Volatile Fatty Acid Uptake Research Articles

Towards maximization of parameters identifiability: Development of the CalOpt tool and its application to the anaerobic digestion model

This work addresses the need for robust approaches to estimate kinetic parameters in continuous anaerobic co-digestion plants. Specifically, it outlines a procedure for including data from batch activity tests on digestate samples, in addition to the poorly informative dataset from conventional monitoring of biogas facilities. To profitably make use of activity tests, the estimation of the biomass composition in digestate samples is required to improve parameter identifiability. To this purpose, an open-source tool (CalOpt) was developed and tested on a pilot-scale co-digester, simultaneously using data from both conventional monitoring and lab-scale activity tests. Targeting the prediction of volatile fatty acids (VFAs) concentrations in the digestate of the continuous co-digester, the kinetic parameters of different microbial groups were selected for calibration based on parameters importance ranking and collinearity index. The results demonstrate that the CalOpt tool significantly improved the fitting of acetic, propionic, and butyric acid concentrations in digestate as well as the methane flowrate from the digester. Furthermore, the study assessed the necessity of modifying the Monod kinetics to account for substrate inhibition by incorporating the Haldane term in the VFAs uptake kinetic model, which proved to be essential for better interpreting batch activity tests.

The impact of auxin analogues on microalgal intracellular component accumulation and nutrient removal for mariculture wastewater treatment basing on bacterial-algal coupling technology

Acidogenic fermentation coupled with microalgae cultivation is a promising approach for mariculture wastewater (MW) treatment. However, the low biomass and lipid production of microalgae restrict its practical application. In this study, auxin analogues including α-naphthylacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D) and indomethacin were applied to the cultivation of Chlorella pyrenoidosa under mixotrophic condition. The effects of auxin analogues on microalgal growth, intracellular component accumulation and nutrient removal were evaluated. The optimal growth and intracellular composition accumulation of Chlorella pyrenoidosa were obtained at 2 mg L−1 NAA, 1 mg L−1 indomethacin, and 1 mg L−1 2,4-D. NAA was more efficient than indomethacin and 2,4-D for lipid production, and the maximum lipid accumulation reached 52% with 2 mg L−1 NAA. Auxin analogues enhanced the uptake of volatile fatty acids, ammonium and phosphate from acidogenic fermentation effluent, attributed to the improvement of microalgal growth. This study provided a new insight for enhancing mariculture wastewater treatment with mixotrophic microalgae.

Carbon uptake bioenergetics of PAOs and GAOs in full-scale enhanced biological phosphorus removal systems

This work analyzed, for the first time, the bioenergetics of PAOs and GAOs in full-scale wastewater treatment plants (WWTPs) for the uptake of different carbon sources. Fifteen samples were collected from five full-scale WWTPs. Predominance of different PAOs, i.e., Ca. Accumulibacter (0.00–0.49%), Tetrasphaera (0.37–3.94%), Microlunatus phosphovorus (0.01–0.18%), etc., and GAOs, i.e., Ca. Competibacter (0.08–5.39%), Defluviicoccus (0.05–5.34%), Micropruina (0.17–1.87%), etc., were shown by 16S rRNA gene amplicon sequencing. Despite the distinct PAO/GAO community compositions in different samples, proton motive force (PMF) was found as the key driving force (up to 90.1%) for the uptake of volatile fatty acids (VFAs, acetate and propionate) and amino acids (glutamate and aspartate) by both GAOs and PAOs at the community level, contrasting the previous understanding that Defluviicoccus have a low demand of PMF for acetate uptake. For the uptake of acetate or propionate, PAOs rarely activated F1, F0- ATPase (< 11.7%) or fumarate reductase (< 5.3%) for PMF generation; whereas, intensive involvements of these two pathways (up to 49.2% and 61.0%, respectively) were observed for GAOs, highlighting a major and community-level difference in their VFA uptake biogenetics in full-scale systems. However, different from VFAs, the uptake of glutamate and aspartate by both PAOs and GAOs commonly involved fumarate reductase and F1, F0-ATPase activities. Apart from these major and community-level differences, high level fine-scale micro-diversity in carbon uptake bioenergetics was observed within PAO and GAO lineages, probably resulting from their versatilities in employing different pathways for reducing power generation. Ca. Accumulibacter and Halomonas seemed to show higher dependency on the reverse operation of F1, F0-ATPase than other PAOs, likely due to the low involvement of glyoxylate shunt pathway. Unlike Tetrasphaera, but similar to Ca. Accumulibacter, Microlunatus phosphovorus took up glutamate and aspartate via the proton/glutamate-aspartate symporter driven by PMF. This feature was testified using a pure culture of Microlunatus phosphovorus stain NM-1. The major difference between PAOs and GAOs highlights the potential to selectively suppress GAOs for community regulation in EBPR systems. The finer-scale carbon uptake bioenergetics of PAOs or GAOs from different lineages benefits in understanding their interactions in community assembly in complex environment.

