Biosynthesis of poly(3-hydroxybutyrate) by N,N-dimethylformamide degrading strain Paracoccus sp. PXZ: A strategy for resource utilization of pollutants.

N,N-dimethylformamide is a toxic chemical solvent, which widely exists in industrial wastewater. Nevertheless, the relevant methods merely achieved non-hazardous treatment of N,N-dimethylformamide. In this study, one efficient N,N-dimethylformamide degrading strain was isolated and developed for pollutant removal coupling with poly(3-hydroxybutyrate) (PHB) accumulation. The functional host was characterized as Paracoccus sp. PXZ, which could consume N,N-dimethylformamide as the nutrient substrate for cell reproduction. Whole-genome sequencing analysis confirmed that PXZ simultaneously possesses the essential genes for poly(3-hydroxybutyrate) synthesis. Subsequently, the approaches of nutrient supplementation and various physicochemical variables to strengthen poly(3-hydroxybutyrate) production were investigated. The optimal biopolymer concentration was 2.74 g·L-1 with a poly(3-hydroxybutyrate) proportion of 61%, showing a yield of 0.29 g-PHB·g-1-fructose. Furthermore, N,N-dimethylformamide served as the special nitrogen matter that could realize a similar poly(3-hydroxybutyrate) accumulation. This study provided a fermentation technology coupling with N,N-dimethylformamide degradation, offering a new strategy for resource utilization of specific pollutants and wastewater treatment.

Succession of microbial communities reveals the inevitability of anammox core in the development of anammox processes.

The lack of anammox seeds is regarded as the bottleneck of anammox-based processes. Although the interactions in anammox consortia have attracted increasing attention, little is known about the influence of inoculated sludge populations on the growth of anammox bacteria. In this study, four sludge of distinct communities mixed with anammox sludge (the relative abundance of Ca. Kuenenia was 1.96 %) were used as the seeds, respectively for the start-up of anammox processes. Notably, all these mixed microbial communities tend to form a similar microbial community, defined as the anammox core, containing anammox-bacteria (22.9 ± 5.9 %), ammonia-oxidizing-bacteria (0.8 ± 0.7 %), nitrite-oxidizing-bacteria (0.2 ± 0.2 %), Chloroflexi-bacteria (0.7 ± 0.4 %), and heterotrophic-denitrification-bacteria (0.3 ± 0.2 %). It also elucidated that the communities of Nitrosomonas-dominated sludge were the closest to the anammox core, and achieved the highest nitrogen-removal rate of 0.73 kg-N m-3 d-1. This study sheds light on the solution to the shortage of anammox seeds in the full-scale wastewater treatment application.

Coupling continuous poly(3-hydroxybutyrate) synthesis with piperazine-contained wastewater treatment: Fermentation performance and microbial contamination deciphering.

Open poly(3-hydroxybutyrate) (PHB) fermentation is of great potential, and batch PHB synthesis with piperazine as the nitrogen switch has been realized. However, it is vital to explore the feasibility of continuous PHB fermentation with piperazine-contained wastewater remediation collaboratively. Here, an aerobic membrane bioreactor was constructed for consecutive PHB synthesis. The removal efficiency of piperazine decreased from 100 % to 82.6 % after three cycles, meanwhile, the PHB concentration was 0.39 g·L-1, 0.18 g·L-1, and undetected for each cycle. Microbial community analysis showed that Proteobacteria, Actinobacteriota, and Bacteroidota were the main contaminating microbes. Furthermore, three metagenome-assembled genomes related to Flavobacterium collumnare, Herbaspirillum aquaticum, and Microbacterium enclense were identified as the dominant contaminating strains. These microbes obtained nitrogenous substrates transformed by Paracoccus sp. TOH, such as amino acids and dissolved organic matter, as nutrient for accumulation. This study verified the practicability of coupling continuous PHB synthesis with industrial wastewater treatment and revealed the derivation mechanism of contaminating species, which could provide a reference for the targeted nitrogen release gene knockout of functional PHB fermentation chassis.

Bioconversion of waste activated sludge hydrolysate into polyhydroxyalkanoates using Paracoccus sp. TOH: Volatile fatty acids generation and fermentation strategy.

The expensive carbon matrix is a bottleneck restricting the industrialization of polyhydroxyalkanoates (PHAs). Volatile fatty acids (VFAs) derived from waste activated sludge via anaerobic fermentation might be alternative carbon matters for PHAs synthesis. In this study, the effect of enzymes on VFAs yields and the feasibility of the produced VFAs for PHAs fermentation by Paracoccus sp. TOH were investigated. The optimum cumulative VFAs concentration reached 4076.6 mg-COD·L-1 in the lysozyme treatment system. Correspondingly, the highest poly(3-hydroxybuturate-co-3-hydroxyvalerate) (PHBV) concentration (119.1 mg·L-1) containing 20.3 mol% 3-hydroxyvalerate was obtained. It proved that Paracoccus sp. TOH possesses the capability for PHBV accumulation. The functional hydrolytic-acidogenic microorganisms, such as Clostridium sensu stricto and Bacteroides sp. were accumulated. The functional genes encoding hydrolysis, carbohydrates metabolism, VFAs generation were enriched. This study offered a possible strategy for VFAs production and verified the feasibility of sludge hydrolysate as a high-quality carbon substrate for PHAs fermentation.

