Symbiotic virus-bacteria interactions in biological treatment of coking wastewater manipulating bacterial physiological activities.

Biological treatment is commonly used in coking wastewater (CWW) treatment. Prokaryotic microbial communities in CWW treatment have been comprehensively studied. However, viruses, as the critical microorganisms affecting microbial processes and thus engineering parameters, still remain poorly understood in CWW treatment context. Employing viromics sequencing, the composition and function of the viral community in CWW treatment were discovered, revealing novel viral communities and key auxiliary metabolic functions. Caudovirales appeared to be the predominant viral order in the oxic-hydrolytic-oxic (OHO) CWW treatment combination, showing relative abundances of 62.47 %, 56.64 % and 92.20 % in bioreactors O1, H and O2, respectively. At the family level, Myoviridae, Podoviridae and Siphoviridae mainly prevailed in bioreactors O1 and H while Phycodnaviridae dominated in O2. A total of 56.23-92.24% of novel viral contigs defied family-level characterization in this distinct CWW habitat. The virus-host prediction results revealed most viruses infecting the specific functional taxa Pseudomonas, Acidovorax and Thauera in the entire OHO combination, demonstrating the viruses affecting bacterial physiology and pollutants removal from CWW. Viral auxiliary metabolic genes (AMGs) were screened, revealing their involvement in the metabolism of contaminants and toxicity tolerance. In the bioreactor O1, AMGs were enriched in detoxification and phosphorus ingestion, where glutathione S-transferase (GSTs) and beta-ketoadipyl CoA thiolase (fadA) participated in biodegradation of polycyclic aromatic hydrocarbons and phenols, respectively. In the bioreactors H and O2, the AMGs focused on cell division and epicyte formation of the hosts, where GDPmannose 4,6-dehydratase (gmd) related to lipopolysaccharides biosynthesis was considered to play an important role in the growth of nitrifiers. The diversities of viruses and AMGs decreased along the CWW treatment process, pointing to a reinforced virus-host adaptive strategy in stressful operation environments. In this study, the symbiotic virus-bacteria interaction patterns were proposed with a theoretical basis for promoting CWW biological treatment efficiency. The findings filled the gaps in the virus-bacteria interactions at the full-scale CWW treatment and provided great value for understanding the mechanism of biological toxicity and sludge activity in industrial wastewater treatment.

Boosting Potassium Storage by Integration Advantageous of Defect Engineering and Spatial Confinement: A Case Study of Sb2 Se3.

The potassium ion batteries (KIBs) based on conversion/alloying reaction mechanisms show high theoretical capacity. However, their applications are hampered by the poor cyclability resulting from the inherent large volume variations and the sluggish kinetics during K+ repeated insertion/extraction process. Herein, taken Sb2 Se3 as a model material, by rational design, nickel doped-carbon coated Sb2 Se3 nanorods (denoted as (Sb0.99 Ni0.01 )2 Se3 @C) are prepared through combined strategies of the conductive encapsulation and crystal structure modification. The carbon coating acts as an efficient buffer layer that maintains superior structural stability upon cycling. The introduction of Ni atoms can enhance electrical conductivity, leading to outstanding rate performance, which are confirmed by density functional theory calculation. The (Sb0.99 Ni0.01 )2 Se3 @C displays excellent reversible capacity (410 mAh g-1 at 0.1 A g-1 after 100 cycles) and unprecedented rate capability (140 mAh g-1 at 10 A g-1 ). Furthermore, KFeHCF//(Sb0.99 Ni0.01 )2 Se3 @C full cell exhibits a high specific capacity (408 mAh g-1 at 0.1 A g-1 ), superior rate capability (260 mAh g-1 at 2 A g-1 ). This work can open up a new avenue for the design of stable conversion/alloying-based anodes for high energy density KIBs.

Boosting Potassium Storage Performance of the Cu2S Anode via Morphology Engineering and Electrolyte Chemistry.