Investigation of fiber utilization in the rumen of dairy cows based on metagenome-assembled genomes and single-cell RNA sequencing

BackgroundDairy cows utilize human-inedible, low-value plant biomass to produce milk, a low-cost product with rich nutrients and high proteins. This process largely relies on rumen microbes that ferment lignocellulose and cellulose to produce volatile fatty acids (VFAs). The VFAs are absorbed and partly metabolized by the stratified squamous rumen epithelium, which is mediated by diverse cell types. Here, we applied a metagenomic binning approach to explore the individual microbes involved in fiber digestion and performed single-cell RNA sequencing on rumen epithelial cells to investigate the cell subtypes contributing to VFA absorption and metabolism.ResultsThe 52 mid-lactating dairy cows in our study (parity = 2.62 ± 0.91) had milk yield of 33.10 ± 6.72 kg. We determined the fiber digestion and fermentation capacities of 186 bacterial genomes using metagenomic binning and identified specific bacterial genomes with strong cellulose/xylan/pectin degradation capabilities that were highly associated with the biosynthesis of VFAs. Furthermore, we constructed a rumen epithelial single-cell map consisting of 18 rumen epithelial cell subtypes based on the transcriptome of 20,728 individual epithelial cells. A systematic survey of the expression profiles of genes encoding candidates for VFA transporters revealed that IGFBP5+ cg-like spinous cells uniquely highly expressed SLC16A1 and SLC4A9, suggesting that this cell type may play important roles in VFA absorption. Potential cross-talk between the microbiome and host cells and their roles in modulating the expression of key genes in the key rumen epithelial cell subtypes were also identified.ConclusionsWe discovered the key individual microbial genomes and epithelial cell subtypes involved in fiber digestion, VFA uptake and metabolism, respectively, in the rumen. The integration of these data enables us to link microbial genomes and epithelial single cells to the trophic system.1dVKuikNrJ1xsEB1JvqD2DVideo abstract

Defluviicoccus vanus Glycogen-Accumulating Organisms (DvGAOs) Are Less Competitive Than Polyphosphate-Accumulating Organisms (PAOs) at High Temperature

Glycogen-accumulating organisms (GAOs) have been previously observed to be more competitive in anaerobic volatile fatty acid (VFA) uptake as compared to polyphosphate-accumulating organisms (PAOs) at high temperatures in enhanced biological phosphorus removal (EBPR) systems. However, previous evidence regarding the impact of temperature on GAOs has exclusively focused on Competibacter. Defluviicoccus vanus GAOs (DvGAOs) make up another very important GAO group and have been found in EBPR plants; however, their competitiveness at high temperature for anaerobic VFA uptake was previously unknown. The rate of uptake of VFAs by both DvGAOs reactors enriched with acetate or propionate was much lower than those of Candidatus Accumulibacter phosphatis PAOs operated under similar conditions, showing that PAOs displayed a competitive advantage at high temperature, unlike previous observations with Competibacter. This study provides new insight into our understanding of GAOs in EBPR processes, where the impact of temperature on PAO-GAO competition is highly dependent on the phylogenetic identity of the GAO present in the system. New strategies for promoting EBPR performance at high temperatures are proposed on the basis of the results of this work.

