Influence of drinking water biofilm microbiome on water quality: Insights from a real-scale distribution system.

Biofilms, constituting over 95 % of the biomass in drinking water distribution systems, form an ecosystem impacting both the aesthetic and microbiological quality of water. This study investigates the microbiome of biofilms within a real-scale drinking water distribution system in eastern Spain, utilizing amplicon-based metagenomics. Forty-one biofilm samples underwent processing and sequencing to analyze both bacterial and eukaryotic microbiomes, with an assessment of active biomass. Genus-level analysis revealed considerable heterogeneity, with Desulfovibrio, Ralstonia, Bradyrhizobium, Methylocystis, and Bacillus identified as predominant genera. Notably, bacteria associated with corrosion processes, including Desulfovibrio, Sulfuricella, Hyphomicrobium, and Methylobacterium, were prevalent. Potentially pathogenic bacteria such as Helicobacter, Pseudomonas, and Legionella were also detected. Among protozoa, Opisthokonta and Archaeplastida were the most abundant groups in biofilm samples, with potential pathogenic eukaryotes (Acanthamoeba, Naegleria, Blastocystis) identified. Interestingly, no direct correlation between microbiota composition and pipe materials was observed. The study suggests that the usual concentration of free chlorine in bulk water proved insufficient to prevent the presence of undesirable bacteria and protozoa in biofilms, which exhibited a high concentration of active biomass.

Continuous microalgae cultivation for wastewater treatment - Development of a process strategy during day and night.

Conventional wastewater treatment (WWT) is not able to recycle nutrients from the wastewater (WW) directly. Microalgae integrate the valuable nutrients nitrogen and phosphorus within their biomass very efficiently, making them predestined for an application in WWT. Nevertheless, microalgae-based processes are driven by natural sunlight as energy source, making a continuous process mode during day and night difficult. The aim of this study was therefore to investigate metabolic activities of the continuously cultivated microalgae Chlorella vulgaris at light and dark periods (16 h,8 h) with focus on nutrient uptake during night from a synthetic WW. Varying the dilution rate D (D = 0.0-1.0 d-1 in 0.1 d-1-steps) causes different limitations for algae growth. Nutrient limitations at low D's cause maximum accumulation of intracellular storage components (sum of carbohydrates and lipids) of ~70 % of dry biomass, starch is converted to lipids at the absence of light. From middle to high D's, the growth rate is determined by light limitation, reducing the intracellular storage components to ~20 % of dry biomass. Complete nutrient uptake is measurable up to D = 0.5 d-1, marking the maximum operating point for wastewater purification. At that point, cells are characterised by high protein (up to 57%DBM) and pigment (up to 6.9%DBM) quotas. During the night, the build-up of proteins at the degradation of intracellular storage components is furthermore visible. Applying the concept of active biomass (cells without storage components), a constant cellular protein (~68%ABM) and nitrogen quota (11.94%ABM) was revealed. A nitrogen spiking experiment clearly showed nitrogen uptake and proliferation during the night period. Based on the experimental data, a window of operation for a continuous WWT process was designed, allowing the hypothesis that continuous WWT using microalgae during day and night operation is possible without the supplementation of artificial light. This revealed the system's capacity to treat WW throughout 24 h applying cell recycling and storage of carbohydrate-rich biomass. At the end of the night, protein-rich biomass is available for further valorisation.

Investigation of an intermittently-aerated moving bed biofilm reactor in rural wastewater treatment under low dissolved oxygen and C/N condition.

An intermittently-aerated moving bed biofilm reactor (MBBR) was proposed for nitrogen and carbon removal from low C/N synthetic rural wastewater. In purposes of low energy consumption and costs, the intermittent aeration modes were changed and the dissolved oxygen was reduced gradually during the operation. The results showed that effluent concentrations of ammonia nitrogen and chemical oxygen demand were lower than 15 and 50 mg/L, respectively, even under microaerobic condition (0.1-1.0 mg/L). Meanwhile, the simultaneous nitrification-denitrification was achieved by intermittent aeration. The activity of functional bacteria was still high and the proportion of autotrophic biomass increased significantly under intermittent micro-aeration mode, which improved the nitrification performance. Aerobic denitrifier Hydrogenophaga, anoxic denitrifier Thiothrix, and heterotrophic nitrifier such as Rhodobacter were enriched in the intermittently micro-aerated MBBR, which will provide an applicable solution for rural wastewater treatment under low C/N and costs.

Active biomass estimation based on ASM1 and on-line OUR measurements for partial nitrification processes in sequencing batch reactors.

The main challenge for partial nitrification is to reach stable nitrite accumulation, which strongly depends on the nitrite-oxidizing bacteria (NOB) growth in the reactor. The on-line estimation of active biomass may enhance the decision-making process to maintain a high nitrite accumulation in the reactor. In this work, we propose an active biomass estimator based on ASM1 and on-line oxygen uptake rate measurements (OUR-E) in a sequencing batch reactor. In order to validate the OUR-E, two operating scenarios were applied during 200 days of operation: unfavorable (sludge retention time (SRT) = 40 d, pH = 7.6, dissolved oxygen (DO) = 2 mg/L) and favorable for partial nitrification (SRT = 10 d, pH = 8.5, DO = 2 mg/L). Furthermore, a second estimation method based on off-line measurements of N-species concentrations (Nsp-E) was implemented to evaluate the performance of the OUR-E. The OUR-E was able to predict a reduction in the NOB active fraction from 10.3% to 1.6% with nitrite accumulation over 80% when we shifted the operating scenario. Although both estimators predicted similar results, the OUR-E showed a better prediction quality than the Nsp-E, according to Theil's coefficient of inequality.

