Volatile fatty acid production from food waste: The effect of retention time and lipid content.

The bioconversion of food waste to renewable products has an important role in alleviating the environmental burden of food wastage. This study evaluates the effect of solids retention time (1.5, 4, and 7 days) and lipid content (up to 30 % DS) on the solid's destruction efficiency and VFA yield from food waste fermentation. Although SRT below 4 days and lipid content beyond 20 % reduced the solids destruction efficiency (SRT -12 %, lipids -13 %), the VFA yield improved (SRT 0.36 to 0.48 g CODVFA/TCODFED; lipids 0.17 to 0.39 g CODVFA/TCODFED). This appeared to be a mechanism of improved acidification which doubled to 0.77 gCODVFA/g SCOD at 1.5-day SRT. The introduction of easily degradable organics in waste oils and methanogen inhibition by LCFAs were likely causes of process instability when lipids >20 %. Further research is needed considering the COD fractionation of the feed to maximize recoverable products on a commercial scale.

Revealing the Metabolic Flexibility of "Candidatus Accumulibacter phosphatis" through Redox Cofactor Analysis and Metabolic Network Modeling.

Environmental fluctuations in the availability of nutrients lead to intricate metabolic strategies. "Candidatus Accumulibacter phosphatis," a polyphosphate-accumulating organism (PAO) responsible for enhanced biological phosphorus removal (EBPR) from wastewater treatment systems, is prevalent in aerobic/anaerobic environments. While the overall metabolic traits of these bacteria are well described, the nonavailability of isolates has led to controversial conclusions on the metabolic pathways used. In this study, we experimentally determined the redox cofactor preferences of different oxidoreductases in the central carbon metabolism of a highly enriched "Ca Accumulibacter phosphatis" culture. Remarkably, we observed that the acetoacetyl coenzyme A reductase engaged in polyhydroxyalkanoate (PHA) synthesis is NADH preferring instead of showing the generally assumed NADPH dependency. This allows rethinking of the ecological role of PHA accumulation as a fermentation product under anaerobic conditions and not just a stress response. Based on previously published metaomics data and the results of enzymatic assays, a reduced central carbon metabolic network was constructed and used for simulating different metabolic operating modes. In particular, scenarios with different acetate-to-glycogen consumption ratios were simulated, which demonstrated optima using different combinations of glycolysis, glyoxylate shunt, or branches of the tricarboxylic acid (TCA) cycle. Thus, optimal metabolic flux strategies will depend on the environment (acetate uptake) and on intracellular storage compound availability (polyphosphate/glycogen). This NADH-related metabolic flexibility is enabled by the NADH-driven PHA synthesis. It allows for maintaining metabolic activity under various environmental substrate conditions, with high carbon conservation and lower energetic costs than for NADPH-dependent PHA synthesis. Such (flexible) metabolic redox coupling can explain the competitiveness of PAOs under oxygen-fluctuating environments.IMPORTANCE Here, we demonstrate how microbial storage metabolism can adjust to a wide range of environmental conditions. Such flexibility generates a selective advantage under fluctuating environmental conditions. It can also explain the different observations reported in PAO literature, including the capacity of "Ca Accumulibacter phosphatis" to act like glycogen-accumulating organisms (GAOs). These observations stem from slightly different experimental conditions, and controversy arises only when one assumes that metabolism can operate only in a single mode. Furthermore, we also show how the study of metabolic strategies is possible when combining omics data with functional cofactor assays and modeling. Genomic information can only provide the potential of a microorganism. The environmental context and other complementary approaches are still needed to study and predict the functional expression of such metabolic potential.

Stress-induced assays for polyphosphate quantification by uncoupling acetic acid uptake and anaerobic phosphorus release.

Phosphorus has been successfully eliminated from wastewater by biological techniques of enhanced biological phosphorus removal (EBPR) process, which relies on a specific microbiota of polyphosphate accumulating organisms (PAOs) that accumulate phosphate as polyphosphates (poly-P). Most methods for quantification of poly-P pools suffer from low accuracy and specificity. More powerful and implementable P-analysis tools are required for poly-P quantification, which will help in improved evaluation of processes in laboratory and full-scale EBPR systems. This study developed two methods to quantify poly-P pools by releasing the poly-P from the cell. During experimental optimization, it was observed that two different methods resulted in the highest phosphate release: acetate addition at a pH of 4.8 and exposure to EDTA solution with a concentration of 1% (w/v). Treatment with EDTA resulted in a higher amount of phosphate release from all sludge samples. This was characterized by P-release of 1.5-2.5 times higher than the control tests. In contrast, treatments with acetate addition at a low pH exhibited that P-release depended upon the types of the sludge samples. The highest P-release amount and rate were found in highly-enriched PAO sludge samples, but with fewer influences on the sludge collected from WWTP, which may be attributed to the lower fraction of PAOs in the sludge. Overall, the proposed approaches to quantify the poly-P concentration can be applied in simple, user-friendly, and cost-effective ways.

Effect of Lactate on the Microbial Community and Process Performance of an EBPR System.