Infusion of sodium butyrate promotes rumen papillae growth and enhances expression of genes related to rumen epithelial VFA uptake and metabolism in neonatal twin lambs.

The objective of this study was to evaluate the effect of sodium butyrate (SB) infusion on rumen papillae growth and volatile fatty acid (VFA) uptake and metabolism in neonatal lambs. Seven pairs of newborn twin lambs were used. Within each pair, lambs were assigned to receive an oral infusion of SB at 0.36 g/kg body weight (BW) (SB, n = 7) or the same volume of saline (Con, n = 7). Treatments were administered from 10 to 49 d of age, when all lambs were slaughtered. Results showed that the average daily feed intake (ADFI) of starter, average daily gain (ADG), BW of lambs at ages of 5 and 6 wk in SB group were greater (P < 0.05) than those in Con group. Infusion of SB increased (P < 0.05) the concentrations of acetate, butyrate, and total VFA in the rumen fluid and elevated (P < 0.05) the levels of β-hydroxybutyrate acid (BHBA), insulin-like growth factor-1 (IGF-1), and insulin in plasma. Infusion of SB promoted rumen papillae growth, depicted by higher emptied rumen weight, larger rumen papillae length, width, and surface area, and greater thickness of stratum corneum and total epithelium. Sodium butyrate infusion upregulated (P < 0.05) mRNA expression of cyclin A2, cyclin D1, and cyclin-dependent kinases 6 (CDK6), and downregulated (P < 0.05) mRNA expression of caspase-3 and Bcl-2-associated X protein (Bax) in the rumen epithelia. Moreover, SB infusion also upregulated (P < 0.05) mRNA expression of insulin-like growth factor-1 receptor (IGF-1R), and insulin-like growth factor-binding protein 5 (IGFBP-5), and downregulated (P < 0.05) mRNA expression of insulin-like growth factor-binding protein 3 (IGFBP-3) in the rumen epithelia. Sodium butyrate infusion also enhanced (P < 0.05) gene expressions of monocarboxylate transporter isoform 1 (MCT1), downregulated in adenoma (DRA), 3-hydroxy-3-methylglutaryl-CoA synthase isoform 2 (HMGCS2), and 3-hydroxy-3-methylglutaryl-CoA lyase (HMGCL), while depressed (P < 0.05) mRNA expression of sodium/proton exchanger isoform 2 (NHE2) in the rumen epithelia. Our results suggest that the SB infusion can improve animal performance, promote the ruminal papillae growth, and enhance expression of genes related to ruminal epithelial VFA uptake and metabolism in preweaning twin lambs. These findings provide a better understanding of the molecular mechanism of SB promoting rumen epithelial development and function in preweaning lambs.

Kinetic parameters of volatile fatty acids uptake in the ADM1 as key factors for modeling co-digestion of silages with pig manure, thin stillage and glycerine phase

Abstract The present study extended the Anaerobic Digestion Model No. 1 (ADM1) to simulate co-digestion of silages with pig manure and thin stillage, pig manure and glycerine phase, or thin stillage and glycerine phase under mesophilic conditions. New state variables were added, including glycerol, lactate, valerate and iso-valerate. Furthermore, the model was extended to include both the rapidly and slowly degradable fractions of carbohydrates and proteins. It was found that the model is more sensitive to changes in the constant for slowly degraded carbohydrates than to changes in the constant for rapidly degraded carbohydrates. After parameter calibration with the mixture of silages with pig manure and thin stillage, the ADM1 showed good agreement with measurements of daily biogas and methane production, and of concentrations of individual volatile fatty acids (VFAs) in the effluent. Although, initially, the prediction of biogas production with the silages mixture with thin stillage and glycerine phase was not satisfactory because the calibrated KI_h2_pro value (2.86E-08 kg COD/m3) was too low, adjusting this constant to a value of 9.70E-08 kg COD/m3 enabled this to be predicted with high accuracy.

