Principles, Advances, and Perspectives of Anaerobic Digestion of Lipids.

Several problems associated with the presence of lipids in wastewater treatment plants are usually overcome by removing them ahead of the biological treatment. However, because of their high energy content, waste lipids are interesting yet challenging pollutants in anaerobic wastewater treatment and codigestion processes. The maximal amount of waste lipids that can be sustainably accommodated, and effectively converted to methane in anaerobic reactors, is limited by several problems including adsorption, sludge flotation, washout, and inhibition. These difficulties can be circumvented by appropriate feeding, mixing, and solids separation strategies, provided by suitable reactor technology and operation. In recent years, membrane bioreactors and flotation-based bioreactors have been developed to treat lipid-rich wastewater. In parallel, the increasing knowledge on the diversity of complex microbial communities in anaerobic sludge, and on interspecies microbial interactions, contributed to extend the knowledge and to understand more precisely the limits and constraints influencing the anaerobic biodegradation of lipids in anaerobic reactors. This critical review discusses the most important principles underpinning the degradation process and recent key discoveries and outlines the current knowledge coupling fundamental and applied aspects. A critical assessment of knowledge gaps in the field is also presented by integrating sectorial perspectives of academic researchers and of prominent developers of anaerobic technology.

Multiple and flexible roles of facultative anaerobic bacteria in microaerophilic oleate degradation.

Anaerobic degradation of long-chain fatty acids (LCFA) involves syntrophic bacteria and methanogens, but facultative anaerobic bacteria (FAB) might have a relevant role as well. Here we investigated oleate degradation by a syntrophic synthetic co-culture of Syntrophomonas zehnderi (Sz) and Methanobacterium formicicum (Mf) and FAB (two oleate-degrading Pseudomonas spp. I1 + I2). Sz + Mf were first cultivated in a continuous bioreactor under strict anaerobic conditions. Thereafter, I1 + I2 were inoculated and microaerophilic conditions were provided. Methane and acetate were the main degradation products by Sz + Mf in anaerobiosis and by Sz + Mf + I1 + I2 in microaerophilic conditions. However, acetate production from oleate was higher in microaerophilic conditions (5% O2 ) with the four microorganisms together (0.41 ± 0.07 mmol day-1 ) than in anaerobiosis with Sz + Mf (0.23 ± 0.05 mmol day-1 ). Oleate degradation in batch assays was faster by Sz + Mf + I1 + I2 (under microaerophilic conditions) than by Sz + Mf alone (under strict anaerobic conditions). I1 + I2 were able to grow with oleate and with intermediates of oleate degradation (hydrogen, acetate and formate). This work highlights the importance of FAB, particularly Pseudomonas sp., in anaerobic reactors treating oleate-based wastewater, because they accelerate oleate conversion to methane, by protecting strict anaerobes from oxygen toxicity and also by acting as alternative hydrogen/formate and acetate scavengers for LCFA-degrading anaerobes.

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.

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.

A Review of Computational Modeling in Wastewater Treatment Processes.

Wastewater treatment companies are facing several challenges related to the optimization of energy efficiency, meeting more restricted water quality standards, and resource recovery potential. Over the past decades, computational models have gained recognition as effective tools for addressing some of these challenges, contributing to the economic and operational efficiencies of wastewater treatment plants (WWTPs). To predict the performance of WWTPs, numerous deterministic, stochastic, and time series-based models have been developed. Mechanistic models, incorporating physical and empirical knowledge, are dominant as predictive models. However, these models represent a simplification of reality, resulting in model structure uncertainty and a constant need for calibration. With the increasing amount of available data, data-driven models are becoming more attractive. The implementation of predictive models can revolutionize the way companies manage WWTPs by permitting the development of digital twins for process simulation in (near) real-time. In data-driven models, the structure is not explicitly specified but is instead determined by searching for relationships in the available data. Thus, the main objective of the present review is to discuss the implementation of machine learning models for the prediction of WWTP effluent characteristics and wastewater inflows as well as anomaly detection studies and energy consumption optimization in WWTPs. Furthermore, an overview considering the merging of both mechanistic and machine learning models resulting in hybrid models is presented as a promising approach. A critical assessment of the main gaps and future directions on the implementation of mathematical modeling in wastewater treatment processes is also presented, focusing on topics such as the explainability of data-driven models and the use of Transfer Learning processes.

