Zhi-Pei Liu

The characteristics of a novel heterotrophic nitrification-aerobic denitrification bacterium, Bacillus methylotrophicus strain L7.

Bacillus methylotrophicus strain L7, exhibited efficient heterotrophic nitrification-aerobic denitrification ability, with maximum NH(4)(+)-N and NO(2)(-)-N removal rate of 51.58 mg/L/d and 5.81 mg/L/d, respectively. Strain L7 showed different gaseous emitting patterns from those strains ever described. When (15)NH(4)Cl, or Na(15)NO(2), or K(15)NO(3) was used, results of GC-MS indicated that N(2)O was emitted as the intermediate of heterotrophic nitrification or aerobic denitrification, while GC-IRMS results showed that N(2) was produced as end product when nitrite was used. Single factor experiments suggested that the optimal conditions for heterotrophic nitrification were sodium succinate as carbon source, C/N 6, pH 7-8, 0 g/L NaCl, 37 °C and a wide range of NH(4)(+)-N from 80 to 1000 mg/L. Orthogonal tests showed that the optimal conditions for aerobic denitrification were C/N 20, pH 7-8, 10 g/L NaCl and DO 4.82 mg/L (shaking speed 50 r/min) when nitrite was served as substrate.

Novel Partial Reductive Pathway for 4-Chloronitrobenzene and Nitrobenzene Degradation in Comamonas sp. Strain CNB-1

ABSTRACT Comamonas sp. strain CNB-1 grows on 4-chloronitrobenzene (4-CNB) and nitrobenzene as sole carbon and nitrogen sources. In this study, two genetic segments, cnbB-orf2-cnbA and cnbR-orf1-cnbCaCbDEFGHI, located on a newly isolated plasmid, pCNB1 (ca. 89 kb), and involved in 4-CNB/nitrobenzene degradation, were characterized. Seven genes (cnbA, cnbB, cnbCa, cnbCb, cnbD, cnbG, and cnbH) were cloned and functionally expressed in recombinant Escherichia coli, and they were identified as encoding 4-CNB nitroreductase (CnbA), 1-hydroxylaminobenzene mutase (CnbB), 2-aminophenol 1,6-dioxygenase (CnbCab), 2-amino-5-chloromuconic semialdehyde dehydrogenase (CnbD), 2-hydroxy-5-chloromuconic acid (2H5CM) tautomerase, and 2-amino-5-chloromuconic acid (2A5CM) deaminase (CnbH). In particular, the 2A5CM deaminase showed significant identities (31 to 38%) to subunit A of Asp-tRNAAsn/Glu-tRNAGln amidotransferase and not to the previously identified deaminases for nitroaromatic compound degradation. Genetic cloning and expression of cnbH in Escherichia coli revealed that CnbH catalyzed the conversion of 2A5CM into 2H5CM and ammonium. Four other genes (cnbR, cnbE, cnbF, and cnbI) were tentatively identified according to their high sequence identities to other functionally identified genes. It was proposed that CnbH might represent a novel type of deaminase and be involved in a novel partial reductive pathway for chloronitrobenzene or nitrobenzene degradation.

Functional Identification of Novel Genes Involved in the Glutathione-Independent Gentisate Pathway in Corynebacterium glutamicum

ABSTRACT Corynebacterium glutamicum used gentisate and 3-hydroxybenzoate as its sole carbon and energy source for growth. By genome-wide data mining, a gene cluster designated ncg12918-ncg12923 was proposed to encode putative proteins involved in gentisate/3-hydroxybenzoate pathway. Genes encoding gentisate 1,2-dioxygenase (ncg12920) and fumarylpyruvate hydrolase (ncg12919) were identified by cloning and expression of each gene in Escherichia coli. The gene of ncg12918 encoding a hypothetical protein (Ncg12918) was proved to be essential for gentisate-3-hydroxybenzoate assimilation. Mutant strain RES167Δncg12918 lost the ability to grow on gentisate or 3-hydroxybenzoate, but this ability could be restored in C. glutamicum upon the complementation with pXMJ19-ncg12918. Cloning and expression of this ncg12918 gene in E. coli showed that Ncg12918 is a glutathione-independent maleylpyruvate isomerase. Upstream of ncg12920, the genes ncg12921-ncg12923 were located, which were essential for gentisate and/or 3-hydroxybenzoate catabolism. The Ncg12921 was able to up-regulate gentisate 1,2-dioxygenase, maleylpyruvate isomerase, and fumarylpyruvate hydrolase activities. The genes ncg12922 and ncg12923 were deduced to encode a gentisate transporter protein and a 3-hydroxybenzoate hydroxylase, respectively, and were essential for gentisate or 3-hydroxybenzoate assimilation. Based on the results obtained in this study, a GSH-independent gentisate pathway was proposed, and genes involved in this pathway were identified.

