Microbial community-based protein from soybean-processing wastewater as a sustainable alternative fish feed ingredient.

As the global demand for food increases, aquaculture plays a key role as the fastest growing animal protein sector. However, existing aquafeeds contain protein ingredients that are not sustainable under current production systems. We evaluated the use of microbial community-based single cell protein (SCP), produced from soybean processing wastewater, as a partial fishmeal protein substitute in juvenile Asian seabass (Lates calcarifer). A 24-day feeding trial was conducted with a control fishmeal diet and a 50% fishmeal replacement with microbial community-based SCP as an experimental group, in triplicate tanks containing 20 fish each. Both diets met the protein, essential amino acids (except for lysine), and fat requirements for juvenile Asian sea bass. The microbial composition of the SCP was dominated by the genera Acidipropionibacterium and Propioniciclava, which have potential as probiotics and producers of valuable metabolites. The growth performance in terms of percent weight gain, feed conversion ratio (FCR), specific growth rate (SGR), and survival were not significantly different between groups after 24 days. The experimental group had less variability in terms of weight gain and FCR than the control group. Overall, microbial community-based protein produced from soybean processing wastewater has potential as a value-added feed ingredient for sustainable aquaculture feeds.

Microbial community-based production of single cell protein from soybean-processing wastewater of variable chemical composition.

The use of food-processing wastewaters to produce microbial biomass-derived single cell protein (SCP) is a sustainable way to meet the global food demand. Microbial community-based approaches to SCP production have the potential benefits of lower costs and greater resource recovery compared to pure cultures, yet they have received scarce attention. Here, SCP production from soybean-processing wastewaters using their existent microbial communities was evaluated. Six sequencing batch reactors of 4.5-L working volume were operated at 30 °C for 34 d in cycles consisting of 3-h anaerobic and 9-h aerobic phases. Four reactors received no microbial inoculum and the remaining two were amended with 1.5 L of a mixed culture from a prior SCP production cycle. Reactors produced more SCP when fed with wastewaters of higher soluble total Kjeldahl nitrogen (sTKN) content. The protein yield in biomass ranged from 0.53 to 3.13 g protein/g sTKN, with a maximum protein content of 50 %. The average removal of soluble chemical oxygen demand (sCOD) and soluble total nitrogen (sTN) was 92 % and 73 %, respectively. Distinct microbial genera were enriched in all six bioreactors, with Azospirillum, Rhodobacter, Lactococcus, and Novosphingobium dominating. The study showed that constituents in soybean wastewater can be converted to SCP and demonstrated the effect of variable influent wastewater composition on SCP production.

Controlling anammox speciation and biofilm attachment strategy using N-biotransformation intermediates and organic carbon levels.

Conventional nitrogen removal in wastewater treatment requires a high oxygen and energy input. Anaerobic ammonium oxidation (anammox), the single-step conversion of ammonium and nitrite to nitrogen gas, is a more energy and cost effective alternative applied extensively to sidestream wastewater treatment. It would also be a mainstream treatment option if species diversity and physiology were better understood. Anammox bacteria were enriched up to 80%, 90% and 50% relative abundance, from a single inoculum, under standard enrichment conditions with either stepwise-nitrite and ammonia concentration increases (R1), nitric oxide supplementation (R2), or complex organic carbon from mainstream wastewater (R3), respectively. Candidatus Brocadia caroliniensis predominated in all reactors, but a shift towards Ca. Brocadia sinica occurred at ammonium and nitrite concentrations > 270 mg NH4-N L-1 and 340 mg NO2-N L-1 respectively. With NO present, heterotrophic growth was inhibited, and Ca. Jettenia coexisted with Ca. B. caroliniensis before diminishing as nitrite increased to 160 mg NO2-N L-1. Organic carbon supplementation led to the emergence of heterotrophic communities that coevolved with Ca. B. caroliniensis. Ca. B. caroliniensis and Ca. Jettenia preferentially formed biofilms on surfaces, whereas Ca. Brocadia sinica formed granules in suspension. Our results indicate that multiple anammox bacteria species co-exist and occupy sub-niches in anammox reactors, and that the dominant population can be reversibly shifted by, for example, changing nitrogen load (i.e. high nitrite concentration favors Ca. Brocadia caroliniensis). Speciation has implications for wastewater process design, where the optimum cell immobilization strategy (i.e. carriers vs granules) depends on which species dominates.

Global warming readiness: Feasibility of enhanced biological phosphorus removal at 35 °C.

