Transcriptomic Insights into Metabolism-Dependent Biosynthesis of Bacterial Nanocellulose.

Bacterial nanocellulose (BNC) is an attractive green-synthesized biomaterial for biomedical applications and various other applications. However, effective engineering of BNC production has been limited by our poor knowledge of the related metabolic processes. In contrast to the traditional perception that genome critically determines biosynthesis behaviors, here we discover that the glucose metabolism could also drastically affect the BNC synthesis in Gluconacetobacter hansenii. The transcriptomic profiles of two model BNC-producing strains, G. hansenii ATCC 53582 and ATCC 23769, which have highly similar genomes but drastically different BNC yields, were compared. The results show that their BNC synthesis capacities were highly related to metabolic activities such as ATP synthesis, ion transport protein assembly, and carbohydrate metabolic processes, confirming an important role of metabolism-related transcriptomes in governing the BNC yield. Our findings provide insights into the microbial biosynthesis behaviors from a transcriptome perspective, potentially guiding cellular engineering for biomaterial synthesis.

[Bacterial Community Structure and Antibiotic Resistance Gene Changes in IFAS+Magnetic Coagulation Process Wastewater Treatment Plant in Cold Regions].

The main objective of this study was to explore the changes in bacterial communities and antibiotic resistance genes (ARGs) in an integrated fixed-film activated sludge (IFAS)+magnetic coagulation process wastewater treatment plant (WWTP) in Xinjiang. The bacterial communities and ARGs in the influent, suspended activated sludge, attached biofilm, and effluent were studied using 16S rRNA gene sequencing and metagenomic sequencing. The results showed that the average relative abundances of Chloroflexi and Nitrospirae in activated sludge were 3.50% and 0.03%, respectively, and their relative abundances in biofilm reached 10.02% and 2.12%, respectively. The average removal rates of NH4+-N and TN increased from 91.89% and 66.76% to 97.71% and 91.90% after the reformation of this wastewater treatment plant, respectively, indicating that IFAS enhanced the biological nitrogen removal capacity of wastewater treatment plants in cold regions. The average relative abundances of Ferruginibacter and Rhodoferax related to iron redox in the biological treatment section were 5.24% and 3.72%, respectively, and the relative abundance of Rhodoferax in effluent reached 9.48%, indicating that the magnetic powder had an impact on the bacterial community. The IFAS wastewater treatment plant had an obvious removal effect on ARGs, and the relative abundance of ARGs decreased from 191.08×10-3‰ in the influent to 32.58×10-3‰ in the effluent. The relative abundance of ARGs in activated sludge was 63.25×10-3‰-72.38×10-3‰, which was significantly higher than 41.31×10-3‰ in biofilm. However, the relative abundances of dominant subtypes of ARGs such as sul2, floR, and rpoB2 in biofilm were 5.77×10-3‰, 2.52×10-3‰, and 2.03×10-3‰, respectively, which were higher than the 3.15×10-3‰-3.57×10-3‰, 1.73×10-3‰-2.24×10-3‰, and 1.28×10-3‰-1.76×10-3‰ in activated sludge. The network analysis indicated that Caldilineaceae_norank and Trichococcus were respectively positively correlated with sul2 and floR. These results can provide theoretical reference for the optimal operation and ARGs control of WWTPs in cold regions.

pH-adjustment strategy for volatile fatty acid production from high-strength wastewater for biological nutrient removal.

Volatile fatty acid (VFA) production from three types of high-strength organic wastewater (cassava thin stillage, starch wastewater and yellow-wine processing wastewater) were compared. The results showed that cassava thin stillage was the most suitable substrate, based on its high specific VFA production (0.68 g chemical oxygen demand (COD)/g initial soluble chemical oxygen demand (SCOD)) and yield (0.72 g COD/g SCOD) as well as low nutrient content in the substrate and fermented liquid. The acid fermented cassava thin stillage was evaluated and compared with sodium acetate in a sequencing batch reactor system. Total nitrogen removal efficiency was higher with fermented cassava thin stillage than with the sodium acetate. The effects of pH and a pH-adjustment strategy on VFA production and composition were determined using cassava thin stillage. At an initial pH range of 7-11, a relatively high VFA concentration of about 9 g COD/L was obtained. The specific VFA production (g COD/g initial SCOD) increased from 0.27 to 0.47 to 0.67 at pH 8 and from 0.26 to 0.68 to 0.81 at pH 9 (initial pH, interval pH, and constant pH adjustment, respectively). The dominant VFA species changed significantly with the increasing frequency of the pH adjustment. Further studies will examine the metabolic pathways responsible for VFA composition.