Transition metal sulfides (TMSs) have been demonstrated as attractive anodes for potassium-ion batteries (KIBs) due to the high capacity, abundant resource, and excellent redox reversibility. Unfortunately, practical implementation of TMSs to KIBs is still hindered by the unsatisfactory cyclability and rate performance which result from the vast volume variation during charge/discharge processes. Herein, a uniform nitrogen-doped carbon coated Cu2S hollow nanocube (Cu2S@NC) is designed as an anode material for the KIB, which displays an outstanding cycle performance (317 mAh g-1 after 1200 cycles at 1 A g-1) and excellent rate capacity (257 mAh g-1 at 6 A g-1) in a half-cell. The hollow nanosized structure can both shorten the diffusion length of potassium ions/electrons and buffer the volume expansion upon cycling. Besides, the high concentration electrolyte is beneficial to form the stable solid electrolyte interphase (SEI) film, reducing the interface impedance and enhancing the cycling stability. Ex situ transmission electron microscopy (TEM) and ex situ X-ray diffraction (XRD) reveal the reaction mechanism of Cu2S@NC.

Changes in nitrogen removal and microbiota of anammox biofilm reactors under tetracycline stress at environmentally and industrially relevant concentrations.

Anammox-related processes are often applied for the wastewater treatment which contains both ammonium and antibiotics. Herein, the long-term effects of tetracycline (TC), at environmentally and industrially relevant concentrations, on the performance, anammox activity and microbial community of anammox reactors were investigated for 518 days. The control reactor (without TC exposure) was stable for nitrogen removal during the long-term operation (a nitrogen removal rate of 0.56 ±â€¯0.05 kg-N·m-3·d-1). In the TC-added reactor, the nitrogen removal efficiency increased slightly at low TC levels (1-100 µg/L), whereas poor anammox performance occurred at high TC concentration (1000 µg/L). Furthermore, the concentrations of extracellular polymeric substances (EPS) were much higher at 10 µg/L than those in the control reactor (P < 0.01), whereas rapidly decreased at 1000 µg-TC/L. Furthermore, the reactor performance was highly consistent with the variations of the heme c contents. Consistently, exposure to TC changed the abundance of anammox bacteria, e.g., an increase in Candidatus Jettenia abundance occurred from 2.20 ±â€¯0.97% (0-10 µg/L) to 12.13 ±â€¯1.66% (100 µg/L). Similarly, the genus Denitratisoma, the most predominant denitrification bacteria, also had a higher abundance at a TC concentration of 100 µg/L (15.60 ±â€¯6.42%) than other TC concentrations (5.40 ±â€¯2.50% and 7.65 ±â€¯0.55% at concentrations of 10 and 1000 µg/L, respectively). The results can explain why the exposure of anammox bacteria to a lower TC concentration (100 µg/L) resulted in a better nitrogen removal rate. In contrast, exposure to a high TC level (1000 µg/L) led to a decline in the abundance of anammox bacteria and denitrifiers (1.53 ±â€¯0.64% and 8.18 ±â€¯0.63%, respectively) but an increased abundance in the nitrifier population (8.07 ±â€¯1.21%; P < 0.01). Therefore, this study can aid in the design and operation of anammox-based processes treating sewage and industrial wastewater.

Roles of quorum sensing in biological wastewater treatment: A critical review.

Quorum sensing (QS) and quorum quenching (QQ) are increasingly reported in biological wastewater treatment processes because of their inherent roles in biofilm development, bacterial aggregation, granulation, colonization, and biotransformation of pollutants. As such, the fundamentals and ubiquitous nature of QS bacteria are critical for fully understanding the process of the wastewater treatment system. In this article, the details of QS-based strategies related to community behaviors and phenotypes in wastewater treatment systems were reviewed. The molecular aspects and coexistence of QS and QQ bacteria were also mentioned, which provide evidence that future wastewater treatment will indispensably rely on QS-based strategies. In addition, recent attempts focusing on the use of QQ for biofilm or biofouling control were also summarized. Nevertheless, there are still several challenges and knowledge gaps that warrant future targeted research on the ecological niche, abundance, and community of QS- and QQ-bacteria in environmental settings or engineered systems.

Linking Exoproteome Function and Structure to Anammox Biofilm Development.