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.

Endurance of methanogenic archaea in anaerobic bioreactors treating oleate-based wastewater.

Methanogenic archaea are reported as very sensitive to lipids and long chain fatty acids (LCFA). Therefore, in conventional anaerobic processes, methane recovery during LCFA-rich wastewater treatment is usually low. By applying a start-up strategy, based on a sequence of step feeding and reaction cycles, an oleate-rich wastewater was efficiently treated at an organic loading rate of 21 kg COD m(-3) day(-1) (50 % as oleate), showing a methane recovery of 72 %. In the present work, the archaeal community developed in that reactor is investigated using a 16S rRNA gene approach. This is the first time that methanogens present in a bioreactor converting efficiently high loads of LCFA to methane are monitored. Denaturing gradient gel electrophoresis profiling showed that major changes on the archaeal community took place during the bioreactor start-up, where phases of continuous feeding were alternated with batch phases. After the start-up, a stable archaeal community (similarity higher than 84 %) was observed and maintained throughout the continuous operation. This community exhibited high LCFA tolerance and high acetoclastic and hydrogenotrophic activity. Cloning and sequencing results showed that Methanobacterium- and Methanosaeta-like microorganisms prevailed in the system and were able to tolerate and endure during prolonged exposure to high LCFA loads, despite the previously reported LCFA sensitivity of methanogens.

Bioaugmentation of sewage sludge with Trametes versicolor in solid-phase biopiles produces degradation of pharmaceuticals and affects microbial communities.

The use of sludge (biosolids) in land application may contribute to the spread of organic micropollutants as wastewater treatments do not completely remove these compounds. Therefore, the development of alternative strategies for sludge treatment is a matter of recent concern. The elimination of pharmaceuticals at pre-existent concentrations from sewage sludge was assessed, for the first time, in nonsterile biopiles by means of fungal bioaugmentation with Trametes versicolor (BTV-systems) and compared with the effect of autochthonous microbiota (NB-systems). The competition between the autochthonous fungal/bacterial communities and T. versicolor was studied using denaturing gradient gel electrophoresis (DGGE) and the cloning/sequencing approach. An inhibitory effect exerted by T. versicolor over bacterial populations was suggested. However, after 21 days, T. versicolor was no longer the main taxon in the fungal communities. The elimination profiles revealed an enhanced removal of atorvastatin-diclofenac-hydrochlorothiazide (during the whole treatment) and ranitidine-fenofibrate (at short periods) in the BTV biopiles in respect to NB biopiles, coincident with the presence of the fungus. For ibuprofen-clarithromycin-furosemide, the elimination profiles were similar irrespective of the system, and with carbamazepine no significant degradation was obtained. The results suggest that a fungal treatment with T. versicolor could be a promising process for the remediation of some pharmaceuticals in complex matrices such as biosolids.

Corksorb Enhances Alkane Degradation by Hydrocarbonoclastic Bacteria.

Biosorbent materials are effective in the removal of spilled oil from water, but their effect on hydrocarbonoclastic bacteria is not known. Here, we show that corksorb, a cork-based biosorbent, enhances growth and alkane degradation by Rhodococcus opacus B4 (Ro) and Alcanivorax borkumensis SK2 (Ab). Ro and Ab degraded 96 ± 1% and 72 ± 2%, respectively, of a mixture of n-alkanes (2 g L-1) in the presence of corksorb. These values represent an increase of 6 and 24%, respectively, relative to the assays without corksorb. The biosorbent also increased the growth of Ab by 51%. However, no significant changes were detected in the expression of genes involved in alkane uptake and degradation in the presence of corksorb relative to the control without the biosorbent. Nevertheless, transcriptomics analysis revealed an increased expression of rRNA and tRNA coding genes, which confirms the higher metabolic activity of Ab in the presence of corksorb. The effect of corksorb is not related to the release of soluble stimulating compounds, but rather to the presence of the biosorbent, which was shown to be essential. Indeed, scanning electron microscopy images and downregulation of pili formation coding genes, which are involved in cell mobility, suggest that cell attachment on corksorb is a determinant for the improved activity. Furthermore, the existence of native alkane-degrading bacteria in corksorb was revealed, which may assist in situ bioremediation. Hence, the use of corksorb in marine oil spills may induce a combined effect of sorption and stimulated biodegradation, with high potential for enhancing in situ bioremediation processes.