Determination and evaluation of kinetic parameters of activated sludge biomass from a sludge reduction system treating real sewage by respirometry testing.

Oxic-settling-anoxic (OSA) process is one of the promising variants that produces lower amounts of sludge and has been applied to aerobic and nutrient removal systems. The only consequence on this modification is that supplementary research is advisable to fully understand the mechanism, which eventually leads to the development of a more realistic model. This study evaluated the characteristics of an OSA process as a sludge reduction system by calibration of kinetic coefficients of ASM1 model with some modifications. A series of respirometric tests were designed for the assessment of microbial kinetics and for further clarification of sludge reduction mechanism. The calibration results depicted that the decay rate is the most variable kinetic parameter depending on the system configuration. It was determined that this kinetic coefficient increased significantly after the system was modified into OSA configuration while other model parameters were almost kept constant. This may be referred to a change either in the microbial population or in the metabolism of the community. The active biomass ratio in the CAS reactor was found to be around 75%, while it was almost 2 times lower in the side-stream reactor. All results led to a conclusion that OSA process is encouraging endogenous decay and consequently lowers biomass viability in the reactor and achieves excess sludge reduction in the system. All experimental results confirmed that side-stream reactor accelerates decay rate of the community and causes the introduction of sludge with low viability to the main reactor.

Degradation of the unbiodegradable particulate fraction (XU) from different activated sludges during batch digestion tests at ambient temperature.

One strategy for the management of excess sludge in small wastewater treatment plants (WWTPs) consists in minimizing the excess sludge production by operating the WWTP at very long solids retention times (SRTs > 30 days). A number of recent studies have suggested that sludge minimization at very long SRT results from the degradation of the unbiodegradable particulate fraction (XU) (influent unbiodegradable compounds and endogenous decay products). But the biodegradability of the unbiodegradable particulate fraction has only been evaluated during batch digestion test performed at ambient temperature with sludge fed with synthetic wastewaters. It is not clear to what extent observations made for sludge fed with synthetic influents can be transposed to sludge fed with real influent. The current study thus focused on evaluating the biodegradability of the unbiodegradable particulate fraction for sludge fed with real wastewater. Batch digestion tests (400 days, ambient temperature) were conducted with three different sludges fed with either synthetic or real influents and exposed to aerobic or intermittent aeration conditions. Our results indicate that volatile suspended solids (VSS) decreased even after complete decay of the active biomass (i.e., after 30 days of aerobic batch digestion) indicating that the unbiodegradable particulate fraction is biodegradable. However, very low degradation rates of the unbiodegradable particulate fraction were monitored after day 30 of digestion (0.7-1.7·10(-3) d(-1)). These values were in the lower range of previously published values for synthetic wastewaters (1-7.5·10(-3) d(-1)). The low values determined in our study indicate that the rate could decrease over time or that sludge composition influences the degradability of the unbiodegradable particulate fraction. But our results also demonstrate that extracellular polymeric substances (EPS) have a minor impact on the biodegradability of the unbiodegradable particulate fraction. Overall bound EPS were indeed biodegradable under all conditions and thus did not accumulate in the unbiodegradable particulate fraction. Different bound EPS pools (e.g., cation bound EPS) were associated with specific degradation behaviors. Besides improved mechanistic understanding of sludge degradation processes, our results have implications for the development of decentralized wastewater treatment technologies with on-site reduction of excess sludge.

Improvement on the yield of polyhydroxyalkanotes production from cheese whey by a recombinant Escherichia coli strain using the proton suicide methodology.

In this work Escherichia coli strain CML3-1 was engineered through the insertion of Cupriavidus necator P(3HB)-synthesis genes, fused to a lactose-inducible promoter, into the chromosome, via transposition-mediated mechanism. It was shown that polyhydroxyalkanotes (PHAs) production by this strain, using cheese whey, was low due to a significant organic acids (OA) synthesis. The proton suicide method was used as a strategy to obtain an E. coli mutant strain with a reduced OA-producing capacity, aiming at driving bacterial metabolism toward PHAs synthesis. Thirteen E. coli mutant strains were obtained and tested in shake flask assays, using either rich or defined media supplemented with lactose. P8-X8 was selected as the best candidate strain for bioreactor fed-batch tests using cheese whey as the sole carbon source. Although cell growth was considerably slower for this mutant strain, a lower yield of OA on substrate (0.04 Cmol(OA)/Cmol(lac)) and a higher P(3HB) production (18.88 g(P(3HB))/L) were achieved, comparing to the original recombinant strain (0.11 Cmol(OA)/Cmol(lac) and 7.8 g(P(3HB))/L, respectively). This methodology showed to be effective on the reduction of OA yield by consequently improving the P(3HB) yield on lactose (0.28 Cmol (P(3HB))/Cmol(lac) vs 0.10 Cmol(P(3HB))/Cmol(lac) of the original strain).