Extracellular proteins are of paramount importance in the cell-cell interactions of anammox biofilms. However, the inherent aggregation mechanisms of anammox have largely remained elusive. Herein, using a quartz sand extraction protocol and follow-up iTRAQ-based quantitative proteomics, we identified 367 extracellular proteins from initial colonizers, mature biofilm, and detached biofilm. The extracellular proteins were mainly membrane-associated. Most of the recovered proteins (226, 72.5%) originated from the phylum Planctomycetes. In summary, 215 and 190 of the 367 proteins recovered were up- and/or downregulated at least 1.2-fold during the biofilm formation and detachment periods, respectively. These differentially expressed proteins were dominantly involved in metal ion binding, which was regarded as strong evidence for their abilities to enhance ionic bridges in extracellular polymeric substances (EPS). Scanning electron microscopy-energy-dispersive X-ray spectroscopy (SEM-EDX) analysis of the biofilms further showed substantial levels of calcium and iron minerals. Critically, representative Sec-dependent secretory proteins affiliated with coccoid Planctomycetes, rod-shaped Proteobacteria, and filamentous Chloroflexi (11, 4, and 2 with differential expression, respectively) were found to have typical and abundant inner ß-sheet structures, wherein hydrophobic moieties can promote anammox aggregation. Overall, these findings highlight links between differentially expressed protein functions and morphologic traits of anammox consortia during biofilm development.

Day/night temperature differences (DNTD) trigger changes in nutrient removal and functional bacteria in membrane bioreactors.

Temperature is a well-known environmental stress that influences both microbial metabolism and community structure in the biological wastewater treatment systems. In this study, responses of biological performance and sludge microbiota to the long-term day/night temperature differences (DNTD) were investigated in membrane bioreactors (MBRs). The results showed that the functional bacteria could sustained their ecological functions at low DNTD (20/30 °C), resulting in relatively stable performance with respect to nutrient removal. However, when the activated sludge was subjected to a high DNTD (17/33 °C), the effluent concentrations of COD, TN and TP were significantly higher in MBR-B than that in MBR-A. In addition, more severe membrane fouling occurred under the perturbation of high DNTD as revealed by the transmembrane pressure (TMP) profile, which was mainly attributed to the accumulation of extracellular polymeric substances (EPS). The results of 16S rRNA gene sequencing showed that DNTD showed negligible effect on the bacterial community structures. Nonetheless, the functional bacteria responded differently to DNTD, which were in accordance with the bioreactor performances. Specifically, Nitrospina (NOB) and Tetrasphaera (PAOs) appeared to be sensitive to both low and high DNTD. In contrast, a low DNTD showed marginal effects on the denitrifiers, while a high DNTD significantly decreased their abundances. More strikingly, filamentous bulking bacteria were found to be well-adapted to DNTD, indicating their tolerance to the daily temperature fluctuation. This study will advance our knowledge regarding the response of microbial ecology of activated sludge to daily temperature variations in full-scale MBRs.

A Bacterial Multidomain NAD-Independent d-Lactate Dehydrogenase Utilizes Flavin Adenine Dinucleotide and Fe-S Clusters as Cofactors and Quinone as an Electron Acceptor for d-Lactate Oxidization.

