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Applied Microbiology and Biotechnology (v.88, #6)

Novel soluble expression technologies derived from unique properties of halophilic proteins by Hiroko Tokunaga; Tsutomu Arakawa; Masao Tokunaga (pp. 1223-1231).

Recombinant production of mammalian cytoplasmic proteins plays a major role in developing pharmaceutical products. Here we describe two expression technologies using unique nature of halophilic bacteria. One of such properties of halophilic bacteria is accumulation of compatible solutes in the cytoplasm. As the compatible solutes enhance protein solubility and folding, one might utilize these bacteria for cytoplasmic soluble expression of recombinant proteins, as described in this review. Another uniqueness is high reversibility of thermally unfolded halophilic proteins. Here we show that one such protein, β-lactamase (BLA), is highly soluble both in the native and thermally unfolded states and reversibly refolds after thermal melting. This makes BLA as a potential fusion partner for soluble expression of target proteins. The BLA fusion technology is also introduced in the review.

Keywords: Halophilic protein; Halophilic bacteria; Fusion protein; Betalactamase; Soluble expression; Recombinant protein

Engineered polyketide biosynthesis and biocatalysis in Escherichia coli by Xue Gao; Peng Wang; Yi Tang (pp. 1233-1242).

Polyketides are important bioactive natural products biosynthesized by bacteria, fungi, and plants. The enzymes that synthesize polyketides are collectively referred to as polyketide synthases (PKSs). Because many of the natural hosts that produce polyketides are difficult to culture or manipulate, establishing a universal heterologous host that is genetically tractable has become an important goal toward the engineered biosynthesis of polyketides and analogues. Here, we summarize the recent progresses in engineering Escherichia coli as a heterologous host for reconstituting PKSs of different types. Our increased understanding of PKS enzymology and structural biology, combined with new tools in protein engineering, metabolic engineering, and synthetic biology, has firmly established E. coli as a powerful host for producing polyketides.

Keywords: Biosynthesis; Megasynthase; Heterologous host

Current progress in the study on biosynthesis and regulation of ganoderic acids by Liang Shi; Ang Ren; Dashuai Mu; Mingwen Zhao (pp. 1243-1251).

Ganoderic acids (GAs) isolated from Ganoderma lucidum, which have shown remarkable pharmacological activities and a variety of therapeutic effects on a number of human diseases, have provided an important resource for the development of new medicines. The yield of GAs in field cultivation is still limited, which is mainly due to a scarcity of information regarding its biosynthesis pathway and its regulation. Here, we review the recent publication that has been made in the biosynthesis and regulation of GAs. From these studies, researchers have identified part of the biosynthesis pathway of GAs named mevalonate pathway. They have cloned and characterized the genes involved in the biosynthesis pathway. Additionally, they found that expression of the genes involved in GA biosynthesis is closely related to the impact of environmental factors through transcriptional profiling analysis. Moreover, this review focuses on suggesting new directions for studying GAs and attempts to gain some insights for better understanding of the biosynthesis and regulation of GAs.

Keywords: Ganoderma lucidum ; Ganoderic acid; Gene regulation; Environmental factors; Biosynthetic pathway

Membrane bioreactors and their uses in wastewater treatments by Pierre Le-Clech (pp. 1253-1260).

With the current need for more efficient and reliable processes for municipal and industrial wastewaters treatment, membrane bioreactor (MBR) technology has received considerable attention. After just a couple of decades of existence, MBR can now be considered as an established wastewater treatment system, competing directly with conventional processes like activated sludge treatment plant. However, MBR processes still suffer from major drawbacks, including high operational costs due to the use of anti-fouling strategies applied to the system to maintain sustainable filtration conditions. Moreover, this specific use of membranes has not reached full maturity yet, as MBR suppliers and users still lack experience regarding the long-term performances of the system. Still, major improvements of the MBR design and operation have been witnessed over the recent years, making MBR an option of choice for wastewater treatment and reuse. This mini-review reports recent developments and current research trends in the field.

Keywords: Membrane bioreactor; Wastewater; Recycling; Fouling; Aerobic; Anaerobic

Sources of novel antibiotics—aside the common roads by Stefano Donadio; Sonia Maffioli; Paolo Monciardini; Margherita Sosio; Daniela Jabes (pp. 1261-1267).

