Innovative start-up strategies for optimal methane production from lipids in anaerobic bioreactors

Lipids and long-chain fatty acids (LCFA) are energy-rich compounds that can be used as carbon and energy source by anaerobic microbial communities. Theoretically, large amounts of methane, a valuable energy carrier, may be generated during this process. However, operational problems, mainly associated with LCFA accumulation onto the sludge, have limited the use of anaerobic technology to produce methane from LCFA. In this work, two novel start-up strategies were tested for optimal methane production from LCFA: (i) bioreactor start-up using a intermittent feeding strategy, and (ii) bioreactor bioaugmentation with a LCFA-degrading bacterium. Intermittent feeding start-up resulted in efficient continuous methane production from high LCFA loads (up to 21 kgCOD m-3 day-1, 50% COD as oleate (unsaturated LCFA, C18:1)). Alternating continuous bioreactor feeding and batch degradation periods, during bioreactor start-up, was crucial for sludge acclimation and contributed to the development of a metabolically specialized anaerobic microbial community that was able to efficiently convert oleate to methane. After intermittent feeding bioreactor start-up, methane yields higher than 70% were achieved, and neither LCFA nor VFA accumulated in the system. Bioaugmentation experiments were performed using Syntrophomonas zehnderi, a bacterium able to degrade saturated and unsaturated LCFA [1]. Anaerobic sludge amended with active and inactive S. zehnderi was incubated with 1 mM oleate as sole carbon and energy source. Methane production from oleate in bioaugmented batches was faster and high methane yields (89±5%) were achieved. This work highlights the importance of the start-up strategy for the development of balanced syntrophic communities specialized in methane production from LCFA. Intermittent feeding and bioaugmentation with LCFA-degrading bacteria may be applied as alternative or complementary strategies.Fundação para a Ciência e a Tecnologia (FCT) - FCOMP-01-0124-FEDER-014784, SFRH/BD/24256/2005.FEDER - COMPETE programEuropean Social Fund (ESF

New perspectives for methane production from oleate : bioaugmentation of anaerobic sludge with Syntrophomonas zehnderi

Biogas production from waste lipids is a promising technology for sustainable energy production. In anaerobic bioreactors, lipids and long-chain fatty acids (LCFA) are easily removed from the liquid medium, mainly by adsorption. However, further LCFA degradation is rate-limiting and possible dependent on the development of syntrophic communities. Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA genes was used to follow the changes in bacterial communities during continuous and fed-batch reactors operation with oleate, an unsaturated LCFA. A specific dominant DGGE-band corresponding to bacteria deeply clustering with Syntrophomonas zehnderi (99% identity) was found in all the sludges that could degrade oleate, thus suggesting the involvement of this bacterium in unsaturated LCFA catabolism. Therefore, the potential of S. zehnderi as bioaugmenting strain for improving methane production from oleate was further studied in batch assays. Oleate was added to the medium at a final concentration of 1 mM and the assays were performed with and without the solid microcarrier sepiolite. Methane production was faster in the bioaugmented assays, and this effect was more pronounced in the presence of sepiolite. The positive effects of sepiolite can be related to a decrease in oleate toxicity towards the acetoclastic methanogens, or to an improvement of the syntrophic relationships. Bioaugmentation with S. zehnderi might be a suitable strategy for accelerating LCFA conversion to methane in anaerobic bioreactors, shortening the start-up period of high rate continuous processes or recover LCFA-inhibited sludge

Enhancing methane production from fat by bioaugmenting Syntrophomonas zehnderi to anaerobic sludge

Fundação para a Ciência e a Tecnologia (FCT) - bolsa SFRH/BD/24256/200

Anaerobic treatment of LCFA-rich wastewater: assessing the bioaugmentation potential of Syntrophomonas zehnderi