Paracoccus halophilus sp. nov., isolated from marine sediment of the South China Sea, China, and emended description of genus Paracoccus Davis 1969.

An aerobic bacterial isolate, strain HN-182(T), was isolated from sediments of the South China Sea. Cells of strain HN-182(T) are coccoid to short rods, Gram-negative, non-spore-forming and non-motile. Strain HN-182(T) is heterotrophic and grows well on marine broth (Difco 2216), and is not capable of growing autotrophically on reduced sulfur. Grows at temperatures ranging from 7 to 42 degrees C (optimum at 25 degrees C), but not at 4 or 45 degrees C, and at pH 5.0-9.0 (optimum at pH 7.0), but not at pH 4.5 or 9.5. NaCl is required for growth [0.5-8.5% (w/v)] with an optimum of 4.5%. Cells are positive for catalase, oxidase and urease activities. Nitrate is not reduced. Strain HN-182(T) contains ubiquinone-10 as sole respiratory quinone. The major polar lipids are phosphatidylglycerol, diphosphatidylglycerol, phosphatidylcholine, an unidentified phospholipid and an unidentified glycolipid. Major cellular fatty acids are C18:1omega7c (60.7 %), C16:0 (12.5%) and C18:0 (8.1%). DNA G+C content is 67.2 mol% (by T(m)). The analysis of 16S rRNA gene sequences indicated that strain HN-182(T) was related to members of the genus Paracoccus, with similarities ranging from 91.2 to 96.7% (highest to Paracoccus versutus) and a close relationship with Paracoccus sulfuroxidans, indicating that strain HN-182(T) is a member of Paracoccus. Based on these results, it is concluded that strain HN-182(T) represents a novel species of the genus Paracoccus, for which the name Paracoccus halophilus sp. nov. is proposed. The type strain is HN-182(T) (=CGMCC 1.6117(T)=JCM 14014(T)).

Flavobacterium caeni sp. nov., isolated from a sequencing batch reactor for the treatment of malachite green effluents

A Gram-stain-negative, heterotrophic, aerobic, non-spore-forming and non-motile bacterial strain, designated LM5(T), was isolated from activated sludge from a sequencing batch reactor for the treatment of effluents contaminated by malachite green. The taxonomy of strain LM5(T) was studied by phenotypic and phylogenetic methods. Strain LM5(T) formed orange colonies on R2A and YP plates. Cells were rods, 0.4-0.6 microm in diameter and 0.8-1.2 microm in length. Growth occurred at 10-35 degrees C (optimum, 20-25 degrees C), at pH 5.5-9.5 (optimum, pH 6.5-7.5) and in the presence of 0-2 % (w/v) NaCl (optimum, 0.5 %). Oxidase and catalase activities were present. Flexirubin-type pigments were present, but extracellular glycans were absent. MK-6 was the major respiratory quinone. The major fatty acids were iso-C(15 : 0) (28.3 %) and iso-C(17 : 1)omega9c (13.8 %). Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain LM5(T) was a member of the genus Flavobacterium with highest sequence similarity to Flavobacterium soli DS-6(T) (93.2 %) and Flavobacterium lindanitolerans IP-10(T) (92.9 %). Together with F. lindanitolerans IP-10(T), strain LM5(T) formed a distinct lineage in the phylogenetic tree. The DNA G+C content was 52+/-0.6 mol% (HPLC), which is significantly higher than that of other species of the genus Flavobacterium (30-41 mol%). Based on phylogenetic and phenotypic evidence, strain LM5(T) is considered to represent a novel species of the genus Flavobacterium, for which the name Flavobacterium caeni sp. nov. is proposed; the type strain is LM5(T) (=CGMCC 1.7031(T)=NBRC 104239(T)).

