A. J. Cavaleiro

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.

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.

Biochemical methane potential of raw and pre-treated meat-processing wastes

Raw and pre-treated greaves and rinds, two meat-processing wastes, were assessed for biochemical methane potential (BMP). Combinations of temperature (25, 55, 70 and 120 °C), NaOH (0.3 g g(-1) waste volatile solids) and lipase from Candida rugosa (10 U g(-1) fat) were applied to promote wastes hydrolysis, and the effect on BMP was evaluated. COD solubilisation was higher (66% for greaves; 55% for rinds) when greaves were pre-treated with NaOH at 55 °C and lipase was added to rinds after autoclaving. Maximum fat hydrolysis (52-54%) resulted from NaOH addition, at 55 °C for greaves and 25 °C for rinds. BMP of raw greaves and rinds was 707±46 and 756±56 L CH4 (at standard temperature and pressure) kg(-1)VS, respectively. BMP of rinds improved 25% by exposure to 70 °C; all other strategies tested had no positive effect on BMP of both wastes, and anaerobic biodegradability was even reduced by the combined action of base and temperature.

Microbial and operational response of an anaerobic fixed bed digester to oleic acid overloads

The effect of oleic acid overloads on biomass accumulation and activity in an anaerobic filter was investigated. An anaerobic fixed-bed reactor specially designed to allow the regular withdrawal of accumulated biomass was used for that purpose. Organic and hydraulic shocks were performed during four days, by stepwise increasing the substrate concentration from 4000 to 20 000 mg COD/l or by reducing the hydraulic retention time from 16 to 3.2 h. During the organic shock, operational performance was more affected than in the hydraulic one, which was the result of the higher degree of inhibition detected in the acetoclastic, hydrogenophilic and syntrophic activities. The ratio adhered/total biomass remained between 17 and 32% during the hydraulic shock, and between 13 and 60% during the organic shock, suggesting a more stable biofilm during the hydraulic shock. A long time (900 h) after the hydraulic shock, hydrogenophilic and syntrophic activities recovered to higher values than before the shock, but after the organic shock only acetoclastic activity recovered pre-shock values. Hydraulic shock induced an increase in tolerance to oleic acid toxicity, evidenced by an increase in the toxicity limit (IC50) from 140 30 to 215 25 mg/l.

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.

Anaerobic digestion of lipid-rich waste

Lipids present in waste and wastewater, also referred as fat, oil, and grease (FOG), can be efficiently converted to methane. This fact constitutes an opportunity for conserving the high energy content of waste lipids, thus facilitating its storage and future use as fuel, electricity, and heat. In anaerobic bioreactors, long-chain fatty acids (LCFAs) are released during hydrolysis of FOG. LCFAs tend to form stable emulsions, adhere to all available surfaces, and adsorb on the microbial cell walls leading to foam formation, sludge flotation, and washout, as well as temporary inhibition of microbes. These problems can be prevented if a correct balance between LCFA accumulation and biodegradation is assured, by sequential feeding and degradation steps. Appropriate reactor operation is the key strategy to prevent the excessive accumulation of LCFA and to stimulate microbial acclimation, especially during the start-up phase. After successful acclimation, a continuously feeding operation is possible, provided that there is proper process control through an adequate monitoring protocol. In addition to adequate operation, a suitable reactor design is recommended. Among other technologies, the inverted anaerobic sludge blanket (IASB) was recently developed for the direct treatment of FOG-containing wastewater. This chapter reports a protocol with a detailed operation and monitoring strategy for achieving effective methane production from FOG-containing waste and wastewater and presents a brief description of the basic concepts behind the development of the reactor

Conversion of Cn-unsaturated into Cn-2-saturated LCFA can occur uncoupled from methanogenesis in anaerobic bioreactors

Fat, oils, and grease present in complex wastewater can be readily converted to methane, but the energy potential of these compounds is not always recyclable, due to incomplete degradation of long chain fatty acids (LCFA) released during lipids hydrolysis. Oleate (C18:1) is generally the dominant LCFA in lipid-containing wastewater, and its conversion in anaerobic bioreactors results in palmitate (C16:0) accumulation. The reason why oleate is continuously converted to palmitate without further degradation via β-oxidation is still unknown. In this work, the influence of methanogenic activity in the initial conversion steps of unsaturated LCFA was studied in 10 bioreactors continuously operated with saturated or unsaturated C16- and C18-LCFA, in the presence or absence of the methanogenic inhibitor bromoethanesulfonate (BrES). Saturated Cn-2-LCFA accumulated both in the presence and absence of BrES during the degradation of unsaturated Cn-LCFA, and represented more than 50% of total LCFA. In the presence of BrES further conversion of saturated intermediates did not proceed, not even when prolonged batch incubation was applied. As the initial steps of unsaturated LCFA degradation proceed uncoupled from methanogenesis, accumulation of saturated LCFA can be expected. Analysis of the active microbial communities suggests a role for facultative anaerobic bacteria in the initial steps of unsaturated LCFA biodegradation. Understanding this role is now imperative to optimize methane production from LCFA.

Microbial remediation of organometals and oil hydrocarbons in the marine environment

Marine environments are exposed to pollution that mostly results from human activities. Organometals and oil hydrocarbons are among the most hazardous pollutants. In surface waters and along the water column, these compounds are more easily degraded than in sediments, especially under anoxic conditions, where they are highly persistent. Due to their negative impact in living organisms, decontamination of polluted marine sites with minimum collateral impacts is imperative. Bioremediation strategies, benefiting from the ability of aerobic and anaerobic microorganisms to degrade organometals or oil hydrocarbons to simpler and less toxic derivatives, represent an alternative to traditional physicochemical decontamination methods. Different bioremediation strategies have been applied in marine environments, including monitored natural recovery, biostimulation, bioaugmentation and phytoremediation. Individual microbial agents or mixed microbial consortia able to remediate these pollutants in marine environments have been identified, and the most relevant mechanisms of biodegradation of pollutants are characterised.

The Role of Marine Anaerobic Bacteria and Archaea in Bioenergy Production

The development of products from marine bioresources is gaining importance in the biotechnology sector. The global market for Marine Biotechnology products and processes was, in 2010, estimated at € 2.8 billion with a cumulative annual growth rate of 5–10% (Borresen et al., Marine biotechnology: a new vision and strategy for Europe. Marine Board Position Paper 15. Beernem: Marine Board-ESF, 2010).