Bacterial membrane-associated NAD-independent d-lactate dehydrogenase (Fe-S d-iLDH) oxidizes d-lactate into pyruvate. A sequence analysis of the enzyme reveals that it contains an Fe-S oxidoreductase domain in addition to a flavin adenine dinucleotide (FAD)-containing dehydrogenase domain, which differs from other typical d-iLDHs. Fe-S d-iLDH from Pseudomonas putida KT2440 was purified as a His-tagged protein and characterized in detail. This monomeric enzyme exhibited activities with l-lactate and several d-2-hydroxyacids. Quinone was shown to be the preferred electron acceptor of the enzyme. The two domains of the enzyme were then heterologously expressed and purified separately. The Fe-S cluster-binding motifs predicted by sequence alignment were preliminarily verified by site-directed mutagenesis of the Fe-S oxidoreductase domain. The FAD-containing dehydrogenase domain retained 2-hydroxyacid-oxidizing activity, although it decreased compared to the full Fe-S d-iLDH. Compared to the intact enzyme, the FAD-containing dehydrogenase domain showed increased catalytic efficiency with cytochrome c as the electron acceptor, but it completely lost the ability to use coenzyme Q10 Additionally, the FAD-containing dehydrogenase domain was no longer associated with the cell membrane, and it could not support the utilization of d-lactate as a carbon source. Based on the results obtained, we conclude that the Fe-S oxidoreductase domain functions as an electron transfer component to facilitate the utilization of quinone as an electron acceptor by Fe-S d-iLDH, and it helps the enzyme associate with the cell membrane. These functions make the Fe-S oxidoreductase domain crucial for the in vivo d-lactate utilization function of Fe-S d-iLDH.IMPORTANCE Lactate metabolism plays versatile roles in most domains of life. Lactate utilization processes depend on certain enzymes to oxidize lactate to pyruvate. In recent years, novel bacterial lactate-oxidizing enzymes have been continually reported, including the unique NAD-independent d-lactate dehydrogenase that contains an Fe-S oxidoreductase domain besides the typical flavin-containing domain (Fe-S d-iLDH). Although Fe-S d-iLDH is widely distributed among bacterial species, the investigation of it is insufficient. Fe-S d-iLDH from Pseudomonas putida KT2440, which is the major d-lactate-oxidizing enzyme for the strain, might be a representative of this type of enzyme. A study of it will be helpful in understanding the detailed mechanisms underlying the lactate utilization processes.

Coexistence of two d-lactate-utilizing systems in Pseudomonas putida KT2440.

It is advantageous for rhizosphere-dwelling microorganisms to utilize organic acids such as lactate. Pseudomonas putida KT2440 is one of the most widely studied rhizosphere-dwelling model organisms. The P. putida KT2440 genome contains an NAD-dependent d-lactate dehydrogenase encoding gene, but mutation of this gene does not play a role in d-lactate utilization. Instead, it was found that d-lactate utilization in P. putida KT2440 proceeds via a multidomain NAD-independent d-lactate dehydrogenase with a C-terminal domain containing several Fe-S cluster-binding motifs (Fe-S d-iLDH) and glycolate oxidase, which is widely distributed in various microorganisms. Both Fe-S d-iLDH and glycolate oxidase were identified to be membrane-bound proteins. Neither Fe-S d-iLDH nor glycolate oxidase is constitutively expressed but both of them can be induced by either enantiomer of lactate in P. putida KT2440. This study shows a case in which an environmental microbe contains two types of enzymes specific for d-lactate utilization.

A novel biocatalyst for efficient production of 2-oxo-carboxylates using glycerol as the cost-effective carbon source.

BACKGROUND: The surplus of glycerol has increased remarkably as a main byproduct during the biofuel's production. Exploiting an alternative route for glycerol utilization is significantly important for sustainability of biofuels. RESULTS: A novel biocatalyst that could be prepared from glycerol for producing 2-oxo-carboxylates was developed. First, Pseudomonas putida KT2440 was reconstructed by deleting lldR to develop a mutant expressing the NAD-independent lactate dehydrogenases (iLDHs) constitutively. Then, the Vitreoscilla hemoglobin (VHb) was heterologously expressed to further improve the biotransformation activity. The reconstructed strain, P. putida KT2440 (ΔlldR)/pBSPPcGm-vgb, exhibited high activities of iLDHs when cultured with glycerol as the carbon source. This cost-effective biocatalyst could efficiently produce pyruvate and 2-oxobutyrate from dl-lactate and dl-2-hydroxybutyrate with high molar conversion rates of 91.9 and 99.8 %, respectively. CONCLUSIONS: The process would not only be a promising alternative for the production of 2-oxo-carboxylates, but also be an example for preparation of efficient biocatalysts for the value-added utilization of glycerol.

