Summary of discussion on nuclear enzymes

A novel approach to the identification of microorganisms that accumulate high density microbial storage products based on density separation, denaturing gradient gel electrophoresis (DGGE), and DNA sequencing was developed and applied to bench and pilot scale enhanced biological phosphorus removal (EBPR) systems. Polyphosphate (PP), glycogen, and polyhydroxyalkanoates (PHAs), are all of higher density than a typical bacterial cell. PP-accumulating organisms (PAOs), the organisms responsible for EBPR, accumulate all three of these storage products. Density separation in a homogenous solution of Percoll produced a high-density biomass fraction with a relatively high concentration of PAOs, as determined by Neisser staining. DNA was extracted from these fractions, amplified, and separated by DGGE. DGGE profiles demonstrated some bacterial strains were present at a greater concentration in the high density fractions than in low density fractions. These strains were considered PAO candidates. 5 of 12 PAO candidates from high density fractions were gamma Proteobacteria and only 1 was a beta Proteobacterium. 2 PAO candidates were most similar to recently identified gamma Proteobacteria sequences obtained by DGGE analysis of a deteriorated benchtop EBPR system.

This study reports a proof-of concept study to demonstrate the novel approach of phenotyping microbial communities in enhanced biological phosphorus removal (EBPR) systems using single cell Raman microspectroscopy and link it with phylogentic structures. We use hierarchical clustering analysis (HCA) of single-cell Raman spectral fingerprints and intracellular polymer signatures to separate and classify the functionally relevant populations in EBPR systems, namely polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs), as well as other microbial populations. We then investigated the link between Raman-based community phenotyping and 16S rRNA gene-based phylogenetic characterization of four lab-scale EBPR systems with varying solid retention time (SRT) to gain insights into possible genotype-function relationships. Combined and simultaneous phylogenetic and phenotypic evaluation of EBPR ecosystems revealed SRT-dependent phylogenetic and phenotypic characteristics of the PAOs and GAOs, and their association with EBPR performance. The phenotypic diversity and plasticity of PAO populations, which otherwise could not be obtained with phylogenetic analysis alone, showed complex but potentially crucial association with EBPR process stability.

Polyhydroxyalkanoate (PHA) as bioplastic is considered a replacement for conventional plastic due to its more beneficial properties. The ability of PHA to biodegrade in a shorter period is a major advantage. Different sewage water samples were collected from the Budha Nala near the Maheru regions of Punjab. PHA-producing bacteria were isolated using minimal salt media supplemented with Nile blue. Further screening was carried out using Sudan Black B stain and Nile red stain. The positive isolates were characterized for gram reaction, motility, and biochemical tests. The individual isolates were later screened for maximum PHA accumulation using minimal salt supplemented with glucose. The extracted PHA was characterized using FTIR, XRD, SEM, UV spectroscopy, NMR, and TGA. Twenty-six different PHA-producing bacteria were isolated on minimal salt media supplemented with Nile blue. Upon Sudan Black B stain and Nile red stain, nineteen isolates showed black granules and orange fluorescence bodies under 100X magnification that confirmed polyhydroxyalkanoates. The biochemical tests partially characterized isolates belonging to the Bacillus genus. All the isolates produced PHA in granular form, however, isolate P-3 showed maximum production of 0.068 g/L. The extracted PHA was characterized using FTIR and XRD for its chemical and crystallinity studies and the UV spectroscopy confirmed the extracted PHA by analyzing absorption spectra at 235 nm of standard crotonic acid and sulfuric acid conversion of PHA to crotonic acid. The isolated P-3, Bacillus paranthracis RSKS-3 is the first reported bacterium to produce polyhydroxyalkanoates. Further studies is necessary to optimize the production efficiency of the bacterium for maximum PHA yield.

