Dual auxotrophy coupled red labeling strategy for efficient genome editing in Saccharomyces cerevisiae.

The homologous recombination strategy has a long history of editing Saccharomyces cerevisiae target genes. The application of CRISPR/Cas9 strategy to editing target genes in S. cerevisiae has also received a lot of attention in recent years. All findings seem to indicate that editing relevant target genes in S. cerevisiae is an extremely easy event. In this study, we systematically analyzed the advantages and disadvantages of homologous recombination (HR) strategy, CRISPR/Cas9 strategy, and CRISPR/Cas9 combined homology-mediated repair (CRISPR/Case9-HDR) strategy in knocking out BY4742 ade2. Our data showed that when the ade2 was knocked out by HR strategy, a large number of clones appeared to be off-target, and 10 %-80 % of the so-called knockout clones obtained were heteroclones. When the CRISPR/Cas9 strategy was applied, 60% of clones were off-target and the rest were all heteroclones. Interestingly, most of the cells were edited successfully, but at least 60 % of the clones were heteroclones, when the CRISPR/Cas9-HDR strategy was employed. Our results clearly showed that the emergence of heteroclone seems inevitable regardless of the strategies used for editing BY4742 ade2. Given the characteristics of BY4742 defective in ade2 showing red on the YPD plate, we attempted to build an efficient yeast gene editing strategy, in which the CRISPR/Cas9 combines homology-mediated repair template carrying an ade2 expression cassette, BY4742(ade2Δ0) as the start strain. We used this strategy to successfully achieve 100 % knockout efficiency of trp1, indicating that technical challenges of how to easily screen out pure knockout clones without color phenotype have been solved. Our data showed in this study not only establishes an efficient yeast gene knockout strategy with dual auxotrophy coupled red labeling but also provides new ideas and references for the knockout of target genes in the monokaryotic mycelium of macrofungi.

Cloning, Expression and Characterization of Two Beta Carbonic Anhydrases from a Newly Isolated CO2 Fixer, Serratia marcescens Wy064.

Bacterial strains from karst landform soil were enriched via chemostat culture in the presence of sodium bicarbonate. Two chemolithotrophic strains were isolated and identified as Serratia marcescens Wy064 and Bacillus sp. Wy065. Both strains could grow using sodium bicarbonate as the sole carbon source. Furthermore, the supplement of the medium with three electron donors (Na2S, NaNO2, and Na2S2O3) improved the growth of both strains. The activities of carbonic anhydrase (CA) and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) could be detected in the crude enzyme of strain Wy064, implying that the strain Wy064 might employ Calvin cycle to fix CO2. S. marcescens genome mining revealed four potential CA genes designated CA1-CA4. The proteins encoded by genes CA1-3 were cloned and expressed in Escherichia coli. The purified recombinant enzymes of CA1 and CA3 exhibited CO2 hydration activities, whereas enzyme CA2 was expressed in inclusion bodies. A CO2 hydration assay demonstrated that the specific activity of CA3 was significantly higher than that of CA1. The maximum CO2 hydration activities for CA1 and CA3 were observed at pH 7.5 and 40 °C. The activities of CA1 and CA3 were significantly enhanced by several metal ions, especially Zn2+, which resulted in 21.1-fold and 26.1-fold increases of CO2 hydration activities, respectively. The apparent K m and V max for CO2 as substrate were 27 mM and 179 WAU/mg for CA1, and 14 mM and 247 WAU/mg for CA3, respectively. Structure modeling combined with sequence analysis indicated that CA1 and CA3 should belong to the Type II ß-CA.

GANT-61 and GDC-0449 induce apoptosis of prostate cancer stem cells through a GLI-dependent mechanism.