Molecular assessment of complex microbial communities degrading long chain fatty acids in methanogenic bioreactors.

Microbial diversity of anaerobic sludge after extended contact with long chain fatty acids (LCFA) was studied using molecular approaches. Samples containing high amounts of accumulated LCFA were obtained after continuous loading of two bioreactors with oleate or with palmitate. These sludge samples were then incubated in batch assays to allow degradation of the biomass-associated LCFA. In addition, sludge used as inoculum for the reactors was also characterized. Predominant phylotypes of the different samples were monitored using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene fragments. Fingerprinting analysis showed changes in bacterial and archaeal communities during LCFA accumulation and degradation. Full-length 16S rRNA gene sequences of 22 clones, representing the predominant bacteria and archaea, were determined. Most bacterial clones (80%) clustered within the Clostridiaceae. Two major groups of methanogens were identified: hydrogen- and formate-utilizing organisms, closely related to Methanobacterium, and acetoclastic organisms closely related to Methanosaeta and Methanosarcina. Quantification by FISH and real-time PCR showed that the relative abundance of archaea increased during degradation of biomass-accumulated LCFA. These results provide insight into the importance and dynamics of balanced communities of bacteria and methanogens in LCFA-accumulation/degradation cycles.

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.

Thermophilic co-digestion of organic fraction of municipal solid wastes with FOG wastes from a sewage treatment plant: reactor performance and microbial community monitoring.

Working at thermophilic conditions instead of mesophilic, and also the addition of a co-substrate, are both the ways to intend to improve the anaerobic digestion of the source-collected organic fraction of municipal solid wastes (SC-OFMSW). Addition of sewage treatment plant fat, oil and grease wastes (STP-FOGW), that are nowadays sent to landfill, would represent an opportunity to recover a wasted methane potential and, moreover, improve the whole process. In this study, after a first period feeding only SC-OFMSW, a co-digestion step was performed maintaining thermophilic conditions. During the co-digestion period enhancements in biogas production (52%) and methane yield (36%) were achieved. In addition, monitoring of microbial structure by using PCR-DGGE and cloning techniques showed that bacterial community profiles clustered in two distinct groups, before and after the extended contact with STP-FOGW, being more affected by the STP-FOGW addition than the archaeal one.

Engineered heat treated methanogenic granules: a promising biotechnological approach for extreme thermophilic biohydrogen production.

In the present study, two granular systems were compared in terms of hydrogen production rate, stability and bacterial diversity under extreme thermophilic conditions (70 degrees C). Two EGSB reactors were individually inoculated with heat treated methanogenic granules (HTG) and HTG amended with enrichment culture with high capacity of hydrogen production (engineered heat treated methanogenic granules - EHTG), respectively. The reactor inoculated with EHTG (R(EHTG)) attained a maximum production rate of 2.7l H(2)l(-1)day(-1) in steady state. In comparison, the R(HTG) containing the HTG granules was very unstable, with low hydrogen productions and only two peaks of hydrogen (0.8 and 1.5l H(2)l(-1)day(-1)). The presence of active hydrogen producers in the R(EHTG) system during the reactor start-up resulted in the development of an efficient H(2)-producing bacterial community. The results showed that "engineered inocula" where known hydrogen producers are co-inoculated with HTG is an efficient way to start up biohydrogen-producing reactors.

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.

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

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.