Long-chain fatty acids (LCFA) are commonly present in fatty-wastewaters. Complete LCFA degradation depends on the coordinated activity of syntrophic bacteria, which convert LCFA to acetate and hydrogen, and methanogenic archaea, that utilize these substrates, making the overall conversion energetically possible. LCFA-degrading bacteria are fastidious microorganisms with low predominance in bioreactors. Thus, addition of LCFA-degrading bacteria to anaerobic sludge can possibly improve LCFA biodegradation and enhance methane production. In this work, a co-culture of Syntrophomonas zehnderi and Methanobacterium formicicum was added to non-acclimated granular sludge. Two sets of bottles were prepared, with and without sepiolite, a solid microcarrier. Sludge was bioaugmented with co-culture and supplemented with 1 mM oleate. Blanks (without oleate) and controls (with inactivated co-culture) were also prepared. Methane, VFA and LCFA were quantified. Addition of S. zehnderi enhanced LCFA degradation, both in the assays prepared with and without microcarrier. In the bottles containing bioaugmented sludge and no microcarrier, acetate accumulated in the medium indicating a fast LCFA β-oxidation: after 15 days of incubation, maximum acetate concentrations (approx. 5 mM) were attained and 77% of the added oleate could be accounted for the acetate and methane measured. In non-bioaugmented sludge, acetate accumulation started later and, after 15 days of incubation, was not higher than 1.5 mM. In bottles containing microcarrier methane was produced at a higher rate. In this case only residual acetate concentrations were measured, indicating balanced syntrophic relations, maybe due to stimulation of bacteria-archaea relation by the microcarrier. Methane production from oleate was most favored in bottles supplemented with the syntrophic co-culture and containing microcarrier: 71% of the added oleate was recovered as methane after 12 days of incubation and a maximum methane production rate of 1.12 mMCH4day-1 was observed. Bioaugmentation with S. zehnderi enhances oleate biodegradation and can be potentially useful for a faster reactor start-up

Methane production from oleate : assessing the bioaugmentation potential of Syntrophomonas zehnderi

The potential for improving long-chain fatty acids (LCFA) conversion to methane was evaluated by bioaugmenting a non-acclimated anaerobic granular sludge with Syntrophomonas zehnderi. Batch bioaugmentation assays were performed with and without the solid microcarrier sepiolite, using 1 mM oleate as sole carbon and energy source. When S. zehnderi was added to the anaerobic sludge methane production from oleate was faster. High methane yields, i.e. 89 ± 5% and 72 ± 1%, were observed in bioaugmented assays in the absence and presence of sepiolite, respectively. Sepiolite stimulated a faster methane production from oleate and prevented the accumulation of acetate. Acetoclastic activity was affected by oleate in the absence of sepiolite, where methane production rate was 26% lower than in assays with microcarrier.Fundação para a Ciência e a Tecnologia (FCT) - SFRH/BD/24256/200

Methane production from fat : assessing the bioaugmentation potential of syntrophomonas zehnderi

Fundação para a Ciência e a Tecnologia (FCT

Bioaugmentation of anaerobic sludge with Syntrophomonas zehnderi as a Prospect for enhanced methane production from oleate

Manuscript revisedBioaugmentation of anaerobic sludge with long-chain fatty acids (LCFA)-degrading bacteria can be a feasible strategy to enhance methane production from LCFA. This hypothesis was studied in batch assays with Syntrophomonas zehnderi as bioaugmenting strain. This bacterium is able to degrade a wide range of saturated and unsaturated LCFA, and its presence has been reported in several oleate-fed bioreactors. Bioaugmentation assays were performed in the presence and absence of the solid microcarrier sepiolite, and with oleate (unsaturated C18:1 LCFA, 1 mM) as substrate. S. zehnderi addition enhanced methane production from oleate, leading to high methane yields in the bioaugmented assays, i.e. 895% and 721% in the absence and presence of sepiolite, respectively. Comparing bioaugmented assays, a two times faster methane production was observed in the presence of sepiolite, where maximum cumulative methane production was attained after 15 days of incubation. Also, acetate accumulation was prevented by the microcarrier, probably due to a decrease of acetoclastic methanogens inhibition by oleate. Bioaugmentation of anaerobic bioreactors with S. zehnderi or other syntrophic LCFA-degraders can be potentially useful for faster reactor start-up or recovery of LCFA-inhibited processes

Predominance of Syntrophomonas zehnderi in oleate-fed bioreactors and its potential as bioaugmenting strain

Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA genes was used to follow the changes in bacterial communities during anaerobic continuous and fed-batch reactors operation with oleate, an unsaturated long-chain fatty acid (LCFA). Throughout continuous operation, bacterial profiles of the sludge showed an average similarity with the inoculum of 9±5%. Bacterial profiles were also altered during fed-batch operation; similarity between sludges before and after fed-batch cycles was 50%. A predominant DGGE-band was identified in all the DGGE profiles of sludges contacting with oleate. 16S rRNA gene sequences retrieved from the different sludges, and corresponding to this position in the DGGE profiles, where found to be closely related to Syntrophomonas zehnderi (99% identity). S. zehnderi is known to degrade saturated and unsaturated LCFA [1], and could potentially be used to bioaugment anaerobic sludge for improving methane recovery from LCFA. This hypothesis was tested in batch assays performed with and without the solid microcarrier sepiolite. Oleate was added to the medium at a final concentration of 1 mM. Methane production and volatile fatty-acids were monitored throughout the experiment and LCFA were quantified at the end of the assays. The results obtained show that methane production from oleate was enhanced through S. zehnderi addition to the anaerobic sludge. After 15 days of incubation, methane yield in bioaugmented assays with sepiolite was as high as 71±3%, whereas in non-bioaugmented bottles only 27±1% of the theoretical methane could be accounted. Methane yield in bioaugmented bottles without sepiolite was also lower, i.e. 36±12%. The use of a microcarrier might facilitate interspecies metabolite exchange, favoring faster methane production. This approach can be potentially useful for a faster reactor start-up or recovery of an LCFA-inhibited anaerobic bioreactor. [1] Sousa DZ, Smidt H, Alves MM & Stams AJM (2007) Int J Syst Ev

Bioaugmentation strategies to enhance long chain fatty acids (LCFA) conversion to methane

Bioaugmentation of bioreactors with LCFA-degrading bacteria is a possibility for improving methane production from lipid-rich wastes/wastewaters. Cavaleiro et al. [1] has shown that methane production from oleate (unsaturated LCFA) is faster and more efficient in batch tests bioaugmented with Syntrophomonas zehnderi, a bacterium that is able to degrade a wide range of both saturated and unsaturated LCFA [2]. In this work, anaerobic sludge bioaugmentation with S. zehnderi was studied in order to evaluate: (I) the recovery of bioreactors after an episode of LCFA overload; (II) the potential for decreasing reactor start-up periods. The potential of using S. zehnderi for recovering LCFA-overloaded sludge was tested using anaerobic sludge collected from a oleate-fed bioreactor at three different operation times. Bioaugmentation batches were prepared with LCFA loaded biomasses in the presence of S. zehnderi. Controls were set using inactivated S. zehnderi. Methane yields of 72, 53 and 40% were obtained from the first, second and third collected sludge samples respectively. However, addition of S. zehnderi did not significantly improve LCFA conversion loaded-sludges as similar yields were achieved in non-bioaugmented controls. Fed-batch bioreactor start-up, using a non-acclimated sludge, was attempted in the presence of S. zehnderi. Assays were conducted in the presence and absence of both a solid microcarier (sepiolite) and a substoichiometric amount of ferric hydroxide. Blank (no oleate) and control assays (inactivated S. zehnderi) were also prepared. Bioaugmentation assays with sepiolite and ferric hydroxide showed the highest methane yield, with an observed methane yield 16% higher than in non-bioaugmented controls. The potential of bioaugmenting S. zehnderi as means to recover methanogenic activity of LCFA-loaded biomass was not demonstrated. However faster reactor start-up could be accomplished since higher methane yield was achieved in bioaugmented fed-batch assays in the presence of sepiolite with ferric hydroxide. [1] Cavaleiro, A.J., Sousa, D.Z. and Alves, M.M. 2010. Water Res 44:4940-7. [2] Sousa, D.Z., Smidt, H., Alves, M.M. and Stams, A.J.M. 2007. Int J Syst Evol Microbiol 57:609-15. Acknowledgments: This study has been funded by FEDER, through the COMPETE program, and by Portuguese funds, through Portuguese Foundation for Science and Technology (FCT), in the frame of the project FCOMP-01-0124-FEDER-014784. References