[Kinetic simulation of enhanced biological phosphorus removal with fermentation broth as carbon source].

As a high-quality carbon source, fermentation broth could promote the phosphorus removal efficiency in enhanced biological phosphorus removal (EBPR). The transformation of substrates in EBPR fed with fermentation broth was well simulated using the modified activated sludge model No. 2 (ASM2) based on the carbon source metabolism. When fermentation broth was used as the sole carbon source, it was found that heterotrophic bacteria acted as a promoter rather than a competitor to the phosphorus accumulating organisms (PAO). When fermentation broth was used as a supplementary carbon source of real municipal wastewater, the wastewater composition was optimized for PAO growth; and the PAO concentration, which was increased by 3.3 times compared to that in EBPR fed with solely real municipal wastewater, accounting for about 40% of the total biomass in the reactor.

[Mechanisms of the improvement in dewaterability of alkaline fermented sludge by simultaneous ammonium and phosphate recovery].

Simultaneous ammonium and phosphate recovery could notably improve the dewaterability of alkaline fermented sludge, the mechanisms of which, however, remains unclear at present. The influence of zeta potential, divalent ions, extracellular polymeric substances (EPS), dissolved polymers and struvite were studied in batch experiments. Under the optimal ammonium and phosphate recovery condition [i. e. pH =10.0, n(P)/n(N) = 1.3 mol x mol(-1), n(Mg)/n(N) = 1.9 mol x mol(-1)], it was found that magnesium ion could not only decrease the absolute value of zeta potential to 14 mV, but also reduce the monovalent to divalent ions ratio to 9 mol x mol(-1), which promoted the dewaterability of alkaline fermented sludge; also, the dissolved polymers and EPS, especially the dissolved protein and the loosely bound EPS, reduced remarkably. Results showed that all the factors above promoted sludge dewatering. Furthermore, the struvite formed during ammonium and phosphate recovery improved the dewaterability.

[Kinetic model of enhanced biological phosphorus removal with mixed acetic and propionic acids as carbon sources. (I): Model constitution].

Based on activated sludge model No. 2 (ASM2), the anaerobic/aerobic kinetic model of phosphorus-accumulating organisms (PAO) was established with mixed short-chain fatty acids (SCFAs) as the base substance in enhanced biological phosphorus removal process. The characteristic of the PAO model was that the anaerobic metabolism rates of glycogen degradation, poly-beta-hydroxyalkanoates synthesis and polyphosphate hydrolysis were expressed by SCFAs uptake equation, and the effects of anaerobic maintenance on kinetics and stoichiometry were considered. The PAO kinetic model was composed of 3 soluble components, 4 particulate components and a pH parameter, which constituted the matrix of stoichiometric coefficients. On the basis of PAO model, the GAO kinetic model was established, which included 7 processes, and phosphorus content influenced the aerobic metabolism only.

[Kinetic model of enhanced biological phosphorus removal with mixed acetic and propionic acids as carbon sources. (II): Process simulation].

Two groups of sequencing batch reactors were used to study the metabolism substrate transformation of phosphorus-accumulating organisms (PAO) and glycogen-accumulating organisms (GAO) fed with mixed acetic and propionic acids. Seven stoichiometry parameters and 24 kinetic parameters were contained in the PAO and GAO kinetic model, and stoichiometry parameters were deduced from the stoichiometry models, while kinetic parameters were determined by experimental results. The kinetic model parameters of stoichiometry and kinetics were determined according the experiments and the literature. Subsequently, the substrate transformations of PAO and GAO were calculated by the Matlab software. The model curves matched the SBR experimental data well, indicating that the kinetic model based on SCFAs metabolism could be used to simulate PAO and GAO in anaerobic-aerobic conditions.