Recent research has shown enhanced biological phosphorus removal (EBPR) from municipal wastewater at warmer temperatures around 30 °C to be achievable in both laboratory-scale reactors and full-scale treatment plants. In the context of a changing climate, the feasibility of EBPR at even higher temperatures is of interest. We operated two lab-scale EBPR sequencing batch reactors for > 300 days at 30 °C and 35 °C, respectively, and followed the dynamics of the communities of polyphosphate accumulating organisms (PAOs) and competing glycogen accumulating organisms (GAOs) using a combination of 16S rRNA gene metabarcoding, quantitative PCR and fluorescence in situ hybridization analyses. Stable and nearly complete phosphorus (P) removal was achieved at 30 °C; similarly, long term P removal was stable at 35 °C with effluent PO43-_P concentrations < 0.5 mg/L on half of all monitored days. Diverse and abundant Candidatus Accumulibacter amplicon sequence variants were closely related to those found in temperate environments, suggesting that EBPR at this temperature does not require a highly specialized PAO community. A slow-feeding strategy effectively limited the carbon uptake rates of GAOs, allowing PAOs to outcompete GAOs at both temperatures. Candidatus Competibacter was the main GAO, along with cluster III Defluviicoccus members. These organisms withstood the slow-feeding regime, suggesting that their bioenergetic characteristics of carbon uptake differ from those of their tetrad-forming relatives. Comparative cycle studies revealed higher carbon and P cycling activity of Ca. Accumulibacter when the temperature was increased from 30 °C to 35 °C, implying that the lowered P removal performance at 35 °C was not a direct effect of temperature, but a result of higher metabolic rates of carbon (and/or P) utilization of PAOs and GAOs, the resultant carbon deficiency, and escalated community competition. An increase in the TOC-to-PO43--P ratio (from 25:1 to 40:1) effectively eased the carbon deficiency and benefited PAOs. In general, a slow-feeding strategy and sufficiently high carbon input benefited a high and stable EBPR at 35 °C, representing basic conditions suitable for full-scale treatment plants experiencing higher water temperatures.

Metabolic Traits of Candidatus Accumulibacter clade IIF Strain SCELSE-1 Using Amino Acids As Carbon Sources for Enhanced Biological Phosphorus Removal.

Despite recent evidence from full-scale plants suggesting that Candidatus Accumulibacter may be capable of using amino acids, this metabolic trait has never been confirmed in a bioreactor experiment. Here we show that an enriched culture of Ca. Accumulibacter clade IIF strain SCELSE-1 could metabolize 11 of 20 α-amino acids, with aspartate, glutamate, asparagine, and glutamine resulting in the highest phosphorus removal. The anaerobic uptake of aspartate and glutamate was achieved through a glutamate/aspartate-proton symporter fully powered by the proton motive force (PMF). Under anaerobic conditions aspartate was deaminized and routed into core carbon metabolic pathways to form polyhydroxyalkanoates (PHA). The lack of genes encoding NADH dependent isocitrate dehydrogenase in the Ca. Accumulibacter genome resulted in a kinetic barrier for glutamate to be channelled to the TCA cycle. Glutamate was stored as glutamate polymer. When amino acids (aspartate or glutamate) and acetate were supplied together, Ca. Accumulibacter took up both carbon sources simultaneously, with the uptake rate of each carbon source largely preserved. Overall energy savings (up to 17%) were achieved under mixed carbon scenarios, due to the ability of Ca. Accumulibacter to rearrange its anaerobic carbon metabolism based on the reducing power, PMF and ATP balance.

Polyphosphate-accumulating organisms in full-scale tropical wastewater treatment plants use diverse carbon sources.

Enhanced biological phosphorus removal (EBPR) is considered challenging in the tropics, based on a great number of laboratory-based studies showing that the polyphosphate-accumulating organism (PAO) Candidatus Accumulibacter does not compete well with glycogen accumulating organisms (GAOs) at temperatures above 25 °C. Yet limited information is available on the PAO community and the metabolic capabilities in full-scale EBPR systems operating at high temperature. We studied the composition of the key functional PAO communities in three full-scale wastewater treatment plants (WWTPs) with high in-situ EBPR activity in Singapore, their EBPR-associated carbon usage characteristics, and the relationship between carbon usage and community composition. Each plant had a signature community composed of diverse putative PAOs with multiple operational taxonomic units (OTUs) affiliated to Ca. Accumulibacter, Tetrasphaera spp., Dechloromonas and Ca. Obscuribacter. Despite the differences in community composition, ex-situ anaerobic phosphorus (P)-release tests with 24 organic compounds from five categories (including four sugars, three alcohols, three volatile fatty acids (VFAs), eight amino acids and six other carboxylic acids) showed that a wide range of organic compounds could potentially contribute to EBPR. VFAs induced the highest P release (12.0-18.2 mg P/g MLSS for acetate with a P release-to-carbon uptake (P:C) ratio of 0.35-0.66 mol P/mol C, 9.4-18.5 mg P/g MLSS for propionate with a P:C ratio of 0.38-0.60, and 9.5-17.3 mg P/g MLSS for n-butyrate), followed by some carboxylic acids (10.1-18.1 mg P/g MLSS for pyruvate, 4.5-11.7 mg P/g MLSS for lactate and 3.7-12.4 mg P/g MLSS for fumarate) and amino acids (3.66-7.33 mg P/g MLSS for glutamate with a P:C ratio of 0.16-0.43 mol P/mol C, and 4.01-7.37 mg P/g MLSS for aspartate with a P:C ratio of 0.17-0.48 mol P/mol C). P-release profiles (induced by different carbon sources) correlated closely with PAO community composition. High micro-diversity was observed within the Ca. Accumulibacter lineage, which represented the most abundant PAOs. The total population of Ca. Accumulibacter taxa was highly correlated with P-release induced by VFAs, highlighting the latter's importance in tropical EBPR systems. There was a strong link between the relative abundance of individual Ca. Accumulibacter OTUs and the extent of P release induced by distinct carbon sources (e.g., OTU 81 and amino acids, and OTU 246 and ethanol), suggesting niche differentiation among Ca. Accumulibacter taxa. A diverse PAO community and the ability to use numerous organic compounds are considered key factors for stable EBPR in full-scale plants at elevated temperatures.