Aberrant reactivation of the Sonic Hedgehog (SHH) signaling pathway promotes prostate cancer (PC) growth and progression by regulating cancer-related genes through its downstream effectors GLI1 and GLI2. Therefore, targeting the SHH-GLI pathway provides an alternative approach to avoid cancer progression. The aim of this study was to delineate the underlying molecular mechanisms by which GDC-0449 (a SMO receptor inhibitor) and GANT-61 (a GLI transcription factor inhibitor) regulate cellular proliferation and self-renewal in human PC stem cells (ProCSCs). Inhibition of the SHH signaling pathway by GANT-61 induced apoptosis with more efficacy than by GDC-0449 in ProCSCs and PC cell lines. GLI1 and GLI2 expression, promoter-binding activity and GLI-responsive luciferase reporter activity were all decreased with either GDC-0449 or GANT-61 treatment. Expression of Fas, DR4, DR5, and cleavage of caspase-3 and PARP were increased, whereas levels of PDGFR-α and Bcl-2 were reduced. Double knockout of GLI1 and GLI2 using shRNA abolished the effects observed with either GDC-0449 or GANT-61 treatment. Collectively, our results showed that GANT-61 and GDC-0449 induced ProCSC apoptosis by directly or indirectly inhibiting the activities of the GLI family transcription factors, may enhance the efficacy of PC treatment.

Importance of a Laccase Gene (Lcc1) in the Development of Ganoderma tsugae.

In this study, a novel laccase gene (Lcc1) from Ganoderma tsugae was isolated and its functions were characterized in detail. The results showed that Lcc1 has the highest expression activity during mycelium development and fruit body maturation based on the analysis of Lcc1 RNA transcripts at different developmental stages of G. tsugae. To investigate the exact contribution of Lcc1 to mycelium and fruit body development in G. tsugae, Lcc1 transgenic strains were constructed by targeted gene replacement and over-expression approaches. The results showed that the lignin degradation rate in Lcc1 deletion mutant was much lower than the degradation efficiency of the wild-type (WT), over-expression and rescue strains. The lignin degradation activity of G. tsugae is dependent on Lcc1 and the deletion of Lcc1 exerted detrimental influences on the development of mycelium branch. Furthermore, the study uncovered that Lcc1 deletion mutants generated much shorter pale grey fruit bodies, suggesting that Lcc1 contributes directly to pigmentation and stipe elongation during fruit body development in G. tsugae. The information obtained in this study provides a novel and mechanistic insight into the specific role of Lcc1 during growth and development of G. tsugae.

Molecular analysis and biochemical characteristics of degenerated strains of Cordyceps militaris.

Cordyceps militaris has commercially been cultivated, but its degenerated subcultures have gradually resulted in the reduced production. In this study, the biological characteristics and DNA change of degenerated strains of C. militaris were analyzed in detail. The results showed that the degenerated strains exhibited the lower growth rate, and the deficiency in fruit body formation and pigment production. The degradation of strains was not attributable to DNA changes identified by RAPD and SRAP. Compared to normal strains, the biochemical indexes of degradation strains and normal strains showed that the carotenoid content of degradation strains was significantly lower, the activities of cellulase and amylase of degradation strains were slight lower, and the EPS content was lower, but the IPS was higher. All these results suggested that the degradation of C. militaris may be caused by the inhibition or in harmony of metabolite synthesis involved in the metabolic regulation, which should be further verified.

A new NAD(H)-dependent meso-2,3-butanediol dehydrogenase from an industrially potential strain Serratia marcescens H30.

The budC gene coding for a new meso-2,3-butanediol dehydrogenase (BDH) from Serratia marcescens H30 was cloned and expressed in Escherichia coli BL21(DE3), purified, and characterized for its properties. The recombinant BDH with a molecular weight of 27.4 kDa exhibited a reversible transformation between acetoin and 2,3-butanediol. In the presence of NADH, BDH could catalyze the reduction of diacetyl and (3R)-acetoin to (3S)-acetoin and meso-2,3-butanediol, respectively, while (3S)-acetoin as a substrate could be further transformed into (2S, 3S)-2,3-butanediol at pH 9.0. For diol oxidation reactions, (3R)-acetoin and (3S)-acetoin were obtained when meso-2,3-butanediol and (2S,3S)-2,3-butanediol were used as the substrates with BDH and NAD(+). (2R,3R)-2,3-butanediol was not a substrate for the BDH at all. The low K m value (4.1 mM) in meso-2,3-butanediol oxidation reaction and no activity for diacetyl, acetoin, and 2,3-butanediol as the substrates with NADP(+)/NADPH suggested that the budC gene product belongs to a NAD(H)-dependent meso-2,3-BDH. Maximum activities for diacetyl and (3S/3R)-acetoin reduction were observed at pH 8.0 and pH 5.0 while for meso-2,3-butanediol oxidation it was pH 8.0. However, the optimum temperature for oxidation and reduction reactions was about 40 °C. In addition, the BDH activity for meso-2,3-butanediol oxidation was enhanced in the presence of Fe(2+) and for diacetyl and (3S/3R)-acetoin reduction in the presence of Mg(2+) and Mn(2+), while several metal ions inhibited its activity, particularly Fe(3+) for reduction of diacetyl and acetoin. Sequence analysis showed that the BDH from S. marcescens H30 possessed two conserved sequences including the coenzyme binding motif (GxxxGxG) and the active-site motif (YxxxK), which are present in the short-chain dehydrogenase/reductase superfamily.

