D. Z. Sousa

Waste lipids to energy: how to optimize methane production from long‐chain fatty acids (LCFA)

The position of high‐rate anaerobic technology (HR‐AnWT) in the wastewater treatment and bioenergy market can be enhanced if the range of suitable substrates is expanded. Analyzing existing technologies, applications and problems, it is clear that, until now, wastewaters with high lipids content are not effectively treated by HR‐AnWT. Nevertheless, waste lipids are ideal potential substrates for biogas production, since theoretically more methane can be produced, when compared with proteins or carbohydrates. In this minireview, the classical problems of lipids methanization in anaerobic processes are discussed and new concepts to enhance lipids degradation are presented. Reactors operation, feeding strategies and prospects of technological developments for wastewater treatment are discussed. Long‐chain fatty acids (LCFA) degradation is accomplished by syntrophic communities of anaerobic bacteria and methanogenic archaea. For optimal performance these syntrophic communities need to be clustered in compact aggregates, which is often difficult to achieve with wastewaters that contain fats and lipids. Driving the methane production from lipids/LCFA at industrial scale without risk of overloading and inhibition is still a challenge that has the potential for filling a gap in the existing processes and technologies for biological methane production associated to waste and wastewater treatment.

Ecophysiology of syntrophic communities that degrade saturated and unsaturated long-chain fatty acids

Syntrophic relationships are the key for biodegradation in methanogenic environments. We review the ecological and physiological features of syntrophic communities involved in the degradation of saturated and unsaturated long-chain fatty acids (LCFA), as well as their potential application to convert lipids/fats containing waste to biogas. Presently, about 14 species have been described with the ability to grow on fatty acids in syntrophy with methanogens, all belonging to the families Syntrophomonadaceae and Syntrophaceae. The principle pathway of LCFA degradation is through beta-oxidation, but the initial steps in the conversion of unsaturated LCFA are unclear. Communities enriched on unsaturated LCFA also degrade saturated LCFA, but the opposite generally is not the case. For efficient methane formation, the physical and inhibitory effects of LCFA on methanogenesis need to be considered. LCFA adsorbs strongly to biomass, which causes encapsulation of active syntrophic communities and hampers diffusion of substrate and products in and out of the biomass. Quantification of archaea by real-time PCR analysis suggests that potential LCFA inhibitory effect towards methanogens might be reversible. Rather, the conversion of adsorbed LCFA in batch assays was shown to result in a significant increase of archaeal cell numbers in anaerobic sludge samples.

Microbial communities involved in anaerobic degradation of unsaturated or saturated long chain fatty acids

ABSTRACT Anaerobic long-chain fatty acid (LCFA)-degrading bacteria were identified by combining selective enrichment studies with molecular approaches. Two distinct enrichment cultures growing on unsaturated and saturated LCFAs were obtained by successive transfers in medium containing oleate and palmitate, respectively, as the sole carbon and energy sources. Changes in the microbial composition during enrichment were analyzed by denaturing gradient gel electrophoresis (DGGE) profiling of PCR-amplified 16S rRNA gene fragments. Prominent DGGE bands of the enrichment cultures were identified by 16S rRNA gene sequencing. A significant part of the retrieved 16S rRNA gene sequences was most similar to those of uncultured bacteria. Bacteria corresponding to predominant DGGE bands in oleate and palmitate enrichment cultures clustered with fatty acid-oxidizing bacteria within Syntrophomonadaceae and Syntrophobacteraceae families. A low methane yield, corresponding to 9 to 18% of the theoretical value, was observed in the oleate enrichment, and acetate, produced according to the expected stoichiometry, was not further converted to methane. In the palmitate enrichment culture, the acetate produced was completely mineralized and a methane yield of 48 to 70% was achieved from palmitate degradation. Furthermore, the oleate enrichment culture was able to use palmitate without detectable changes in the DGGE profile. However, the palmitate-specialized consortia degraded oleate only after a lag phase of 3 months, after which the DGGE profile had changed. Two predominant bands appeared, and sequence analysis showed affiliation with the Syntrophomonas genus. These bands were also present in the oleate enrichment culture, suggesting that these bacteria are directly involved in oleate degradation, emphasizing possible differences between the degradation of unsaturated and saturated LCFAs.

Thermochemical pre- and biological co-treatments to improve hydrolysis and methane production from poultry litter

The biochemical methane potential (BMP) of raw poultry litter waste was assessed in batch assays. Biological co-treatment with Clostridium cellulolyticum, Caldicellulosiruptor saccharolyticum and Clostridium thermocellum as bioaugmentation strains, and thermochemical pre-treatments with lime and sodium hydroxide performed at different temperatures and pressures were applied as strategies to improve the BMP by favouring the hydrolysis of the cellulolytic material in the waste. Anaerobic digestion of the raw waste allowed a specific methane production of 145 ± 14 LCH(4)kg(-1)VS, with 1% total solids and 0.72 g VS(inoculum)g(-1)VS(waste). The pre- and co-treatments contributed to a significant increase (up to 74%) in the waste solubilisation when using C. saccharolyticum, but methane production did not improve considerably. Therefore, the conversion of soluble organic matter to methane was the limiting step of the anaerobic digestion process of poultry litter waste.