[Kinetic model of enhanced biological phosphorus removal with mixed acetic and propionic acids as carbon sources. (III): Model application].

The kinetic model based on SCFAs metabolism was applied for the prediction of phosphorus-and glycogen-accumulating organisms (PAO and GAO) competition with different carbon sources and m(P)/m(COD) ratios. When acetic acid was used as the sole carbon source, the biomass compositions were almost the same as those before cultivation, and neither PAO nor GAO could be out-competed from EBPR. However, increasing propionic acid in the influent helped PAO to be the predominance organism, and EBPR performance kept excellent when the ratio of propionate to mixed acids (acetate + propionate) was higher than 0.33. It also found that the m(P)/m(COD) ratio should be kept at 0.04-0.10 to avoid phosphorus became a limiting factor for PAO growth. This was because at low m(P)/m(COD) ratios, such as 0.01, GAO would take up 95% of the total (PAO + GAO) biomass.

[Polyhydroxyalkanoate (PHA) synthesis by activated sludge microbes using acetic acid as carbon source].

Polyhydroxyalkanoate (PHA) is a type of biodegradable plastics. The synthesis of PHA by activated sludge has the advantage of easy operation. With acetic acid as the carbon source, the anaerobic synthesis of PHA in the anaerobic-aerobic activated sludge system was compared with the aerobic one in the aerobic sludge system. It was observed that the aerobic PHA synthesis was greater than the anaerobic one. The influences of acetic acid addition frequency, acetic acid concentration and allyl thiourea (ATU) on aerobic PHA synthesis were further investigated. The results showed that the PHA content accounting for volatile suspended solids reached 56.3% under conditions of acetic acid addition with frequency of 3, acetic acid concentration of 2 925 mg/L, and ATU concentration of 6.74 mg/L.

[Effect of different ratios of propionic to acetic acids on long-term cultured active sludge for enhanced biological phosphorus removal].

With the long-term cultured enhanced biological phosphorus removal systems in sequencing batch reactors, the effect of different ratios of propionic acid to acetic acid on the transformations of phosphorus and polyhydoxyalkanoates (PHA), and the metabolism stoichiometry of PHA by phosphorus accumulating organisms were studied in this paper. Results showed that the phosphorus removal efficiency improved with the increase of propionic acid to acetic acid ratio, and the suitable C-mol ratio of propionic acid to acetic acid was 2:1. Stoichiometry study indicated that 0.15 C-mol PHB and 0.33 C-mol (PHV + PH2MV) were synthesized per degraded C-mol acetic acid, and very few PHB and 1.21 C-mol (PHV + PH2MV) were synthesized per degraded C-mol propionic acid. It was also observed that the phosphorus removal efficiency exhibited a good linear relationship with (PHV + PH2MV).

[Biological nitrogen and phosphorus removal in the anaerobic-aerobic-anoxic-aerobic-anoxic-aerobic sequencing batch reactors].

The biological nitrogen and phosphorus removal was investigated in two anaerobic-anoxic-aerobic sequencing batch reactors (SBRs), which were fed with synthetic and municipal wastewater, respectively. The operating cycle of (AO)3 SBR was 1.5 h anaerobic --> 1 h aerobic --> 1 h anoxic -->20 min aerobic --> l h anoxic --> 20 min aerobic. The removal efficiency of COD, total nitrogen and total phosphorus for synthetic wastewater was 88%, 89%, 99%, respectively, while it was 85%, 75% , 99.5% for municipal wastewater. Furthermore, it was observed that though there were much differences in polyhydroxyalkanoates (PHAs) accumulation and phosphorous release in the anaerobic stage between two SBRs, the ratio of the anoxic energy production efficiency to the aerobic one was close to each other (49% versus 50%).

[Long-term and short-term effects of propionic/acetic acid ratios on metabolism of glycogen-accumulating organisms].