Microbial dynamics reveal the adaptation strategies of ecological niche in distinct anammox consortia under mainstream conditions.

The feasibility of anammox-based processes for nitrogen-contained wastewater treatment has been verified with different anammox bacteria, however, the ecological niche of anammox bacteria under mainstream conditions is still elusive. In this study, six sludge samples collected from different habitats were utilized to culture anammox bacteria under mainstream conditions, and two distinct anammox genera (Ca. Kuenenia and Ca. Brocadia) with a relative abundance of 6.31% (C1) and 3.09% (C3), respectively, were identified. Notably, the microbial dynamics revealed that anammox bacteria (AMX), ammonia-oxidizing bacteria (AOB), nitrite-oxidizing bacteria (NOB), Chloroflexi bacteria (CFX), and heterotrophic denitrification bacteria (HDB) were the core members in anammox consortia. However, Ca. Kuenenia and Ca. Brocadia occupied different ecological niches in anammox consortia. The dissolved oxygen and microbial structures of the anammox-continuous stirred tank reactor systems were the main factors to affect their niche differentiation. Meanwhile, comammox might exist in the systems and occupy the ecological niche of AOB in nitrogen cycling. The network analysis suggested that Ignavibacterium could be the associated bacteria in Ca. Kuenenia-dominated consortia, while Ca. Nitrotoga was that in the Ca. Brocadia-dominated consortia. Our findings reveal a valuable reference for the observation of distinct anammox genera under mainstream conditions, which provides theoretical guidance for the engineering application of mainstream anammox-based processes.

Nitrogen contribution and microbial community of size-fractionated anammox sludge in continuous stirred-tank reactors.

In this study, the microbial diversity of size-fractionated anammox sludge in a well-mixed system and their contribution to nitrogen transformation were investigated. Results showed that small granules (0.2-1.0 mm) contributed to the major part of the nitrogen removal rate (56 %) due to its largest mixed liquor volatile suspended solids (1240 ± 80 mg·L-1). However, large granules (>1.0 mm) possessed the highest relative abundances of Ca. Kuenenia stuttgartiensis and specific anammox activity, representing 49.34 % and 24.45 ± 0.01 mg-N·g-1-mixed liquor volatile suspended solids·h-1, respectively. The microbial diversity decreased as the increase of granular size, resulting in microbial community shifting to a simpler model. Metagenomic analysis showed that fine sludge might be the potential major for NO/N2O production in the mature well-mixed system under inorganic conditions. This study provides guidance for the evaluation of nitrogen contribution by anammox size-fractionated sludge and the inhibition of the potential NO/N2O emission in anammox processes.

Poly(3-hydroxybutyrate) biosynthesis under non-sterile conditions: Piperazine as nitrogen substrate control switch.

Poly(3-hydroxybutyrate) (PHB), as a kind of bioplastics for sustainable development, can be synthesized by various microorganisms, however, the high cost of its microbial fermentation is a challenge for its large-scale application. In this study, piperazine degrading strain, Paracoccus sp. TOH, was developed as an excellent chassis for open PHB fermentation with piperazine as controlling element. Whole-genome analysis showed that TOH possesses multi-substrate metabolic pathways to synthesize PHB. Next, TOH could achieve a maximum PHB concentration of 2.42 g L-1, representing a yield of 0.36 g-PHB g-1-glycerol when C/N ratio was set as 60:1 with 10 g L-1 glycerol as substrate. Furthermore, TOH could even synthesize 0.39 g-PHB g-1-glycerol under non-sterile conditions when piperazine was fed with a suitable rate of 1 mg L-1 h-1. 16S rRNA gene sequencing analysis showed that microbial contamination could be effectively inhibited through the regulation of piperazine under non-sterile conditions and TOH dominated the microbial community with a relative abundance of 72.3% at the end of the operational period. This study offers an inspired open PHB fermentation system with piperazine as the control switch, which will realize the goal of efficient industrial biotechnology as well as industrial wastewater treatment.

Promoter engineering enables overproduction of foreign proteins from a single copy expression cassette in Bacillus subtilis.

BACKGROUND: Bacillus subtilis is developed to be an attractive expression host to produce both secreted and cytoplasmic proteins owing to its prominent biological characteristics. Chromosomal integration is a stable expression strategy while the expression level is not ideal compared with plasmid expression. Thus, to meet the requirement of protein overexpression, promoter, as one of the key elements, is important. It is necessary to obtain an ideal promoter for overproduction of foreign proteins from a single copy expression cassette. RESULTS: The activity of promoter Pylb was further enhanced by optimizing the - 35, - 10 core region and upstream sequence (UP) by substituting both sequences with consensus sequences. The final engineered promoter exhibited almost 26-fold in ß-galactosidase (BgaB) activity and 195-fold in super-folded green fluorescent protein (sfGFP) intensity than that of WT. The two proteins account for 43% and 30% of intracellular proteins, respectively. The promoter was eventually tested by successful extracellular overproduction of Methyl Parathion Hydrolase (MPH) and Chlorothalonil hydrolytic dehalogenase (Chd) to a level of 0.3 g/L (144 U/mL) and 0.27 g/L (4.4 U/mL) on shake-flask culture condition. CONCLUSIONS: A strong promoter was engineered for efficient chromosomally integrated expression of heterologous proteins.

