively. Hydrogen production in RHeat was under 300 mL H2 L−1 day−1, with a transient peak of 1,000 mL H2 L−1 day−1 after decreasing HRT. In RBES + Chlo hydrogen production rate did not exceed 300 mL H2 L−1 day−1 and there was granule fragmentation, release of free filaments from aggregates, and decrease of granule density. In RBES, there was an initial period with unstable hydrogen production, but a pulse of BES triggered its production rate to 700 ± 200 mL H2 L−1 day−1. This strategy did not affect granules structure significantly. Bacteria branching within Clostridiaceae and Ruminococcaceae were present in this sludge. This work demonstrates that, methods applied to suppress H2‐consuming microorganisms can cause changes in the macro‐ and microstructure of granular sludge, which can be incompatible with the operation of high‐rate reactors. Biotechnol. Bioeng. 2011; 108:1766–1775.

Genome analyses of the carboxydotrophic sulfate-reducers Desulfotomaculum nigrificans and Desulfotomaculum carboxydivorans and reclassification of Desulfotomaculum caboxydivorans as a later synonym of Desulfotomaculum nigrificans

Desulfotomaculum nigrificans and D. carboxydivorans are moderately thermophilic members of the polyphyletic spore-forming genus Desulfotomaculum in the family Peptococcaceae. They are phylogenetically very closely related and belong to ‘subgroup a’ of the Desulfotomaculum cluster 1. D. nigrificans and D. carboxydivorans have a similar growth substrate spectrum; they can grow with glucose and fructose as electron donors in the presence of sulfate. Additionally, both species are able to ferment fructose, although fermentation of glucose is only reported for D. carboxydivorans. D. nigrificans is able to grow with 20% carbon monoxide (CO) coupled to sulfate reduction, while D. carboxydivorans can grow at 100% CO with and without sulfate. Hydrogen is produced during growth with CO by D. carboxydivorans. Here we present a summary of the features of D. nigrificans and D. carboxydivorans together with the description of the complete genome sequencing and annotation of both strains. Moreover, we compared the genomes of both strains to reveal their differences. This comparison led us to propose a reclassification of D. carboxydivorans as a later heterotypic synonym of D. nigrificans.

Enrichment of syngas-converting communities from a multi-orifice baffled bioreactor

The substitution of natural gas by renewable biomethane is an interesting option to reduce global carbon footprint. Syngas fermentation has potential in this context, as a diverse range of low‐biodegradable materials that can be used. In this study, anaerobic sludge acclimatized to syngas in a multi‐orifice baffled bioreactor (MOBB) was used to start enrichments with CO. The main goals were to identify the key players in CO conversion and evaluate potential interspecies metabolic interactions conferring robustness to the process. Anaerobic sludge incubated with 0.7 × 105 Pa CO produced methane and acetate. When the antibiotics vancomycin and/or erythromycin were added, no methane was produced, indicating that direct methanogenesis from CO did not occur. Acetobacterium and Sporomusa were the predominant bacterial species in CO‐converting enrichments, together with methanogens from the genera Methanobacterium and Methanospirillum. Subsequently, a highly enriched culture mainly composed of a Sporomusa sp. was obtained that could convert up to 1.7 × 105 Pa CO to hydrogen and acetate. These results attest the role of Sporomusa species in the enrichment as primary CO utilizers and show their importance for methane production as conveyers of hydrogen to methanogens present in the culture.

Enhancement of methane production from 1-hexadecene by additional electron donors

1‐Hexadecene‐contaminated wastewater is produced in oil refineries and can be treated in methanogenic bioreactors, although generally at low conversion rates. In this study, a microbial culture able to degrade 1‐hexadecene was enriched, and different stimulation strategies were tested for enhancing 1‐hexadecene conversion to methane. Seven and three times faster methane production was obtained in cultures stimulated with yeast extract or lactate, respectively, while cultures amended with crotonate lost the ability to degrade 1‐hexadecene. Methane production from 1‐hexadecene was not enhanced by the addition of extra hydrogenotrophic methanogens. Bacteria closely related to Syntrophus and Smithella were detected in 1‐hexadecene‐degrading cultures, but not in the ones amended with crotonate, which suggests the involvement of these bacteria in 1‐hexadecene degradation. Genes coding for alkylsuccinate synthase alpha‐subunit were detected in cultures degrading 1‐hexadecene, indicating that hydrocarbon activation may occur by fumarate addition. These findings are novel and show that methane production from 1‐hexadecene is improved by the addition of yeast extract or lactate. These extra electron donors may be considered as a potential bioremediation strategy of oil‐contaminated sites with bioenergy generation through methane production.

Harnessing the Power of PCR Molecular Fingerprinting Methods and Next Generation Sequencing for Understanding Structure and Function in Microbial Communities

Polymerase chain reaction (PCR) is central to methods in molecular ecology. Here, we describe PCR-dependent approaches useful for investigating microbial diversity and its function in various natural, human-associated, and built environment ecosystems. Protocols routinely used for DNA extraction, purification, cloning, and sequencing are included along with various resources for the statistical analysis following gel electrophoresis-based methods (DGGE) and sequencing. We also provide insights into eukaryotic microbiome analysis, sample preservation techniques, PCR troubleshooting, DNA quantification methods, and commonly used ordination techniques.