High solid temperature phased anaerobic digestion from agricultural wastes: Putting several reactors in sequence

High solid anaerobic digestion of agricultural wastes (manure and grass silage) was tested on temperature phased anaerobic digester (TPAD) at lab-scale. A 4days hyperthermophilic hydrolysis was followed by a 15days methanogenesis. The TPAD was divided into 4 or 5 tanks in series without recycling: 2 sectors for hydrolysis (2days HRT each) and 2 or 3 sectors for methanogenesis (5+10 or 3×5days HRT). Two different combinations of temperatures (65°C–37°C, 65°C–55°C) were investigated to assess the process efficiency.For all tested conditions, the TPAD operated steadily with a methane productivity of 235+/−3 mLCH4STPgVS−1. Using four or five reactors in series enabled to identify that during the two first days of hydrolysis, the hydrolysis yield and acidification yield were much higher (22% and 13%, respectively) than during the next two days (2% and 2% respectively). Moreover, concerning methanogenesis it appeared that the retention time may be shortened by 5days with little loss in methane production (11%). In the methanogenic sectors, the uptake of volatile fatty acids and the methane production were higher at 55°C than 37°C.The results suggest that the hydrolytic sector could be limited at 2days HRT, while the methanogenic sector would support a 10days HRT with the feedstocks used.

Sulphide effects on the physiology of Candidatus Accumulibacter phosphatis type I.

Sulphate-rich wastewaters can be generated due to (i) use of saline water as secondary-quality water for sanitation in urban environments (e.g. toilet flushing), (ii) discharge of industrial effluents, (iii) sea and brackish water infiltration into the sewage and (iv) use of chemicals, which contain sulphate, in drinking water production. In the presence of an electron donor and absence of oxygen or nitrate, sulphate can be reduced to sulphide. Sulphide can inhibit microbial processes in biological wastewater treatment systems. The objective of the present study was to assess the effects of sulphide concentration on the anaerobic and aerobic physiology of polyphosphate-accumulating organisms (PAOs). For this purpose, a PAO culture, dominated by Candidatus Accumulibacter phosphatis clade I (PAO I), was enriched in a sequencing batch reactor (SBR) fed with acetate and propionate. To assess the direct inhibition effects and their reversibility, a series of batch activity tests were conducted during and after the exposure of a PAO I culture to different sulphide concentrations. Sulphide affected each physiological process of PAO I in a different manner. At 189mg TS-S/L, volatile fatty acid uptake was 55% slower and the phosphate release due to anaerobic maintenance increased from 8 to 18mg PO4-P/g VSS/h. Up to 8mg H2S-S/L, the decrease in aerobic phosphorus uptake rate was reversible (Ic60). At higher concentrations of sulphide, potassium (>16mg H2S-S/L) and phosphate (>36mg H2S-S/L) were released under aerobic conditions. Ammonia uptake, an indicator of microbial growth, was not observed at any sulphide concentration. This study provides new insights into the potential failure of enhanced biological phosphorus removal sewage plants receiving sulphate- or sulphide-rich wastewaters when sulphide concentrations exceed 8mg H2S-S/L, as PAO I could be potentially inhibited.

Polyhydroxyalkanoate synthesis by mixed microbial consortia cultured on fermented dairy manure: Effect of aeration on process rates/yields and the associated microbial ecology