Biohydrogen production from arabinose and glucose using extreme thermophilic anaerobic mixed cultures.

BackgroundSecond generation hydrogen fermentation technologies using organic agricultural and forestry wastes are emerging. The efficient microbial fermentation of hexoses and pentoses resulting from the pretreatment of lingocellulosic materials is essential for the success of these processes.ResultsConversion of arabinose and glucose to hydrogen, by extreme thermophilic, anaerobic, mixed cultures was studied in continuous (70°C, pH 5.5) and batch (70°C, pH 5.5 and pH 7) assays. Two expanded granular sludge bed (EGSB) reactors, Rarab and Rgluc, were continuously fed with arabinose and glucose, respectively. No significant differences in reactor performance were observed for arabinose and glucose organic loading rates (OLR) ranging from 4.3 to 7.1 kgCOD m-3 d-1. However, for an OLR of 14.2 kgCOD m-3 d-1, hydrogen production rate and hydrogen yield were higher in Rarab than in Rgluc (average hydrogen production rate of 3.2 and 2.0 LH2 L-1 d-1 and hydrogen yield of 1.10 and 0.75 molH2 mol-1substrate for Rarab and Rgluc, respectively). Lower hydrogen production in Rgluc was associated with higher lactate production. Denaturing gradient gel electrophoresis (DGGE) results revealed no significant difference on the bacterial community composition between operational periods and between the reactors. Increased hydrogen production was observed in batch experiments when hydrogen partial pressure was kept low, both with arabinose and glucose as substrate. Sugars were completely consumed and hydrogen production stimulated (62% higher) when pH 7 was used instead of pH 5.5.ConclusionsContinuous hydrogen production rate from arabinose was significantly higher than from glucose, when higher organic loading rate was used. The effect of hydrogen partial pressure on hydrogen production from glucose in batch mode was related to the extent of sugar utilization and not to the efficiency of substrate conversion to hydrogen. Furthermore, at pH 7.0, sugars uptake, hydrogen production and yield were higher than at pH 5.5, with both arabinose and glucose as substrates.

Role of syntrophic microbial communities in high-rate methanogenic bioreactors.

Anaerobic purification is a cost-effective way to treat high strength industrial wastewater. Through anaerobic treatment of wastewaters energy is conserved as methane, and less sludge is produced. For high-rate methanogenesis compact syntrophic communities of fatty acid-degrading bacteria and methanogenic archaea are essential. Here, we describe the microbiology of syntrophic communities in methanogenic reactor sludges and provide information on which microbiological factors are essential to obtain high volumetric methane production rates. Fatty-acid degrading bacteria have been isolated from bioreactor sludges, but also from other sources such as freshwater sediments. Despite the important role that fatty acid-degrading bacteria play in high-rate methanogenic bioreactors, their relative numbers are generally low. This finding indicates that the microbial community composition can be further optimized to achieve even higher rates.

A genomic view on syntrophic versus non-syntrophic lifestyle in anaerobic fatty acid degrading communities.

In sulfate-reducing and methanogenic environments complex biopolymers are hydrolyzed and degraded by fermentative micro-organisms that produce hydrogen, carbon dioxide and short chain fatty acids. Degradation of short chain fatty acids can be coupled to methanogenesis or to sulfate-reduction. Here we study from a genome perspective why some of these micro-organisms are able to grow in syntrophy with methanogens and others are not. Bacterial strains were selected based on genome availability and upon their ability to grow on short chain fatty acids alone or in syntrophic association with methanogens. Systematic functional domain profiling allowed us to shed light on this fundamental and ecologically important question. Extra-cytoplasmic formate dehydrogenases (InterPro domain number; IPR006443), including their maturation protein FdhE (IPR024064 and IPR006452) is a typical difference between syntrophic and non-syntrophic butyrate and propionate degraders. Furthermore, two domains with a currently unknown function seem to be associated with the ability of syntrophic growth. One is putatively involved in capsule or biofilm production (IPR019079) and a second in cell division, shape-determination or sporulation (IPR018365). The sulfate-reducing bacteria Desulfobacterium autotrophicum HRM2, Desulfomonile tiedjei and Desulfosporosinus meridiei were never tested for syntrophic growth, but all crucial domains were found in their genomes, which suggests their possible ability to grow in syntrophic association with methanogens. In addition, profiling domains involved in electron transfer mechanisms revealed the important role of the Rnf-complex and the formate transporter in syntrophy, and indicate that DUF224 may have a role in electron transfer in bacteria other than Syntrophomonas wolfei as well. This article is a part of a Special Issue entitled: 18th European Bioenergetics Conference (Biochim. Biophys. Acta, Volume 1837, Issue 7, July 2014).

Pathways and Bioenergetics of Anaerobic Carbon Monoxide Fermentation

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.

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.

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.