Cloning, expression and characterization of glycerol dehydrogenase involved in 2,3-butanediol formation in Serratia marcescens H30.

The meso-2,3-butanediol dehydrogenase (meso-BDH) from S. marcescens H30 is responsible for converting acetoin into 2,3-butanediol during sugar fermentation. Inactivation of the meso-BDH encoded by budC gene does not completely abolish 2,3-butanediol production, which suggests that another similar enzyme involved in 2,3-butanediol formation exists in S. marcescens H30. In the present study, a glycerol dehydrogenase (GDH) encoded by gldA gene from S. marcescens H30 was expressed in Escherichia coli BL21(DE3), purified and characterized for its properties. In vitro conversion indicated that the purified GDH could catalyze the interconversion of (3S)-acetoin/meso-2,3-butanediol and (3R)-acetoin/(2R,3R)-2,3-butanediol. (2S,3S)-2,3-Butanediol was not a substrate for the GDH at all. Kinetic parameters of the GDH enzyme showed lower K m value and higher catalytic efficiency for (3S/3R)-acetoin in comparison to those for (2R,3R)-2,3-butanediol and meso-2,3-butanediol, implying its physiological role in favor of 2,3-butanediol formation. Maximum activity for reduction of (3S/3R)-acetoin and oxidations of meso-2,3-butanediol and glycerol was observed at pH 8.0, while it was pH 7.0 for diacetyl reduction. The enzyme exhibited relative high thermotolerance with optimum temperature of 60 °C in the oxidation-reduction reactions. Over 60 % of maximum activity was retained at 70 °C. Additionally, the GDH activity was significantly enhanced for meso-2,3-BD oxidation in the presence of Fe(2+) and for (3S/3R)-acetoin reduction in the presence of Mn(2+), while several cations inhibited its activity, particularly Fe(2+) and Fe(3+) for (3S/3R)-acetoin reduction. The properties provided potential application for single configuration production of acetoin and 2,3-butanediol .

A novel breeding strategy for new strains of Hypsizygus marmoreus and Grifola frondosa based on ligninolytic enzymes.

A novel breeding strategy for new strains of Hypsizygus marmoreus and Grifola frondosa using ligninolytic enzymes as markers was evaluated with the detection and analysis of activities and composition of 15 edible fungi. The results showed that the activity and composition of ligninolytic enzyme system varied in response to changes of fungal strains. By analyzing the growth rate of mycelia and their ability to produce ligninolytic enzymes, H. marmoreus and P. geesteranus, G. frondosa and P. sajor-caju were screened for further study. Three colonies of 26 regenerated colonies of H. marmoreus and P. geesteranus protoplast fusion and one colony of 48 regenerated colonies of G. frondosa and P. sajor-caju were selected respectively. At the same time, these four strains were identified using RAPD and ISSR molecular markers. The results showed that the strains HM5G1 and PS7F1 are new strains and have low similarity to parental strains H. marmoreus and G. frondosa. These results are supported by the results of antagonism tests. These two fusants were significantly higher in their ligninolytic enzyme activity than H. marmoreus and G. frondosa. The growth rates of strains HM5G1and PS7F1 were also noticeably higher than those of H. marmoreus and G. frondosa, by 1.36 and 1.5 times respectively. The biological efficiency of the strain HM5G1 was 11.5% higher than that of the parental strain H. marmoreus. This work suggests that it is an efficient way of breeding new strains to use the decolorization of ligninolytic enzymes as a preliminary screening marker.