Three activated sludges enriched with glycogen accumulating organisms (GAO) were acclimatized respectively with different ratios of propionic to acetic acid (i.e. biomass SBR-A, C and E) . The effect of different ratios of propionic/acetic acid on the metabolism of long-term cultivated GAO was investigated. Cultivated with high propionic/acetic acid ratio, GAO consumed less glycogen and synthesized less poly-beta-hydroxyalkanoates (PHA) in the anaerobic phase, and in the aerobic phase accumulated less glycogen and degraded less PHA, and at the same time the microbial growth was lower. When the carbon mole of acetic acid equaled that of propionic acid in the influent, GAO utilized acetic acid faster than propionic acid. Batch tests were carried out with biomass SBR-A and SBR-E to study the transient response of long-term cultivated GAO to short-term change of propionic/acetic acid ratio. The GAO cultivated with a high propionic/acetic acid ratio was able to utilize acetic acid immediately when the concentration of acetic acid in the feed suddenly increased. But when the biomass cultivated with a low propionic/acetic acid ratio was feed with high ratio propionic/acetic acid wastewater, the propionic acid uptake rate was only 41.1% of the rate of the GAO long-term cultivated with high propionic/acetic acid. The sudden increase of propionic/acetic acid ratio could effectively inhibit the metabolism of GAO.

[Addition of propionic acid on simultaneous biological phosphorus and nitrogen removal in anaerobic-low dissolved oxygen system].

Two lab-scale sequencing batch reactors (SBRs) were operated under conditions of anaerobic-low dissolved oxygen (DO) (0.15 - 0.45 mg x L(-1)), and the influences of addition of propionic acid on simultaneous biological nitrogen and phosphorus removal system were investigated. The results showed that the simultaneous nitrification, denitrification and phosphorus removal (SNDPR) occurred in both SBR1 (acetic and propionic acid as mixed carbon source with the carbon molar ratio of 1.5/1) and SBR2 (acetic acid as sole carbon source), and ammonia was completely oxidized during the aerobic period without substantive nitrite accumulation. Compared with SBR2, SBR1 showed less anaerobic phosphorus release and more polyhydroxyvalerate (PHV) synthesis, but the aerobic end phosphorus and nitrate concentrations were lower. The total nitrogen and phosphorus removal efficiencies were higher in SBR1 (68% and 95%, respectively) than in SBR2 (51% and 92%, respectively) suggesting that the addition of propionic acid to SNDPR system would be helpful.

[Effect of SRT on denitrifying phosphorus removal in A/A/O process].

Based on a laboratory-scale anaerobic-anoxic-oxic process acclimated with municipal wastewater, effect of SRT on denitrifying phosphorus removal occurred in the system was investigated. The results indicate that the contribution of anoxic denitrifying phosphorus removal to total phosphorus removal becomes larger with prolonging the sludge retention time. DNPAOs' denitrification and P uptake capacities per anoxic PHAs utilized are quickly increased, while PAOs' P uptake capacity per aerobic PHAs utilized is rarely influenced by SRT. When SRT is set at 12 days, anoxic phosphorus uptake efficiency and biological nutrient removal efficiency are best. The results also show that the COD amountrequired for removing per nitrogen decreases and for removing per phosphorus increases with prolonging the sludge retention time. For the national typical municipal wastewater, the COD amount required for removing per nitrogen and phosphorus under SRT of 12 and 15 days is less than that of under SRT of 8 days, which indicates that anoxic denitrifying phosphorus removal can really decrease the demand of carbon and energy sources.

[Influence of wastewater initial pH on enhanced biological phosphorus removal in sequencing batch reactor (SBR)].