Successful bioremediation of an aged and heavily contaminated soil using a microbial/plant combination strategy.

Bioremediation of an aged and heavily contaminated soil was performed using microbial remediation, phytoremediation, and microbial/phytoremediation. The removal efficiency of polycyclic aromatic hydrocarbons (PAHs) was in the order microbial/phytoremediation>microbial remediation≈phytoremediation>control. The removal percentage of microbial/phytoremediation (69.6%) was twice that of control. Kocuria sp. P10 significantly enhanced PAH removal (P<0.05) and ryegrass growth (P<0.01). Dehydrogenase activity increased steadily and was negatively correlated with total PAH content. Successional changes in soil microbial communities were also detected by pyrosequencing. The results indicated that biodiversity of the soil bacterial community gradually increased with time and was slightly lower in control, as indicated by operational taxonomic unit (OTU) numbers and Shannon-Wiener indices. Proportions of Betaproteobacteria and Gammaproteobacteria were consistently high in all groups. Actinobacteridae were initially predominant (>37.8%) but rapidly decreased to <4%. The proportions of Acidobacteria increased greatly and this increase was positively correlated with PAH removal. These findings indicate a healthy ecological progression and a role of Acidobacteria as an indicator of the process. This study provides new insights into the dynamics of community structure during bioremediation process and a possible basis for ecological assessment for bioremediation on a large scale.

Pilot scale ex-situ bioremediation of heavily PAHs-contaminated soil by indigenous microorganisms and bioaugmentation by a PAHs-degrading and bioemulsifier-producing strain.

This study aims at the remediation of heavily PAH-contaminated soil containing 375 mg of total PAHs per kilogram dry soil. Pilot scale bioremediation experiments were carried out by three approaches with contaminated soil from abandoned sites of Beijing Coking Plant using outdoor pot trials. The first approach was bioaugmentation with a bacterial strain which degrades PAH and produces bioemulsifier, the second approach comprised of biostimulation of indigenous microorganisms with supplementing nutrients and the last approach involved the combination of both biostimulation and bioaugmentation. An on-site land farming group was set as a control in which the total PAHs and 4-6 ring-PAHs were reduced by 23.4% and 10.1%, respectively after 175 days. Meanwhile, in the first approach group, the total PAHs and 4-6 ring-PAHs were reduced by 26.82% and 35.36%, respectively; in the second approach group both percentages were 33.9% and 11.0%, respectively; while in the third approach group, these pollutants were reduced by 43.9% and 55.0%, respectively. The results obtained suggested that biostimulation and bioaugmentation combined could significantly enhance the removal of PAHs in the contaminated soil.

Screening and degrading characteristics and community structure of a high molecular weight polycyclic aromatic hydrocarbon-degrading bacterial consortium from contaminated soil.