Analysis and modification of central carbon metabolism in Hypsizygus marmoreus for improving mycelial growth performance and fruiting body yield.

Hypsizygus marmoreus is one of the main industrially cultivated varieties of edible fungi, with a delicious taste and high nutritional value. However, the long harvest period of 130-150 days greatly limits its large-scale expansion. This study aimed to investigate the effects of central carbon metabolism (CCM) on the mycelial growth performance and fruiting body formation of H. marmoreus. Nine edible fungi with different harvest periods were collected and used to evaluate their intracellular carbon metabolic differences in the CCM, which revealed that the imbalanced distribution of intracellular carbon metabolic levels in the CCM of H. marmoreus might be one of the key factors resulting in a slow mycelial growth rate and a long harvest period. Further analysis by three strategies, including metabolomics, adaptation of different carbon sources, and chemical interference, confirmed that low carbon flux into the pentose phosphate pathway (PPP) limited the supply of raw materials, reduced power, and thus influenced the mycelial growth of H. marmoreus. Furthermore, four transformants with increased expression levels of glucose-6-phosphate dehydrogenase (G6PDH), a key rate-limiting enzyme in the PPP of H. marmoreus, were developed and showed more extracellular soluble protein secretion and higher sugar assimilation rates, as well as improved mycelial growth rates in bottle substrate mixtures. Finally, cultivation experiments indicated that the maturation periods of the fruiting body with ~4-5 days in advance and the maximum fruiting body yield of 574.8 g per bag with an increase of 7.4% were achieved by improving the G6PDH expression level of the PPP in H. marmoreus. This study showed that CCM played an important role in the mycelial growth and development of H. marmoreus, which provided new insights for future advancements in cultivating and breeding edible fungi.

The novel role of fungal intracellular laccase: used to screen hybrids between Hypsizigus marmoreus and Clitocybe maxima by protoplasmic fusion.

Laccase has been proved important in decolorization of Remazol Brilliant Blue R (RBBR), oxidation of 2, 2'-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, lignin degradation and fruiting-body formation. The decolorization of RBBR by laccase was firstly used to screen protoplast fusants. Fusants were obtained by protoplast fusion between the strains of Hypsizigus marmoreus and Clitocybe maxima, and two fusants (IM1 and IIIM5) were screened on PDA medium containing RBBR. These fusants were significant higher in laccase activity than H. marmoreus, nearly 413 and 395 times, respectively. Their hyphal growth rates were also remarkable higher than H. marmoreus, nearly 1.5 and 1.4 times, respectively. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis showed these fusants contained the laccase, and the molecular mass of the laccase was consistent with the laccase of C. maxima, nearly 62 kDa. The pileus color of the IM1 and IIIM5 also showed partial recombined characteristics comparing to the parental strains, while biological efficiency ratios were prominent higher than that of H. marmoreus, up to 14.58 and 10.87 %, respectively. Randomly amplified polymorphic DNA bands of fusants not only were similar to parental bands, but presented new non-parental bands. Using the Unweighted pair-group method together with mathematic averages method to gain a dendrogram, in which the fusants showed intra-cluster variations. Significantly, H. marmoreus was the dominant parent, while C. maxima were distant from the fusants. The differences among IM1, IIIM5 and H. marmoreus, and the similarities among IM1, IIIM5 and C. maxima indicated IM1 and IIIM5 were somatic hybrids of H. marmoreus and C. maxima. Accordingly, it is feasible to use laccase to screen fusants of H. marmoreus and C. maxima.

Effects of Drying Methods on the Physicochemical Aspects and Volatile Compounds of Lyophyllum decastes.

In this study, fresh Lyophyllum decastes was dried using hot air drying (HAD), hot air combined with vacuum drying (HAVD), and vacuum freeze drying (VFD). Additionally, the quality and volatile compounds were analyzed. VFD achieved the best color retention, the highest rehydration capacity, and the slightest damaged tissue structure; however, it recorded the longest drying time and the highest energy consumption. HAD was the most energy-efficient of the three methods. Furthermore, the products with more hardness and elasticity were obtained by HAD and HAVD-this finding was convenient for transportation. In addition, GC-IMS demonstrated that the flavor components had significantly changed after drying. A total of 57 volatile flavor compounds was identified, and the aldehyde, alcohol, and ketone compounds were the primary ingredient of the L. decastes flavor component, whereby the relative content of the HAD sample was apparently higher than HAVD and VFD. Taken together, VFD was better at preserving the color and shape of fresh L. decastes, but HAD was more appropriate for drying L. decastes because of the lower energy consumption, and was more economical. Meanwhile, HAD could be used to produce a more intense aroma.

