A. F. Salvador

Activity and Viability of Methanogens in Anaerobic Digestion of Unsaturated and Saturated Long-Chain Fatty Acids

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.

Carbon nanotubes accelerate methane production in pure cultures of methanogens and in a syntrophic coculture

Carbon materials have been reported to facilitate direct interspecies electron transfer (DIET) between bacteria and methanogens improving methane production in anaerobic processes. In this work, the effect of increasing concentrations of carbon nanotubes (CNT) on the activity of pure cultures of methanogens and on typical fatty acid-degrading syntrophic methanogenic coculture was evaluated. CNT affected methane production by methanogenic cultures, although acceleration was higher for hydrogenotrophic methanogens than for acetoclastic methanogens or syntrophic coculture. Interestingly, the initial methane production rate (IMPR) by Methanobacterium formicicum cultures increased 17 times with 5 g·L-1 CNT. Butyrate conversion to methane by Syntrophomonas wolfei and Methanospirillum hungatei was enhanced (∼1.5 times) in the presence of CNT (5 g·L-1 ), but indications of DIET were not obtained. Increasing CNT concentrations resulted in more negative redox potentials in the anaerobic microcosms. Remarkably, without a reducing agent but in the presence of CNT, the IMPR was higher than in incubations with reducing agent. No growth was observed without reducing agent and without CNT. This finding is important to re-frame discussions and re-interpret data on the role of conductive materials as mediators of DIET in anaerobic communities. It also opens new challenges to improve methane production in engineered methanogenic processes.

Toxicity of long chain fatty acids towards acetate conversion by Methanosaeta concilii and Methanosarcina mazei.

Long‐chain fatty acids (LCFA) can inhibit methane production by methanogenic archaea. The effect of oleate and palmitate on pure cultures of Methanosaeta concilii and Methanosarcina mazei was assessed by comparing methane production rates from acetate before and after LCFA addition. For both methanogens, a sharp decrease in methane production (> 50%) was observed at 0.5 mmol L−1 oleate, and no methane was formed at concentrations higher than 2 mmol L−1 oleate. Palmitate was less inhibitory than oleate, and M. concilii was more tolerant to palmitate than M. mazei, with 2 mmol L−1 palmitate causing 11% and 64% methanogenic inhibition respectively. This study indicates that M. concilii and M. mazei tolerate LCFA concentrations similar to those previously described for hydrogenotrophic methanogens. In particular, the robustness of M. concilii might contribute to the observed prevalence of Methanosaeta species in anaerobic bioreactors used to treat LCFA‐rich wastewater.

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.

Insight into the Role of Facultative Bacteria Stimulated by Microaeration in Continuous Bioreactors Converting LCFA to Methane

Conversion of unsaturated long chain fatty acids (LCFA) to methane in continuous bioreactors is not fully understood. Palmitate (C16:0) often accumulates during oleate (C18:1) biodegradation in methanogenic bioreactors, and the reason why this happens and which microorganisms catalyze this reaction remains unknown. Facultative anaerobic bacteria are frequently found in continuous reactors operated at high LCFA loads, but their function is unclear. To get more insight on the role of these bacteria, LCFA conversion was studied under microaerophilic conditions. For that, we compared bioreactors treating oleate-based wastewater (organic loading rates of 1 and 3 kg COD m-3 d-1), operated under different redox conditions (strictly anaerobic-AnR, -350 mV; microaerophilic-MaR, -250 mV). At the higher load, palmitate accumulated 7 times more in the MaR, where facultative anaerobes were more abundant, and only the biomass from this reactor could recover the methanogenic activity after a transient inhibition. In a second experiment, the abundance of facultative anaerobic bacteria, particularly Pseudomonas spp. (from which two strains were isolated), was strongly correlated ( p < 0.05) with palmitate-to-total LCFA percentage in the biofilm formed in a continuous plug flow reactor fed with very high loads of oleate. This work strongly suggests that microaeration stimulates the development of facultative bacteria that are critical for achieving LCFA conversion to methane in continuous bioreactors. Microbial networks and interactions of facultative and strict anaerobes in microbial communities should be considered in future studies.

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.