Microbial pathogens are becoming increasingly resistant to available treatments, and new antibiotics are badly needed, but the pipeline of compounds under development is scarce. Furthermore, the majority of antibiotics under development are improved derivatives of marketed compounds, which are at best only partially effective against prevailing resistance mechanisms. In contrast, antibiotics endowed with new mechanisms of action are expected to be highly effective against multi-drug resistant pathogens. In this review, examples are provided of new antibiotics classes in late discovery or clinical development, arising from three different avenues: (1) compounds discovered and never brought to market by large pharmaceutical companies; (2) old compounds reanalyzed and rejuvinated with today’s tools; and (3) newly discovered molecules. For each compound, we will briefly describe original discovery, mechanism of action, any known resistance, antimicrobial profile, and current status of development.

Keywords: Antibiotics; Natural products; Drug resistance

Apoptosis of human lung adenocarcinoma A549 cells induced by prodigiosin analogue obtained from an entomopathogenic bacterium Serratia marcescens by Wei Zhou; Zhi-Xiong Jin; Yong-Ji Wan (pp. 1269-1275).

An entomopathogenic bacterial strain SCQ1 was isolated from silkworm (Bombyx mori) and identified as Serratia marcescens via 16S rRNA gene analysis. This strain produces a red pigment that causes acute septicemia of silkworm. The red pigment of strain SCQ1 was identified as prodigiosin analogue (PGA) with various reported biological activities. In this study, we found that low concentration of PGA showed significant anticancer activity in human lung adenocarcinoma A549 cells, but has little effect in human bone marrow stem cells, in vitro. By exposure to different concentrations of PGA for 24 h, morphological changes and the MTT assay showed that A549 cell line was very sensitive to PGA, with IC₅₀ value about 2.2 mg/L. Early stage of apoptosis was detected by flow cytometry while A549 cells were treated with PGA for 4 and 12 h, respectively. The proportion of dead cells was increased with treatment time or the concentrations of PGA, but it was inversely proportional to that of apoptotic cells. These results indicate that PGA obtained from strain SCQ1 induces apoptosis in A549 cells, but the molecular mechanisms of cell death are complicated, and the S. marcescens strain SCQ1 may serve as a source of the anticancer compound, PGA.

Keywords: Prodigiosin analogue; Serratia ; Apoptosis; Anticancer; Silkworm

Biocatalytic synthesis of (S)-4-chloro-3-hydroxybutanoate ethyl ester using a recombinant whole-cell catalyst by Qi Ye; Hou Cao; Guanglou Zang; Lan Mi; Ming Yan; Yan Wang; Yueyuan Zhang; Ximu Li; Jian Li; Lin Xu; Jian Xiong; Pingkai Ouyang; Hanjie Ying (pp. 1277-1285).

A cofactor regeneration system for enzymatic biosynthesis was constructed by coexpressing a carbonyl reductase from Pichia stipitis and a glucose dehydrogenase from Bacillus megaterium in Escherichia coli Rosetta (DE3) PlySs. Transformants containing the polycistronic plasmid pET-PII-SD2-AS1-B exhibited an activity of 13.5 U/mg protein with 4-chloro-3-oxobutanoate ethyl ester (COBE) as the substrate and an activity of 14.4 U/mg protein with glucose as the substrate; NAD(H) was the coenzyme in both cases. Asymmetric reduction of COBE to (S)-4-chloro-3-hydroxybutanoate ethyl ester [(S)-CHBE] with more than 99% enantiomeric excess was demonstrated by transformants. Furthermore, the paper made a comparison of crude enzyme catalysis and whole-cell catalysis in an aqueous monophasic system and a water/organic solvent biphasic system. In the water/n-butyl acetate system, the coexpression system produced 1,398 mM CHBE in the organic phase, which is the highest yield ever reported for CHBE production by NADH-dependent reductases from yeasts. In this case, the molar yield of CHBE was 90.7%, and the total turnover number, defined as moles (S)-CHBE formed per mole NAD+, was 13,980.

Keywords: 4-Chloro-3-oxobutanoate ethyl ester; (S)-4-Chloro-3-hydroxybutanoate ethyl ester; Crude enzyme catalysis; Whole-cell catalysis

Pho regulon promoter-mediated transcription of the key pathway gene aroG ^Fbr improves the performance of an l-phenylalanine-producing Escherichia coli strain by Vera G. Doroshenko; Irina S. Tsyrenzhapova; Alexander A. Krylov; Evgeniya M. Kiseleva; Vladimir Yu. Ermishev; Svetlana M. Kazakova; Irina V. Biryukova; Sergey V. Mashko (pp. 1287-1295).