Quaternary ammonium compounds inhibited phosphorus removal performance and aggravated the spread of resistance genes in enhanced biological phosphorus removal systems

New plants

This study explored the microbial population dynamics of Accumulibacter (Acc) at low temperature and metabolic shift to limiting polyphosphate (Poly-P) in enhanced biological phosphorus removal (EBPR) system. The Accumulibacter-enriched EBPR systems, fed with acetate (HAc) and propionate (HPr) at 10 ± 1 °C respectively, were operated for 60 days in two identical SBR reactors (SBR-1 and SBR-2). The phosphorus removal performance in two systems was stable at 10 ± 1 °C, while the microbial community structure changed. Compared with the population structure in seed sludge, Accumulibacter clades reduced in the HAc system, while Acc I increased significantly in the HPr system. Low temperature was beneficial to the formation of granular sludge in the EBPR system, and the sludge granulation in the HAc system was more homogeneous than that in the HPr system. Accumulibacter in the HPr system can get ATP through glycogen accumulating metabolism (GAM) under limiting Poly-P condition at 10 ± 1 °C, while that in the HAc system cannot. This work suggests that poly-P levels can affect the metabolic pathway of Accumulibacter in EBPR systems under low temperature.

Synthetic plastics are in great demand in society due to their diversified properties, but they cause environmental pollution due to their non-biodegradable nature. Therefore, synthetic plastics are in need to be replaced with biodegradable plastics. Polyhydroxyalkanoates (PHAs), bacterial biopolymers are natural alternative to synthetic plastics. These are present inside the bacterial cytoplasm in granular form. Presently, the production cost of PHA is high due to expensive carbon substrates used in its biosynthesis. Therefore, this study focuses on the cost-effective production of PHA using waste carbon sources. Rice bran and sugarcane molasses were used as the carbon source for PHA production from Bacillus subtilis, Bacillus cereus, Alcaligenes sp. and Pseudomonas aeruginosa. PHA production from these bacterial strains was confirmed through Sudan Black-B screening. With rice bran, as carbon source, the highest PHA yield obtained was for P. aeruginosa, which yielded 93.7% and lowest was 35.5% for B. cereus. Surprisingly, B. cereus produced the highest cell dry mass (0.045 g/L) but its extracted PHA contents were lowest being only 0.02 g/L. Alcaligenes sp. with 0.031 g/L CDM yielded 87.1% PHA. B. subtilis had a CDM 0.029 g/L, 0.02 g/L PHA content and a yield of 69.10%. In the case of sugarcane molasses, P. aeruginosa produced 95% PHA yield, 0.02 g/L CDM, and 0.019 g/L PHA content. Alcaligenes sp. yielded 90.9% PHA, 0.011 g/L CDM, and 0.01 g/L PHA content. B. subtilis produced 91.6% PHA yield, 0.012 g/L CDM, 0.011 g/L PHA content; B. cereus produced 80% PHA yield, 0.015 g/L CDM, 0.012 g/L PHA content at 37°C, pH 7. Higher concentrations of carbon sources increased the CDM and decreased the PHA yield. The maximum yield of PHA was obtained from sugarcane molasses. 24-48 h of incubation was optimal for B. subtilis and B. cereus, while for Alcaligenes and P. aeruginosa incubation time of 48-96 h was desirable for higher PHA yield. The extracted biopolymers were analyzed by Fourier transform infrared spectroscopy (FTIR), which identified the extracted biopolymers as poly-3-hydroxybutyrate P(3HB). The thermal properties of the extracted biopolymers, such as melting temperatures, were analyzed by differential scanning calorimetry (DSC), which confirmed the thermal stability.

This paper describes several techniques and methods for sludge characterization which are recently developed and used in our ongoing research on biological phosphorus removal (BPR): (i) chemical measurement of the poly-hydroxy-alkanoate (PHA) content of the biomass, (ii) bioassay for glycogen determination in BPR systems, (iii) determination of the fraction of denitrifying phosphorus accumulating organisms (DPAOs) in P-removing sludge, (iv) determination of the aerobic and anoxic ATP/NADH2 ratio (δ value) in BPR systems, and (v) determination of the glycogen accumulating organisms (GAOs) / phosphorus accumulating organisms (PAOs) ratio in activated sludge.

