Palmitic acid was the main long chain fatty acids (LCFA) that accumulated onto the anaerobic sludge when oleic acid was fed to an EGSB reactor. The conversion between oleic and palmitic acid was linked to the biological activity. When palmitic acid was fed to an EGSB reactor it represented also the main LCFA that accumulated onto the sludge. The way of palmitic acid accumulation was different in the oleic and in the palmitic acid fed reactors. When oleic acid was fed, the biomass-associated LCFA (83% as palmitic acid) were mainly adsorbed and entrapped in the sludge that became "encapsulated" by an LCFA layer. However, when palmitic acid was fed, the biomass-associated LCFA (the totality as palmitic acid) was mainly precipitated in white spots like precipitates in between the sludge, which remained "non-encapsulated." The two sludges were compared in terms of the specific methanogenic activity (SMA) in the presence of acetate, propionate, butyrate, and H(2)CO(2), before and after the mineralization of similar amounts of biomass-associated LCFA (4.6 and 5.2 g COD-LCFA/g of volatile suspended solids (VSS), for the oleic and palmitic acid fed sludge, respectively). The "non-encapsulated," sludge exhibited a considerable initial methanogenic activity on all the tested substrates, with the single exception of butyrate. However, with the "encapsulated" sludge only methane production from ethanol and H(2)/CO(2) was detected, after a lag phase of about 50 h. After mineralization of the biomass-associated LCFA, both sludges exhibited activities of similar order of magnitude in the presence of the same individual substrates and significantly higher than before. The results evidenced that LCFA accumulation onto the sludge can create a physical barrier and hinder the transfer of substrates and products, inducing a delay on the initial methane production. Whatever the mechanism, metabolic or physical, that is behind this inhibition, it is reversible, being eliminated after the depletion of the biomass-associated LCFA.
SummaryThe 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.
Long-chain fatty acids (LCFA) associated with anaerobic sludge by mechanisms of precipitation, adsorption, or entrapment can be biodegraded to methane. The mineralization kinetics of biomass-associated LCFA were established according to an inhibition model based on Haldane's enzymatic inhibition kinetics. A value around 1,000 mg COD-LCFAÁg VSS À1 was obtained for the optimal specific LCFA content that allowed the maximal mineralization rate. For sludge with specific LCFA contents of 2,838 F 63 and 4,571 F 257 mg COD-LCFAÁg VSS À1 , the specific methanogenic activities in the presence of acetate, butyrate, and H 2 /CO 2 were significantly enhanced after the mineralization of the biomass-associated LCFA. For sludge with a specific LCFA content near the optimal value defined by the kinetic model, the effect of adding VFA to the medium was studied during the mineralization of the biomass-associated LCFA. Different patterns were obtained for each individual substrate. Acetate and butyrate were preferentially consumed by the consortium, but in the case of propionate no evidence of a sequential consumption pattern could be withdrawn. It was concluded that LCFA do not exert a bactericidal neither a permanent toxic effect toward the anaerobic consortia. A discussion is addressed to the relative roles of a reversible inhibitory effect and a transport limitation effect imposed by the LCFA surrounding the cells. B
Oleic acid toxicity and biodegradability were followed during long-term operation of two similar anaerobic fixed-bed units. When treating an oleate based effluent, the sludge from the bioreactor that was acclimated with lipids during the first operation period, showed a higher tolerance to oleic acid toxicity (IC50 = 137 mg/l) compared with the sludge fed with a non-fat substrate (IC50 = 80 mg/l). This sludge showed also the highest biodegradation capacity of oleic acid, achieving maximum methane production rates between 33 and 46 mlCH4(STP)/gVS.day and maximum percentages of methanization between 85 and 98% for the range of concentrations between 500 and 900 mg oleate/l. When oleate was the sole carbon source fed to both digesters, the biomass became encapsulated with organic matter, possibly oleate or an intermediate of its degradation, e.g. stearate that was degraded at a maximum rate of 99 mlCH4(STP)/gVS.day. This suggests the possibility of using adsorption-degradation cycles for the treatment of LCFA based effluents. Both tolerance to toxicity and biodegradability of oleic acid were improved by acclimatization with lipids or oleate below a threshold concentration.
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 CNT. Butyrate conversion to methane by Syntrophomonas wolfei and Methanospirillum hungatei was enhanced (∼1.5 times) in the presence of CNT (5 g·L ), 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.
Whey valorization concerns have led to recent interest on the production of whey beverage simulating kefir. In this study, the structure and microbiota of Brazilian kefir grains and beverages obtained from milk and whole/deproteinised whey was characterized using microscopy and molecular techniques. The aim was to evaluate its stability and possible shift of probiotic bacteria to the beverages. Fluorescence staining in combination with Confocal Laser Scanning Microscopy showed distribution of yeasts in macro-clusters among the grain's matrix essentially composed of polysaccharides (kefiran) and bacteria. Denaturing gradient gel electrophoresis displayed communities included yeast affiliated to Kluyveromyces marxianus, Saccharomyces cerevisiae, Kazachatania unispora, bacteria affiliated to Lactobacillus kefiranofaciens subsp. Kefirgranum, Lactobacillus kefiranofaciens subsp. Kefiranofaciens and an uncultured bacterium also related to the genus Lactobacillus. A steady structure and dominant microbiota, including probiotic bacteria, was detected in the analyzed kefir beverages and grains. This robustness is determinant for future implementation of whey-based kefir beverages.
The aim of the present work was to study the maximum potential methane production in batch assays of sludge samples taken along the operation of two EGSB reactors (RI inoculated with granular sludge and RII inoculated with suspended sludge) fed with increasing oleic acid concentrations between 2 and 8 gCOD/l (HRT = 1 day). After removing the residual substrate, the sludge was incubated in batch vials without any added carbon source. A maximum methane production rate of 152+/-21 mlCH4(STP)/gVS.day was obtained for the suspended sludge taken on day 70, when oleate at a concentration of 2 g COD/l was fed with a co-substrate (50% COD). The maximum plateau achieved in the methane production curve was 1145+/-307 mlCH4(STP)/gVS, obtained for the suspended sludge taken on day 162, when oleate was fed as the sole carbon source at 6 g COD/I. The methanization rate of the adsorbed substrate was enhanced under stirring conditions and was inhibited by adding oleic acid. Extraction and GC analysis confirmed that the main adsorbed substrate was palmitate, and not oleate. Accumulated palmitate adsorbed onto the sludge and further beta-oxidation was inhibited when in the presence of oleic acid. If oleic acid was removed from the medium beta-oxidation proceeded with methane production. Suspended sludge was more efficient than granular sludge.
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.
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