A fungal cellulose nanocrystals-based approach to improve the stability of triterpenes loaded Pickering emulsion.

Depolysaccharide residues of edible fungus Pleurotus eryngii (dePSR-Pe), a mushroom industry waste, have abundant cellulose. In this study, the cellulose nanocrystals of P. eryngii (PeCNs) were extracted by hydrochloric acid. Results showed that the length of PeCNs is 469 ± 76.41 nm with a high aspect ratio of 40-100 and needle morphology. The structural characterization revealed that PeCNs had good thermal stability (approach 300 °C) and high crystallinity (84.2 %). An O/W Pickering emulsion stabilized with PeCNs was prepared to inhibit lipid oxidation and improve the loading capacity of triterpenes of P. coco. Unimodal size distribution of emulsion droplets was obtained under an optimized aqueous-phase condition to form a metastable emulsion, regardless of varying oil-water volume ratio <50/50. In vitro digestion study suggested that triterpenes-loaded Pickering emulsion had 1-3 times higher drug stability than bulk oil. These metastable Pickering emulsions call for fewer nanoparticles and provide a new strategy for the industry application of cellulose nanocrystals at less cost.

A qualitative study of the vocational and psychological perceptions and issues of transdisciplinary nurses during the COVID-19 outbreak.

BACKGROUND: Due to its high infectivity and concealment, the coronavirus disease 2019 (COVID-19) outbreak that occurred in Wuhan attracted global attention. A special nursing group of transdisciplinary nurses (TNs) who had not worked in respiratory medicine, infection departments, or emergency and intensive medicine but who accounted for a large proportion of all nurses also drew our attention. Few studies have examined this special group of TNs. Therefore, this study collected the experiences and views of TNs at the forefront of the COVID-19 outbreak to investigate their potential problems. RESULTS: Twenty-five TNs and 19 nurses with experience in infectious diseases (non-TNs) were enrolled in the study. Compared with non-TNs, TNs showed higher levels of perceived stress and relatively less perceived social support. For TNs, the ambiguous roles, transition of operating mode, unfamiliar work content, and reversal of their daily schedule were the most common vocational problems. Additionally, most TNs had psychological problems such as anxiety, pain and insomnia. The incomprehension of parents, concern for family members and long-term isolation were the most common causes of psychological stress. CONCLUSION: This survey is the first to focus on the group of TNs at the forefront of the COVID-19 outbreak and to investigate their experiences, vocational issues and psychological stresses qualitatively and quantificationally. We found that TNs had more perceived stress and less perceived social support than non-TNs. The vocational and psychological issues of TNs should be highlighted. These findings identify important issues and offer insights into the underlying issues to help TNs ultimately win the battle against novel coronavirus epidemics. METHODS: Semi-structured and face-to-face individual interviews and quantitative assessments were conducted. The Braun Clarke Thematic Analysis method and the strategy outlined by Miles and Huberman were used in the data analysis process of the qualitative study. The perceived stress scale and perceived social support scale were utilized to quantificationally evaluate the perceived stress level and the amount of perceived social support. Both qualitative and quantitative methods were adopted to assess the vocational and psychological perceptions and issues.

The adverse skin reactions of health care workers using personal protective equipment for COVID-19.