Polyhydroxyalkanoates (PHAs) are biodegradable polymers that can substitute for petroleum-based plastics in a variety of applications. One avenue to commercial PHA production involves coupling waste-based synthesis with the use of mixed microbial consortia (MMC). In this regard, production requires maximizing the enrichment of a MMC capable of feast-famine PHA synthesis, with the metabolic response induced through imposition of aerobic-dynamic feeding (ADF) conditions. However, the concept of PHA production in complex matrices remains unrefined; process operational improvements are needed, along with an enhanced understanding of the MMC. Research presented herein investigated the effect of aeration on feast-famine PHA synthesis, with four independent aeration state systems studied; MMC were fed volatile fatty acid (VFA)-rich fermented dairy manure. Regardless of the aeration state, all MMC exhibited a feast-famine response based on observed carbon cycling. Moreover, there was no statistical difference in PHA synthesis rates, with qPHA ranging from 0.10 to 0.19 CmmolPHA gVSS−1 min−1; VFA uptake rates exhibited similar statistical indifferences. PHA production assessments on the enriched MMC resulted in maximum intracellular concentrations ranging from 22.5 to 90.7% (mgPHA mgVSS−1); at maximum concentration, the mean hydroxyvalerate mol content was 73 ± 0.6%. While a typical feast-famine dissolved oxygen (DO) pattern was observed at maximum aeration, less resolution was observed at decreasing aeration rates, suggesting that DO may not be an optimal process monitoring parameter. At lower aeration states, nitrogen cycling patterns, supported by molecular investigations targeting AOBs and NOBs, indicate that NO2 and NO3 sustained feast-famine PHA synthesis. Next-generation sequencing analysis of the respective MMC revealed numerous and diverse genera exhibiting the potential to achieve PHA synthesis, suggesting functional redundancy embedded in the diverse MMC. Ultimately, results demonstrate that aeration can be controlled in waste-based ADF systems to sustain PHA production potential, while enriching for a diverse MMC that exhibits potential functional redundancy. Reduced aeration could also enhance cost competitiveness of waste-based PHA production, with potential further benefits associated with nitrogen treatment.

Influence of CeO2 NPs on biological phosphorus removal and bacterial community shifts in a sequencing batch biofilm reactor with the differential effects of molecular oxygen

The effects of CeO2 nanoparticles (CeO2 NPs) on a sequencing batch biofilm reactor (SBBR) with established biological phosphorus (P) removal were investigated from the processes of anaerobic P release and aerobic P uptake. At low concentration (0.1mg/L), no significant impact was observed on total phosphorus (TP) removal after operating for 8h. However, at a concentration of 20mg/L, TP removal efficiency decreased from 83.68% to 55.88% and 16.76% when the CeO2 NPs were added at the beginning of the anaerobic and aerobic periods, respectively. Further studies illustrated that the inhibition of the specific P release rate was caused by the reversible states of Ce3+ and Ce4+, which inhibited the activity of exopolyphosphatase (PPX) and transformation of poly-β-hydoxyalkanoates (PHA) and glycogen, as well as the uptake of volatile fatty acids (VFAs). The decrease in the specific P uptake rate was mainly attributed to the significantly suppressed energy generation and decreased abundance of Burkholderia caused by excess reactive oxygen species. The removal of chemical oxygen demand (COD) was not influenced by CeO2 NPs under aerobic conditions, due to the increased abundance of Acetobacter and Acidocella after exposure. The inhibitory effects of CeO2 NPs with molecular oxygen were reduced after anaerobic exposure due to the enhanced particle size and the presence of Ce3+.

Modelling phosphorus (P), sulfur (S) and iron (Fe) interactions for dynamic simulations of anaerobic digestion processes

This paper proposes a series of extensions to functionally upgrade the IWA Anaerobic Digestion Model No. 1 (ADM1) to allow for plant-wide phosphorus (P) simulation. The close interplay between the P, sulfur (S) and iron (Fe) cycles requires a substantial (and unavoidable) increase in model complexity due to the involved three-phase physico-chemical and biological transformations. The ADM1 version, implemented in the plant-wide context provided by the Benchmark Simulation Model No. 2 (BSM2), is used as the basic platform (A0). Three different model extensions (A1, A2, A3) are implemented, simulated and evaluated. The first extension (A1) considers P transformations by accounting for the kinetic decay of polyphosphates (XPP) and potential uptake of volatile fatty acids (VFA) to produce polyhydroxyalkanoates (XPHA) by phosphorus accumulating organisms (XPAO). Two variant extensions (A2,1/A2,2) describe biological production of sulfides (SIS) by means of sulfate reducing bacteria (XSRB) utilising hydrogen only (autolithotrophically) or hydrogen plus organic acids (heterorganotrophically) as electron sources, respectively. These two approaches also consider a potential hydrogen sulfide (ZH2S) inhibition effect and stripping to the gas phase (GH2S). The third extension (A3) accounts for chemical iron (III) (SFe3+) reduction to iron (II) (SFe2+) using hydrogen (SH2) and sulfides (SIS) as electron donors. A set of pre/post interfaces between the Activated Sludge Model No. 2d (ASM2d) and ADM1 are furthermore proposed in order to allow for plant-wide (model-based) analysis and study of the interactions between the water and sludge lines. Simulation (A1 – A3) results show that the ratio between soluble/particulate P compounds strongly depends on the pH and cationic load, which determines the capacity to form (or not) precipitation products. Implementations A1 and A2,1/A2,2 lead to a reduction in the predicted methane/biogas production (and potential energy recovery) compared to reference ADM1 predictions (A0). This reduction is attributed to two factors: (1) loss of electron equivalents due to sulfate (SSO4) reduction by XSRB and storage of XPHA by XPAO; and, (2) decrease of acetoclastic and hydrogenotrophic methanogenesis due to ZH2S inhibition. Model A3 shows the potential for iron to remove free SIS (and consequently inhibition) and instead promote iron sulfide (XFeS) precipitation. It also reduces the quantities of struvite (XMgNH4PO4) and calcium phosphate (XCa3(PO4)2) that are formed due to its higher affinity for phosphate anions. This study provides a detailed analysis of the different model assumptions, the effect that operational/design conditions have on the model predictions and the practical implications of the proposed model extensions in view of plant-wide modelling/development of resource recovery strategies.