Two laboratory-scale sequencing batch reactors (SBRs) were operated continuously to investigate the influence of wastewater initial pH on enhanced biological phosphorus removal (SBR1: pH = 6.8; SBR2: pH = 7.6). Results show that SBR2 exhibits greater anaerobic phosphorus release than SBR1. During aerobic stage, SBR2 degrades less polyhydroxyalkanoates (PHA) than SBR1, and the ratio of glycogen synthesis to PHA degradation in SBR2 is much less than that of SBR1, but SBR2 takes up more phosphorus. Further studies show that due to less glycogen synthesis in SBR2 than in SBR1, lower PHA degradation in SBR2 doesn't result in lower phosphorus uptake. The higher phosphorus uptake and PHA utilization efficiency in SBR2 is probably caused by its more phosphorus accumulating organisms (PAO). At the end of aerobic phase, SBR2 has significantly higher phosphorus removal efficiency than SBR1 (93.67% against 65.06%). Thus, the efficiency of enhanced biological phosphorus removal can be significantly improved by controlling the initial pH of wastewater. This method is much more convenient than controlling the entire process pH of wastewater biological treatment.

[Impact of pH on the generation of COD, phosphorous and ammonia-nitrogen during the anaerobic fermentation of excess activated sludge].

There are mainly two kinds of sludge in the municipal wastewater treatment plant, i. e. , primary and secondary sludge. This study investigated the effect of pH, ranging from 4.0-11.0, on hydrolysis in terms of soluble chemical oxygen demand (SCOD) production in the anaerobic solubilization of excess activated sludge at 20-22 degrees C. It was found that when the value of pH was 8.0-10.0, the production quantity of SCOD were higher than pH = 5.0-7.0. Especially when the pH was 10.0 or 11.0, the value of SCOD was almost 10 times of pH = 6.0 during the whole fermentation of 20 days. And volatile fatty acids (VFA) production on the 8th day under alkaline condition was higher than that under acidic condition.

[Research on polyhydroxyalkanoate form a key aspect to enhanced biological phosphorus transformation].

To investigate the influence of PHB and PHV formed on phosphorus (P) release, uptake and removal during enhanced biological phosphorus removal (EBPR), anaerobic/aerobic batch experiments were conducted with biomass acclimated with propionic to acetic acid carbon molar ratios of 0.5 and 2 on two sequencing batch reactors (SBR1 and SBR2). Statistically significant correlations between polyhydroxyalkanoate (PHA) quantity and form and P release/uptake and removal were observed (R2 >0.90). The regression coefficients showed that for biomass cultured with customizing wastewater P release and uptake were both a function of PHB but not of PHV, but higher P removal was largely because of PHV as the predominant type rather than PHV. For biomass cultured with different ratios of propionic to acetic acid, the SBR2 biomass synthesized and utilized more PHB and less PHV and showed higher net P removal (average increase of 16.69%) than SBR1. Thus acetate/propionate content of influent had a major influenceon PHA type and quantity and determine phosphorus (P) release, uptake and removal. Accordingly, PHB and PHV transformations should be taken into account as key aspect for optimizing EBPR.

[Research on substrate transformation mechanism in A2/O process].

Based on a laboratory-scale anaerobic-anoxic-oxic process acclimated with real wastewater, substrate transformation mechanism and the effect of nitrate on substrate transformations occurred in the system were investigated. The results indicated that under the anaerobic condition without nitrate, phosphorus-accumulating organisms (PAOs) could take up 51% of COD consumed and store them to PHAs; anoxic and aerobic specific phosphorus uptake rates were 3.87 mg/(g x h) and 6.54 mg/(g x h), and the anoxic and aerobic ratios of phosphorus uptake and PHAs utilized, nu(P/PHA). were 0.38 and 0.78, respectively. While under the anaerobic condition containing nitrate, only 30.8% of COD consumed was taken up by PAOs and 61.5% was used as the denitrifying carbon source; specific phosphorus uptake rates of anoxic and aerobic sludge were 2.24 mg/(g x h) and 4.58 mg/(g x h), and the nu(P/PHA) under the anoxic and aerobic conditions were 0.35 and 0.77, respectively. Furthermore, in both systems, there was a good linear relationship between COD consumption and phosphate release under the anaerobic condition. The nitrate existed in the anaerobic condition could decrease anaerobic phosphorus release rate and efficiency of PAOs, resulting in a decreased amount of PHAs synthesis and consequently affecting anoxic and aerobic phosphorus uptake rates. However, phosphorus uptake capacity was not influenced by nitrate and the amount of PHAs synthesized in the anaerobic stage.