Utilization of D-Lactate as an Energy Source Supports the Growth of Gluconobacter oxydans.

d-Lactate was identified as one of the few available organic acids that supported the growth of Gluconobacter oxydans 621H in this study. Interestingly, the strain used d-lactate as an energy source but not as a carbon source, unlike other lactate-utilizing bacteria. The enzymatic basis for the growth of G. oxydans 621H on d-lactate was therefore investigated. Although two putative NAD-independent d-lactate dehydrogenases, GOX1253 and GOX2071, were capable of oxidizing d-lactate, GOX1253 was the only enzyme able to support the d-lactate-driven growth of the strain. GOX1253 was characterized as a membrane-bound dehydrogenase with high activity toward d-lactate, while GOX2071 was characterized as a soluble oxidase with broad substrate specificity toward d-2-hydroxy acids. The latter used molecular oxygen as a direct electron acceptor, a feature that has not been reported previously in d-lactate-oxidizing enzymes. This study not only clarifies the mechanism for the growth of G. oxydans on d-lactate, but also provides new insights for applications of the important industrial microbe and the novel d-lactate oxidase.

Efficient production of (R)-2-hydroxy-4-phenylbutyric acid by using a coupled reconstructed D-lactate dehydrogenase and formate dehydrogenase system.

BACKGROUND: (R)-2-hydroxy-4-phenylbutyric acid [(R)-HPBA] is a key precursor for the production of angiotensin-converting enzyme inhibitors. However, the product yield and concentration of reported (R)-HPBA synthetic processes remain unsatisfactory. METHODOLOGY/PRINCIPAL FINDINGS: The Y52L/F299Y mutant of NAD-dependent D-lactate dehydrogenase (D-nLDH) in Lactobacillus bulgaricus ATCC 11842 was found to have high bio-reduction activity toward 2-oxo-4-phenylbutyric acid (OPBA). The mutant D-nLDHY52L/F299Y was then coexpressed with formate dehydrogenase in Escherichia coli BL21 (DE3) to construct a novel biocatalyst E. coli DF. Thus, a novel bio-reduction process utilizing whole cells of E. coli DF as the biocatalyst and formate as the co-substrate for cofactor regeneration was developed for the production of (R)-HPBA from OPBA. The biocatalysis conditions were then optimized. CONCLUSIONS/SIGNIFICANCE: Under the optimum conditions, 73.4 mM OPBA was reduced to 71.8 mM (R)-HPBA in 90 min. Given its high product enantiomeric excess (>99%) and productivity (47.9 mM h(-1)), the constructed coupling biocatalysis system is a promising alternative for (R)-HPBA production.

Draft Genome Sequence of the Gluconobacter oxydans Strain DSM 2003, an Important Biocatalyst for Industrial Use.

Gluconobacter oxydans strain DSM 2003 can efficiently produce some industrially important building blocks, such as (R)-lactic acid and (R)-2-hydroxybutyric acid. Here, we present a 2.94-Mb assembly of its genome sequence, which might provide further insights into the molecular mechanism of its biocatalysis in order to further improve its biotechnological applications.

Highly stereoselective biosynthesis of (R)-α-hydroxy carboxylic acids through rationally re-designed mutation of D-lactate dehydrogenase.

An NAD-dependent D-lactate dehydrogenase (D-nLDH) of Lactobacillus bulgaricus ATCC 11842 was rationally re-designed for asymmetric reduction of a homologous series of α-keto carboxylic acids such as phenylpyruvic acid (PPA), α-ketobutyric acid, α-ketovaleric acid, ß-hydroxypyruvate. Compared with wild-type D-nLDH, the Y52L mutant D-nLDH showed elevated activities toward unnatural substrates especially with large substitutes at C-3. By the biocatalysis combined with a formate dehydrogenase for in situ generation of NADH, the corresponding (R)-α-hydroxy carboxylic acids could be produced at high yields and highly optical purities. Taking the production of chiral (R)-phenyllactic acid (PLA) from PPA for example, 50 mM PPA was completely reduced to (R)-PLA in 90 min with a high yield of 99.0% and a highly optical purity (>99.9% e.e.) by the coupling system. The results presented in this work suggest a promising alternative for the production of chiral α-hydroxy carboxylic acids.

Relative catalytic efficiency of ldhL- and ldhD-encoded products is crucial for optical purity of lactic acid produced by lactobacillus strains.

NAD-dependent l- and d-lactate dehydrogenases coexist in Lactobacillus genomes and may convert pyruvic acid into l-lactic acid and d-lactic acid, respectively. Our findings suggest that the relative catalytic efficiencies of ldhL- and ldhD-encoded products are crucial for the optical purity of lactic acid produced by Lactobacillus strains.