Formate, acetate, and propionate as substrates for sulfate reduction in sub-arctic sediments of Southwest Greenland.

Volatile fatty acids (VFAs) are key intermediates in the anaerobic mineralization of organic matter in marine sediments. We studied the role of VFAs in the carbon and energy turnover in the sulfate reduction zone of sediments from the sub-arctic Godthåbsfjord (SW Greenland) and the adjacent continental shelf in the NE Labrador Sea. VFA porewater concentrations were measured by a new two-dimensional ion chromatography-mass spectrometry method that enabled the direct analysis of VFAs without sample pretreatment. VFA concentrations were low and surprisingly constant (4–6 μmol L−1 for formate and acetate, and 0.5 μmol L−1 for propionate) throughout the sulfate reduction zone. Hence, VFAs are turned over while maintaining a stable concentration that is suggested to be under a strong microbial control. Estimated mean diffusion times of acetate between neighboring cells were <1 s, whereas VFA turnover times increased from several hours at the sediment surface to several years at the bottom of the sulfate reduction zone. Thus, diffusion was not limiting the VFA turnover. Despite constant VFA concentrations, the Gibbs energies (ΔGr) of VFA-dependent sulfate reduction decreased downcore, from −28 to −16 kJ (mol formate)−1, −68 to −31 kJ (mol acetate)−1, and −124 to −65 kJ (mol propionate)−1. Thus, ΔGr is apparently not determining the in-situ VFA concentrations directly. However, at the bottom of the sulfate zone of the shelf station, acetoclastic sulfate reduction might operate at its energetic limit at ~ −30 kJ (mol acetate)−1. It is not clear what controls VFA concentrations in the porewater but cell physiological constraints such as energetic costs of VFA activation or uptake could be important. We suggest that such constraints control the substrate turnover and result in a minimum ΔGr that depends on cell physiology and is different for individual substrates.

The metabolic versatility of PAOs as an opportunity to obtain a highly P-enriched stream for further P-recovery

The effects of two sequencing batch reactor operation strategies for phosphorus stream enrichment over the biological phosphorus removal performance have been studied. The objective of both strategies is of performing an extraction cycle in order to obtain a new stream highly enriched with phosphorus. In the 1st strategy the amount of influent volatile fatty acids (VFAs) is the same in each cycle; while in the 2nd strategy the influent VFAs concentration is increased during phosphorus extraction experiments. Despite the strong decrease of the stored poly-P inside the cells in both strategies after the recovery cycles, the ability of the systems to remove phosphorus was not affected. The Prelease/HAcuptake ratio (changing from 0.73 to 0.21mmolPmmolC−1) together with FISH analyses (around 85% of Accumulibacter through the experimental period) confirmed that a shift from PAM to GAM occurred after phosphorus enrichment in the 2nd strategy experiments. These results suggest that energy required for VFA uptake by polyphosphate-accumulating organisms (PAOs) was not only derived from polyphosphates degradation, but also from glycogen degradation. FISH also revealed that Type II Accumulibacter species are responsible of the metabolic shift. The strategy based on increasing influent VFAs concentration during phosphorus extraction experiments showed a higher extraction efficiency (from 46% to 76%), as higher phosphorus concentration within supernatant can be achieved (from 113.9 to 198.7mgPl−1). Following this strategy, it is possible to concentrate up to 81% of the incoming phosphorus in a single enriched stream. This suggests that, despite the extra addition of carbon source needed (9%), this strategy is more appropriate if phosphorus recovery for reuse purposes is required.