Distributed state-type simulations (based on modeling of individual bacteria as they move through a reactor system) predicted a greater sensitivity of enhanced biological phosphorus removal (EBPR) performance to endogenous degradation than did conventional, "lumped"-type simulations (based on average biomass compositions). Recent research has indicated that the variable hydraulic residence times experienced by individual microbial storage product accumulating bacteria in systems with completely mixed reactors tend to produce populations with diverse microbial storage product contents (distributed states). Endogenous degradation in EBPR systems is of particular interest because the polyphosphate accumulating organisms (PAOs) responsible for EBPR rely on the accumulation of three different storage products that may be endogenously degraded. Simulations indicated that as endogenous degradation rates of microbial storage products were increased, EBPR performance decreased more rapidly according to the distributed approach than according to the lumped approach. State profile analysis demonstrated that as these rates increased, the population fraction with depleted storage products also increased, and this tended to increase the error in calculated biokinetic rates by the lumped approach. Simulations based on recently reported endogenous rate coefficients also suggested large differences between distributed and lumped predictions of EBPR performance. These results demonstrated that endogenous decay processes may play a more important role in EBPR than predicted by the lumped approach. This suggests a need for further research to determine endogenous process rates, and for incorporation of this information to distributed-type simulators, as this should lead to improved accuracy of EBPR simulations.

Rapid quantification of intracellular PHA using infrared spectroscopy: An application in mixed cultures

The microbiology of biological phosphorus removal in activated sludge systems

Members of the genus Candidatus Accumulibacter are important in many wastewater treatment systems performing enhanced biological phosphorus removal (EBPR). The Accumulibacter lineage can be subdivided phylogenetically into multiple clades, and previous work showed that these clades are ecologically distinct. The complete genome of Candidatus Accumulibacter phosphatis strain UW-1, a member of Clade IIA, was previously sequenced. Here, we report a draft genome sequence of Candidatus Accumulibacter spp. strain UW-2, a member of Clade IA, assembled following shotgun metagenomic sequencing of laboratory-scale bioreactor sludge. We estimate the genome to be 80-90% complete. Although the two clades share 16S rRNA sequence identity of >98.0%, we observed a remarkable lack of synteny between the two genomes. We identified 2317 genes shared between the two genomes, with an average nucleotide identity (ANI) of 78.3%, and accounting for 49% of genes in the UW-1 genome. Unlike UW-1, the UW-2 genome seemed to lack genes for nitrogen fixation and carbon fixation. Despite these differences, metabolic genes essential for denitrification and EBPR, including carbon storage polymer and polyphosphate metabolism, were conserved in both genomes. The ANI from genes associated with EBPR was statistically higher than that from genes not associated with EBPR, indicating a high selective pressure in EBPR systems. Further, we identified genomic islands of foreign origins including a near-complete lysogenic phage in the Clade IA genome. Interestingly, Clade IA appeared to be more phage susceptible based on it containing only a single Clustered Regularly Interspaced Short Palindromic Repeats locus as compared with the two found in Clade IIA. Overall, the comparative analysis provided a genetic basis to understand physiological differences and ecological niches of Accumulibacter populations, and highlights the importance of diversity in maintaining system functional resilience.

Process optimization for polyhydroxyalkanoate (PHA) production from waste via microbial enrichment cultures

Mobile gene elements associated with integrons, including as gene cassettes, have been proposed to play an important role in bacterial evolution by providing an extensive genetic resource. This study hypothesized that critical genes for enzymes involved in EBPR systems, including those involved in polyphosphate, PHA and glycogen synthesis, may be present in mobile gene cassettes. Although no such genes were identified in any of the functional and deteriorated enhanced biological phosphorus removal (EBPR) laboratory-scale SBR systems examined here, many of the open reading frames (ORFs) remained unidentified because of the incompleteness of publicly available databases. An ORF of unknown function (SBR6-2) was encountered in deteriorated EBPR system with an unexpectedly high frequency, comprising 35% of the gene cassette-associated sequences for that system.

Discovery of a novel potential polyphosphate accumulating organism without denitrifying phosphorus uptake function in an enhanced biological phosphorus removal process