Candidatus Accumulibacter phosphatis is in general presented as the dominant organism responsible for the biological removal of phosphorus in activated sludge wastewater treatment plants. Lab-scale enhanced biological phosphorus removal (EBPR) studies, usually use acetate as carbon source. However, the complexity of the carbon sources present in wastewater could allow other potential poly-phosphate accumulating organism (PAOs), such as putative fermentative PAOs (e.g., Tetrasphaera), to proliferate in coexistence or competition with Ca. Accumulibacter. This research assessed the effects of lactate on microbial selection and process performance of an EBPR lab-scale study. The addition of lactate resulted in the coexistence of Ca. Accumulibacter and Tetrasphaera in a single EBPR reactor. An increase in anaerobic glycogen consumption from 1.17 to 2.96 C-mol/L and anaerobic PHV formation from 0.44 to 0.87 PHV/PHA C-mol/C-mol corresponded to the increase in the influent lactate concentration. The dominant metabolism shifted from a polyphosphate-accumulating metabolism (PAM) to a glycogen accumulating metabolism (GAM) without EBPR activity. However, despite the GAM, traditional glycogen accumulating organisms (GAOs; Candidatus Competibacter phosphatis and Defluvicoccus) were not detected. Instead, the 16s RNA amplicon analysis showed that the genera Tetrasphaera was the dominant organism, while a quantification based on FISH-biovolume indicated that Ca. Accumulibacter remained the dominant organism, indicating certain discrepancies between these microbial analytical methods. Despite the discrepancies between these microbial analytical methods, neither Ca. Accumulibacter nor Tetrasphaera performed biological phosphorus removal by utilizing lactate as carbon source.

Effects of electron acceptors on sulphate reduction activity in activated sludge processes.

The concentration of sulphate present in wastewater can vary from 10 to 500 mg SO42-/L. During anaerobic conditions, sulphate is reduced to sulphide by sulphate-reducing bacteria (SRB). Sulphide generation is undesired in wastewater treatment plants (WWTPs). Previous research indicated that SRB are inhibited by the presence of electron acceptors (such as O2, NO3 and NO2). However, the contact times and concentrations used in those studies are by far higher than occur in WWTPs. Since sulphide can influence the biological nitrogen and phosphorus removal processes, this research aimed to understand how the different electron acceptors commonly present in biological nutrient removal (BNR) systems can affect the proliferation of SRB. For this purpose, a culture of SRB was enriched in a sequencing batch reactor (approx. 88% of the total bacteria population). Once enriched, the SRB were exposed for 2 h to typical concentrations of electron acceptors like those observed in BNR systems. Their activity was assessed using three different types of electron donors (acetate, propionate and lactate). Oxygen was the most inhibiting electron acceptor regardless the carbon source used. After exposure to oxygen and when feeding acetate, an inactivation time in the sulphate reduction activity was observed for 1.75 h. Once the sulphate reduction activity resumed, only 60% of the original activity was recovered. It is suggested that the proliferation of SRB is most likely to occur in BNR plants with an anaerobic fraction higher than 15% and operating at sludge retention times higher than 20 days (at a temperature of 20 °C). These results can be used to implement strategies to control the growth of sulphate reducers that might compete for organic carbon with phosphate-accumulating organisms.

Denitrification of nitrate and nitrite by 'Candidatus Accumulibacter phosphatis' clade IC.

Phosphate accumulating organisms (PAO) are assumed to use nitrate as external electron acceptor, allowing an efficient integration of simultaneous nitrogen and phosphate removal with minimal organic carbon (COD) requirements. However, contradicting findings appear in literature regarding the denitrification capacities of PAO due to the lack of clade specific highly enriched PAO cultures. Whereas some studies suggest that only PAO clade I may be capable of using nitrate as external electron acceptor for anoxic P-uptake, other studies indicate that PAO clade II may be responsible for anoxic P-removal. In the present study, a highly enriched PAO clade IC culture (>99% according to FISH) was cultivated in an SBR operated under Anaerobic/Oxic conditions and subsequently exposed to Anaerobic/Anoxic/Oxic conditions using nitrate as electron acceptor. Before and after acclimatization to the presence of nitrate, the aerobic and anoxic (nitrate and nitrite) activities of the PAO I culture were assessed through the execution of batch tests using either acetate or propionate as electron donor. In the presence of nitrate, significant P-uptake by PAO I was not observed before or after acclimatization. Using nitrite as electron acceptor, limited nitrite removal rates were observed before acclimatization with lower rates in the acetate fed reactor without P-uptake and slightly higher in the propionate fed reactor with a marginal anoxic P-uptake. Only after acclimatization to nitrate, simultaneous P and nitrite removal was observed. This study suggests that PAO clade IC is not capable of using nitrate as external electron acceptor for anoxic P-removal. The elucidation of the metabolic capacities for individual PAO clades helps in better understanding and optimization of the relation between microbial ecology and process performance in enhanced biological phosphate removal processes.

Metabolic Response of "Candidatus Accumulibacter Phosphatis" Clade II C to Changes in Influent P/C Ratio.

The objective of this study was to investigate the ability of a culture highly enriched with the polyphosphate-accumulating organism, "Candidatus Accumulibacter phosphatis" clade IIC, to adjust their metabolism to different phosphate availabilities. For this purpose the biomass was cultivated in a sequencing batch reactor with acetate and exposed to different phosphate/carbon influent ratios during six experimental phases. Activity tests were conducted to determine the anaerobic kinetic and stoichiometric parameters as well as the composition of the microbial community. Increasing influent phosphate concentrations led to increased poly-phosphate content and decreased glycogen content of the biomass. In response to higher biomass poly-phosphate content, the biomass showed higher specific phosphate release rates. Together with the phosphate release rates, acetate uptake rates also increased up to an optimal poly-phosphate/glycogen ratio of 0.3 P-mol/C-mol. At higher poly-phosphate/glycogen ratios (obtained at influent P/C ratios above 0.051 P-mol/C-mol), the acetate uptake rates started to decrease. The stoichiometry of the anaerobic conversions clearly demonstrated a metabolic shift from a glycogen dominated to a poly-phosphate dominated metabolism as the biomass poly-phosphate content increased. FISH and DGGE analyses confirmed that no significant changes occurred in the microbial community, suggesting that the changes in the biomass activity were due to different metabolic behavior, allowing the organisms to proliferate under conditions with fluctuating phosphate levels.