Modelling the metabolic shift of polyphosphate-accumulating organisms

Enhanced biological phosphorus removal (EBPR) is one of the most important methods of phosphorus removal in municipal wastewater treatment plants, having been described by different modelling approaches. In this process, the PAOs (polyphosphate accumulating organisms) and GAOs (glycogen accumulating organisms) compete for volatile fatty acids uptake under anaerobic conditions. Recent studies have revealed that the metabolic pathways used by PAOs in order to obtain the energy and the reducing power needed for polyhydroxyalkanoates synthesis could change depending on the amount of polyphosphate stored in the cells. The model presented in this paper extends beyond previously developed metabolic models by including the ability of PAO to change their metabolic pathways according to the content of poly-P available. The processes of the PAO metabolic model were adapted to new formulations enabling the change from P-driven VFA uptake to glycogen-driven VFA uptake using the same process equations. The stoichiometric parameters were changed from a typical PAO coefficient to a typical GAO coefficient depending on the internal poly-P with Monod-type expressions. The model was calibrated and validated with seven experiments under different internal poly-P concentrations, showing the ability to correctly represent the PAO metabolic shift at low poly-P concentrations. The sensitivity and error analysis showed that the model is robust and has the ability to describe satisfactorily the change from one metabolic pathway to the other one, thereby encompassing a wider range of process conditions found in EBPR plants.

Dynamic metabolic modelling of volatile fatty acids conversion to polyhydroxyalkanoates by a mixed microbial culture

In this work, we present a dynamic metabolic model that describes the uptake of complex mixtures of volatile fatty acids (VFA) and respective conversion into PHA by mixed microbial cultures (MMC). This model builds upon a previously published flux balance analysis model [1] that identified the minimization of TCA cycle activity as the key metabolic objective to predict PHA storage fluxes and respective composition. The model was calibrated either with experimental data of PHA production from fermented sugar cane molasses or from synthetic mixtures of VFA. All PHA production experiments were performed using a MMC selected with fermented sugar cane molasses under feast and famine regimen. The model was able to capture the process dynamics denoted by an excellent fit between experimental and computed time profiles of concentrations with the regression coefficients always above 0.92. The introduced VFA uptake regulatory factor reflects the decrease of acetyl-CoA and propionyl-CoA available to TCA cycle in conformity with the hypothesis that the minimization of TCA cycle is a key metabolic objective for MMC subjected to feast and famine regimen for the maximization of PHA production.

Assessment of bacterial and structural dynamics in aerobic granular biofilms.

Aerobic granular sludge (AGS) is based on self-granulated flocs forming mobile biofilms with a gel-like consistence. Bacterial and structural dynamics from flocs to granules were followed in anaerobic-aerobic sequencing batch reactors (SBR) fed with synthetic wastewater, namely a bubble column (BC-SBR) operated under wash-out conditions for fast granulation, and two stirred-tank enrichments of Accumulibacter (PAO-SBR) and Competibacter (GAO-SBR) operated at steady-state. In the BC-SBR, granules formed within 2 weeks by swelling of Zoogloea colonies around flocs, developing subsequently smooth zoogloeal biofilms. However, Zoogloea predominance (37–79%) led to deteriorated nutrient removal during the first months of reactor operation. Upon maturation, improved nitrification (80–100%), nitrogen removal (43–83%), and high but unstable dephosphatation (75–100%) were obtained. Proliferation of dense clusters of nitrifiers, Accumulibacter, and Competibacter from granule cores outwards resulted in heterogeneous bioaggregates, inside which only low abundance Zoogloea (<5%) were detected in biofilm interstices. The presence of different extracellular glycoconjugates detected by fluorescence lectin-binding analysis showed the complex nature of the intracellular matrix of these granules. In the PAO-SBR, granulation occurred within two months with abundant and active Accumulibacter populations (56 ± 10%) that were selected under full anaerobic uptake of volatile fatty acids and that aggregated as dense clusters within heterogeneous granules. Flocs self-granulated in the GAO-SBR after 480 days during a period of over-aeration caused by biofilm growth on the oxygen sensor. Granules were dominated by heterogeneous clusters of Competibacter (37 ± 11%). Zoogloea were never abundant in biomass of both PAO- and GAO-SBRs. This study showed that Zoogloea, Accumulibacter, and Competibacter affiliates can form granules, and that the granulation mechanisms rely on the dominant population involved.