In December 2019, a new coronavirus was found in Wuhan, Hubei Province, China, and spread rapidly throughout the country, attracting global attention. On February 11, the World Health Organization (WHO) officially named the disease caused by 2019-nCoV coronavirus disease 2019 (COVID-19). With the increasing number of cases, health care workers (HCWs) from all over China volunteered to work in Hubei Province. Because of the strong infectivity of COVID-19, HCWs need to wear personal protective equipment (PPE), such as N95 masks, latex gloves, and protective clothing. Due to the long-term use of PPE, many adverse skin reactions may occur. Therefore, the purpose of this study is to explore the adverse skin reactions among HCWs using PPE.Questionnaires were used for the research; a quantitative study was carried out to determine the incidence of adverse skin reactions among HCWs using PPE.A total of 61 valid questionnaires were collected. The most common adverse skin reactions among HCWs wearing N95 masks were nasal bridge scarring (68.9%) and facial itching (27.9%). The most common adverse skin reactions among HCWs wearing latex gloves were dry skin (55.7%), itching (31.2%), and rash (23.0%). The most common adverse skin reactions among HCWs wearing protective clothing were dry skin (36.1%) and itching (34.4%).When most HCWs wear PPE for a long period of time, they will experience adverse skin reactions. The incidence of adverse skin reactions to the N95 mask was 95.1%, that to latex gloves was 88.5%, and that to protective clothing was 60.7%.

Barriers to using personal protective equipment by healthcare staff during the COVID-19 outbreak in China.

The spread of coronavirus disease 2019 (COVID-19) around the world has put a heavy burden on human society and is also a great challenge facing medical staff. This study aimed to assess the difficulties faced by health care personnel (HCP) in using personal protective equipment (PPE) in clinical practice during the COVID-19 outbreak in Wuhan, China. One hundred twenty medical staff from the First Affiliated Hospital of Chongqing Medical University presented to the Wuhan First Hospital to provide medical assistance, from whom 20 HCP volunteered to participate in a focus group discussion attended by infection control nurse leaders. Participants' responses and discussions were recorded, and the content was analyzed for themes. Observed difficulties included inappropriate PPE sizes, the design of the PPE and its complexity of use, doubts related to the quality and effectiveness of PPE, potential risks during doffing, space layout between clean and contaminated area, and poor comfort with PPE use. Other factors, such as the support environment, management, processes, preparedness, HCP, and equipment can also have a positive or negative impact on the use of PPE. Future efforts to optimize PPE use should focus on strengthening training for HCP using real items for increasing compliance with standardized protocols, improving PPE design, and performing further research on the risks, benefits, and best practices of PPE use.

N-Acyl-Homoserine Lactone Quorum Sensing Switch from Acidogenesis to Solventogenesis during the Fermentation Process in Serratia marcescens MG1.

N-acyl-homoserine lactone quorum sensing (AHL-QS) has been shown to regulate many physiological behaviors in Serratia marcescens MG1. In the current study, the effects of AHL-QS on the biosynthesis of acid and neutral products by S. marcescens MG1 and its isogenic ∆swrI with or without supplementing exogenous N-hexanoyl-L-homoserine lactone (C6-HSL) were systematically investigated. The results showed that swrI disruption resulted in rapid pH drops from 7.0 to 4.8, which could be restored to wild type by supplementing C6-HSL. Furthermore, fermentation product analysis indicated that ∆swrI could lead to obvious accumulation for acidogenesis products such as lactic acid and succinic acid, especially excess acetic acid (2.27 g/l) produced at the early stage of fermentation, whereas solventogenesis products by ∆swrI appeared to noticeably decrease by an approximate 30% for acetoin during 32-48 h and by an approximate 20% for 2,3-butanediol during 24-40 h, when compared to those by wild type. Interestingly, the excess acetic acid produced could be removed in an AHL-QS-independent manner. Subsequently, quantitative real-time PCR was used to determine the mRNA expression levels of genes responsible for acidogenesis and solventogenesis and showed consistent results with those of product synthesis. Finally, by close examination of promoter regions of the analyzed genes, four putative luxI box-like motifs were found upstream of genes encoding acetyl-CoA synthase, lactate dehydrogenase, α-acetolactate decarboxylase, and Lys-like regulator. The information from this study provides a novel insight into the roles played by AHL-QS in switching from acidogenesis to solventogenesis in S. marcescens MG1.

Piggery wastewater treatment by Acinetobacter sp. TX5 immobilized with spent mushroom substrate in a fixed-bed reactor.