Construction of second generation protease-deficient hosts of Bacillus subtilis for secretion of foreign proteins.

Although one of the major factors limiting the application of Bacillus subtilis as an expression host has been its production of at least eight extracellular proteases, researchers have also noticed that some proteases benefited the secretion of foreign proteins at times. Therefore, to maximize the yield of a foreign protein, the proteases should be selectively inactivated. This raises a new question that how to identify the favorable and unfavorable proteases for a target protein. Here, an evaluation system containing nine mutant strains of B. subtilis 168 was developed to address this question. The mutant strain PD8 has all the eight proteases inactivated whereas each of the other eight mutant strains expresses only one kind of these eight proteases. The target protein is secreted in these nine mutant strains; if the production of target protein in a mutant strain is higher than that in strain PD8, the corresponding protease is regarded as favorable. Accordingly, the optimal protease-deficient host is constructed through inactivating the unfavorable proteases. The effectiveness of this system was confirmed by expressing three foreign proteins. This study provides a strategy for improving the secretion of a foreign protein in B. subtilis through tailoring a personalized protease-deficient host.

Unmarked genetic manipulation in Bacillus subtilis by natural co-transformation.

Bacillus subtilis is well known as both a model organism and as a microbial cell factory. Simple and scarless gene modification is a desirable tool for basic research and industrial applications of B. subtilis. It has been demonstrated that naturally competent strains of B. subtilis can uptake multiple different DNA molecules, a phenomenon called co-transformation. Here, we describe a co-transformation-based method for generating unmarked mutants of B. subtilis. The PCR product containing the desired mutant allele is introduced into B. subtilis through co-transformation of the plasmid pUS20, which harbours a spectinomycin-resistant marker (Spcr). The target mutation is acquired by screening transformants for integration of pUS20 by resistance to spectinomycin. Due to its unstable replication in B. subtilis, pUS20 is easily cured from transformants in the absence of spectinomycin. This method allows for point mutation delivery at frequencies of approximately 30%. Deletions and insertions of long DNA fragments can also be carried out efficiently using this method. Moreover, this method is also successful in Bacillus velezensis, indicating that it may be extended to other Bacillus species that can form natural competence.

Molecular Mechanism and Genetic Determinants of Buprofezin Degradation.

Buprofezin is a widely used insect growth regulator whose residue has been frequently detected in the environment, posing a threat to aquatic organisms and nontarget insects. Microorganisms play an important role in the degradation of buprofezin in the natural environment. However, the relevant catabolic pathway has not been fully characterized, and the molecular mechanism of catabolism is still completely unknown. Rhodococcus qingshengii YL-1 can utilize buprofezin as a sole source of carbon and energy for growth. In this study, the upstream catabolic pathway in strain YL-1 was identified using tandem mass spectrometry. Buprofezin is composed of a benzene ring and a heterocyclic ring. The degradation is initiated by the dihydroxylation of the benzene ring and continues via dehydrogenation, aromatic ring cleavage, breaking of an amide bond, and the release of the heterocyclic ring 2-tert-butylimino-3-isopropyl-1,3,5-thiadiazinan-4-one (2-BI). A buprofezin degradation-deficient mutant strain YL-0 was isolated. A comparative genomic analysis combined with gene deletion and complementation experiments revealed that the gene cluster bfzBA3A4A1A2C is responsible for the upstream catabolic pathway of buprofezin. The bfzA3A4A1A2 cluster encodes a novel Rieske nonheme iron oxygenase (RHO) system that is responsible for the dihydroxylation of buprofezin at the benzene ring; bfzB is involved in dehydrogenation, and bfzC is in charge of benzene ring cleavage. Furthermore, the products of bfzBA3A4A1A2C can also catalyze dihydroxylation, dehydrogenation, and aromatic ring cleavage of biphenyl, flavanone, flavone, and bifenthrin. In addition, a transcriptional study revealed that bfzBA3A4A1A2C is organized in one transcriptional unit that is constitutively expressed in strain YL-1.IMPORTANCE There is an increasing concern about the residue and environmental fate of buprofezin. Microbial metabolism is an important mechanism responsible for the buprofezin degradation in the natural environment. However, the molecular mechanism and genetic determinants of microbial degradation of buprofezin have not been well identified. This work revealed that gene cluster bfzBA3A4A1A2C is responsible for the upstream catabolic pathway of buprofezin in Rhodococcus qingshengii YL-1. The products of bfzBA3A4A1A2C could also degrade bifenthrin, a widely used pyrethroid insecticide. These findings enhance our understanding of the microbial degradation mechanism of buprofezin and benefit the application of strain YL-1 and bfzBA3A4A1A2C in the bioremediation of buprofezin contamination.