Flux balance analysis of mixed microbial cultures: Application to the production of polyhydroxyalkanoates from complex mixtures of volatile fatty acids

Fermented agro-industrial wastes are potential low cost substrates for polyhydroxyalkanoates (PHA) production by mixed microbial cultures (MMC). The use of complex substrates has however profound implications in the PHA metabolism. In this paper we investigate PHA accumulation using a lumped metabolic model that describes PHA storage from arbitrary mixtures of volatile fatty acids (VFA). Experiments were conducted using synthetic and complex VFA mixtures obtained from the fermentation of sugar cane molasses. Metabolic flux analysis (MFA) and flux balance analysis (FBA) were performed at different stages of culture enrichment in order to investigate the effect of VFA composition and time of enrichment in PHA storage efficiency. Substrate uptake and PHA storage fluxes increased over enrichment time by 70% and 73%, respectively. MFA calculations show that higher PHA storage fluxes are associated to an increase in the uptake of VFA with even number of carbon atoms and a more effective synthesis of hydroxyvalerate (HV) precursors from VFA with odd number of carbons. Furthermore, FBA shows that the key metabolic objective of a MMC subjected to the feast and famine regimen is the minimization of the tricarboxylic acid cycle fluxes. The PHA flux and biopolymer composition (hydroxybutyrate (HB): HV) could be accurately predicted in several independent experiments.

Phosphorus release and uptake during start-up of a covered and non-aerated sequencing batch reactor with separate feeding of VFA and sulfate

This study explored a sulfur cycle-associated biological phosphorus (P) removal process in a covered and non-aerated sequencing batch reactor (SBR) fed with volatile fatty acid (VFA) and sulfate separately. During the 60-day start-up, both phosphate release and uptake rates increased, while poly-phosphate cyclically increased and decreased accordingly. The P-release and P-uptake rates were associated with VFA uptake and sulfate reduction. The average ratio of potassium to phosphate during the P-uptake and P-release was also determined to be 0.29-0.31 mol K/mol P, which is close to a reported value (0.33) for biological phosphorus removal. All this evidence confirmed there was biological P removal in this reactor, in which metabolism could be different from conventional biological P removal.

Influence of Substrate Load on Polyhydroxyalkanoates (Pha) Accumulation by Unenriched Mixed Cultures from Excess Sludge Fermentation Liquid

To reduce the production cost of polyhydroxyalkanoates (PHA) and disposal amount of excess sludge simultaneously, volatile fatty acids (VFAs) from alkaline excess sludge fermentation was used as carbon sources to synthesize PHA by unenriched mixed cultures. Released phosphorus and residual ammonium in the fermentative VFAs was controlled by adding magnesium to form struvite precipitation. Four VFAs liquids obtained was used to test the influence of initial carbon load and carbon to nitrogen ratio (C:N ratio) on the VFAs uptake rate, PHA storage rate and biomass growth rate. Results show that higher initial carbon substrate load results in relatively higher VFAs uptake rate and higher PHA storage rate, while higher initial C:N ratio results in relatively lower biomass growth rate from VFAs. VFAs generated from thermophilic alkaline sludge fermentation were a suitable carbon source for PHA production by mixed cultures.