Acinetobacter sp. TX5 immobilized with spent Hypsizygus marmoreus substrate (SHMS) was used to treat the raw piggery wastewater (RPW). In batch experiments, NH4+-N in the diluted RPW decreased from initial 34.95 mg/L to 3.83 mg/L at 8 h with the removal efficiency (RE) being 89%, and the beads immobilized with SHMS were comparable to those immobilized with activated carbon. In continuous experiments, the RE ranged from 74% to 95% for NH4+-N, from 73% to 93% for TN and from 54% to 82% for COD when the RPW was treated in a fixed-bed reactor packed with SHMS-immobilized TX5. The isotope analysis and enzyme purification indicated simultaneous nitrification and denitrification existing in TX5. This is the first time that spent mushroom substrates have been used to immobilize Acinetobacter species to treat the real RPW and a denitrifying nitrite reductase (dNiR) has been purified to make the nitrogen removal pathway in this species clearer.

Enhanced nitrogen removal with spent mushroom compost in a sequencing batch reactor.

In order to remove nitrogen effectively from the wastewater with a low C/N ratio, the feasibility of using spent mushroom compost (SMC) hydrolysates as carbon sources for denitrification was investigated in a sequencing batch reactor (SBR). With SMCs supplement, the SBR performance was improved obviously within the 180days of operation. The total nitrogen removal was promoted from 46.9% to 81-89.4%, and no negative impact induced by different SMCs on the SBR system was observed. The abundance of functional genes including amoA, nirS/K, norB and nosZ in the active sludge was quantified by qPCR, and most of them elevated after SMC was fed. 16S rRNA gene high-throughput sequencing showed that the significant change in microbial community not only promoted pollutants removal but also benefited the stability of the reactor. Therefore, SMC could be an extremely promising carbon source used for nitrogen removal due to its cost-effective and efficient characteristics.

Municipal wastewater treatment by the bioaugmentation of Bacillus sp. K5 within a sequencing batch reactor.

Artificial municipal wastewater was treated successfully by the bioaugmentation of Bacillus sp. K5 capable of simultaneous nitrification and denitrification (SND) within a sequencing batch reactor (SBR). During the long-term operation, the bioaugmentation system exhibited an excellent and steady COD and NH4+-N removal without nitrite and nitrate accumulation. The average removal efficiency for COD and NH4+-N achieved to 98% and 95%, respectively. PCR-DGGE, SEM and FISH revealed that the introduced Bacillus sp. K5 should be an important functional strain and exerted a critical influence on the structure of microbial community. qPCR analysis indicated that the strain K5 facilitated aerobic nutrients removal capabilities and SND might be the primary pathway for the nitrogen removal in the SBR. Overall, the SBR system used in our study should be very promising for the future wastewater treatment.

Mechanism of 2,3-butanediol stereoisomers formation in a newly isolated Serratia sp. T241.

Serratia sp. T241, a newly isolated xylose-utilizing strain, produced three 2,3-butanediol (2,3-BD) stereoisomers. In this study, three 2,3-butanediol dehydrogenases (BDH1-3) and one glycerol dehydrogenase (GDH) involved in 2,3-BD isomers formation by Serratia sp. T241 were identified. In vitro conversion showed BDH1 and BDH2 could catalyzed (3S)-acetoin and (3R)-acetoin into (2S,3S)-2,3-BD and meso-2,3-BD, while BDH3 and GDH exhibited the activities from (3S)-acetoin and (3R)-acetoin to meso-2,3-BD and (2R,3R)-2,3-BD. Four encoding genes were assembled into E. coli with budA (acetolactate decarboxylase) and budB (acetolactate synthase), responsible for converting pyruvate into acetoin. E. coli expressing budAB-bdh1/2 produced meso-2,3-BD and (2S,3S)-2,3-BD. Correspondingly, (2R,3R)-2,3-BD and meso-2,3-BD were obtained by E. coli expressing budAB-bdh3/gdh. These results suggested four enzymes might contribute to 2,3-BD isomers formation. Mutants of four genes were developed in Serratia sp. T241. Δbdh1 led to reduced concentration of meso-2,3-BD and (2S,3S)-2,3-BD by 97.7% and 87.9%. (2R,3R)-2,3-BD with a loss of 73.3% was produced by Δbdh3. Enzyme activity assays showed the decrease of 98.4% and 22.4% by Δbdh1 and Δbdh3 compared with the wild strain. It suggested BDH1 and BDH3 played important roles in 2,3-BD formation, BDH2 and GDH have small effects on 2,3-BD production by Serratia sp. T241.