Inoculation with efficient microbes had been proved to be the most important way for the bioremediation of polluted environments. For the treatment of abandoned site of Beijing Coking Chemical Plant contaminated with high level of high-molecular-weight polycyclic aromatic hydrocarbons (HMW-PAHs), a bacterial consortium capable of degrading HMW-PAHs, designated 1-18-1, was enriched and screened from HMW-PAHs contaminated soil. Its degrading ability was analyzed by high performance liquid chromatography (HPLC), and the community structure was investigated by construction and analyses of the 16S rRNA gene clone libraries (A, B and F) at different transfers. The results indicated that 1-18-1 was able to utilize pyrene, fluoranthene and benzo[a]pyrene as sole carbon and energy source for growth. The degradation rate of pyrene and fluoranthene reached 82.8% and 96.2% after incubation for 8 days at 30 degrees C, respectively; while the degradation rate of benzo[a]pyrene was only 65.1% after incubation for 28 days at 30 degrees C. Totally, 108, 100 and 100 valid clones were randomly selected and sequenced from the libraries A, B, and F. Phylogenetic analyses showed that all the clones could be divided into 5 groups, Bacteroidetes, alpha-Proteobacteria, Actinobacteria, beta-Proteobacteria and gamma-Proteobacteria. Sequence similarity analyses showed total 39 operational taxonomic units (OTUs) in the libraries. The predominant bacterial groups were alpha-Proteobacteria (19 OTUs, 48.7%), gamma-Proteobacteria (9 OTUs, 23.1%) and beta-Proteobacteria (8 OTUs, 20.5%). During the transfer process, the proportions of alpha-Proteobacteria and beta-Proteobacteria increased greatly (from 47% to 93%), while gamma-Proteobacteria decreased from 32% (library A) to 6% (library F); and Bacteroidetes group disappeared in libraries B and F.

Algoriphagus aquatilis sp. nov., isolated from a freshwater lake

A Gram-negative, non-spore-forming, non-motile bacterium, strain A8-7(T), was isolated from fresh water of a slightly alkaline lake, Longhu Lake, in Daqing, north-east China, and its taxonomic position was studied by using a polyphasic approach. Strain A8-7(T) was aerobic, heterotrophic and positive for catalase and oxidase. It grew at 20-37 degrees C (optimum 30 degrees C) and pH 5.5-10.5 (optimum pH 7.5) and in the presence of 0-3 % (w/v) NaCl. It formed pink-pigmented, smooth and circular colonies, 1-2 mm in diameter, on R3A-V agar plates after incubation at 30 degrees C for 3 days. Cells of strain A8-7(T) were rods, 0.2-0.4 mum wide and 1.6-4.0 mum long. The major fatty acids (>10 %) were iso-C(15 : 0) (40.3 %) and summed feature 3 (C(16 : 1)omega7c and/or iso-C(15 : 0) 2-OH; 12.1 %). The menaquinone was MK-7. The DNA G+C content was 43 mol% (T(m)). Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain A8-7(T) was phylogenetically related to members of the genus Algoriphagus, with sequence similarities of 92.6-95.2 %, the highest sequence similarity being to the sequence from Algoriphagus mannitolivorans IMSNU 14012(T). On the basis of phenotypic, chemotaxonomic and phylogenetic data, strain A8-7(T) was considered to represent a novel species of the genus Algoriphagus, for which the name Algoriphagus aquatilis sp. nov. is proposed. The type strain is A8-7(T) (=CGMCC 1.7030(T) =NBRC 104237(T)).

Gene cloning and characterization of a cold-adapted β-glucosidase belonging to glycosyl hydrolase family 1 from a psychrotolerant bacterium Micrococcus antarcticus.

The gene bglU encoding a cold-adapted β-glucosidase (BglU) was cloned from Micrococcus antarcticus. Sequence analysis revealed that the bglU contained an open reading frame of 1419 bp and encoded a protein of 472 amino acid residues. Based on its putative catalytic domains, BglU was classified as a member of the glycosyl hydrolase family 1 (GH1). BglU possessed lower arginine content and Arg/(Arg+Lys) ratio than mesophilic GH1 β-glucosidases. Recombinant BglU was purified with Ni2+ affinity chromatography and subjected to enzymatic characterization. SDS-PAGE and native staining showed that it was a monomeric protein with an apparent molecular mass of 48 kDa. BglU was particularly thermolabile since its half-life time was only 30 min at 30°C and it exhibited maximal activity at 25°C and pH 6.5. Recombinant BglU could hydrolyze a wide range of aryl-β-glucosides and β-linked oligosaccharides with highest activity towards cellobiose and then p-nitrophenyl-β-d-glucopyranoside (pNPG). Under the optimal conditions with pNPG as substrate, the K(m) and k(cat) were 7 mmol/L and 7.85 × 103/s, respectively. This is the first report of cloning and characterization of a cold-adapted β-glucosidase belonging to GH1 from a psychrotolerant bacterium.