DAHP synthase (EC 4.1.2.15) is one of the key enzymes involved in aromatic amino acid biosynthesis in Escherichia coli. An approximately twofold decrease in DAHP synthase activity level was detected in the late growth phase of the l-phenylalanine (Phe)-producing E. coli strain, in which this enzyme encoded by aroG4 is resistant to feedback inhibition. An additional copy of aroG4 that is controlled by promoters of E. coli phoA or pstS genes was integrated into the chromosome of the Phe producer. The choice of promoter was based on the detected activation of the Pho regulon that occurs in response to the depletion of soluble inorganic orthophosphate (P_i) in the medium, provided that the optical density of the Phe-producing culture did not exceed 70% of its maximum value. Pho-mediated aroG4 transcription increased both the accumulation of Phe and the level of DAHP synthase activity in the late stage of batch cultivation on glucose in P_i-limited conditions. Disruption of rpoS led to the improved performance of a P phoA-aroG4 strain. The pstS promoter that is recognized by the σ⁷⁰/σ^S-associated core RNA polymerase resulted in the stable maintenance of DAHP synthase activity during long-drawn fed-batch cultivation of the RpoS⁺ strain carrying the P pstS-aroG4.

Keywords: Amino acid production; phoA promoter; pstS promoter; Phosphate-limited growth; RpoS-dependent expression

A new α-galactosidase from symbiotic Flavobacterium sp. TN17 reveals four residues essential for α-galactosidase activity of gastrointestinal bacteria by Junpei Zhou; Pengjun Shi; Huoqing Huang; Yanan Cao; Kun Meng; Peilong Yang; Rui Zhang; Xiaoyan Chen; Bin Yao (pp. 1297-1309).

The α-galactosidase gene, galA17, was cloned from Flavobacterium sp. TN17, a symbiotic bacterium isolated from the gut of Batocera horsfieldi larvae. The 2,205-bp full-length gene encodes a 734-residue polypeptide (GalA17) containing a putative 28-residue signal peptide and a catalytic domain belonging to glycosyl hydrolase family 36 (GH 36). The deduced amino acid sequence of galA17 was most similar to a putative α-galactosidase from Pedobacter sp. BAL39 (EDM38577; 66.6% identity) and a characterized α-galactosidase from Carnobacterium piscicola BA (AAL27305; 30.1%). Phylogenetic analysis revealed that GalA17 was similar to GH 36 α-galactosidases from symbiotic bacteria sharing two putative catalytic motifs, KWD and SDXXDXXXR, in which D480, S548, D549, and R556 were essential for α-galactosidase activity based on site-directed mutagenesis. Purified recombinant GalA17 showed apparent optimal activity at pH 5.5 and 45°C; exhibited strong resistance to digestion by trypsin, α-chymotrypsin, collagenase, and proteinase K; and efficiently hydrolyzed several synthetic and natural substrates (p-nitrophenyl-α-d-galactopyranoside, stachyose, melibiose, raffinose, soybean meal, locust bean gum, and guar gum).

Keywords: Batocera horsfieldi ; Flavobacterium sp. TN17; α-Galactosidase; Symbiotic bacteria; Site-directed mutagenesis

Cloning and characterization of thermotolerant xylitol dehydrogenases from yeast Pichia angusta by Dipanwita Biswas; Manish Datt; Kaliannan Ganesan; Alok K. Mondal (pp. 1311-1320).

Pichia angusta (syn. Hansenula polymorpha) represents one of the rare yeast that can grow and ferment both xylose and glucose at higher temperature (50°C). However, little is known about the enzymes involved in xylose utilization from this species. Previous studies indicated the presence of one xylose reductase and two xylitol dehydrogenase genes in P. angusta. In this study, we have expressed both xylitol dehydrogenases (PaXdh1p and PaXdh2p) in Escherichia coli and purified them as 6X-Histidine-tagged proteins. Biochemical characterization of the recombinant proteins reveals that both PaXdh1p and PaXdh2p are thermotolerant enzymes. PaXdh2p contains a catalytic and a structural Zn atom. However, the structural Zn atom is not present in PaXdh1p. Both enzymes also differ in their affinity for the substrate as well as in the catalytic efficiency. Through mutagenesis and modeling approaches, we have also identified residues important for catalysis and substrate binding.

Keywords: Xylitol dehydrogenase; Medium-chain alcohol dehydrogenase; Thermotolerant enzyme; Pichia angusta

Flocculation gene variability in industrial brewer’s yeast strains by Sebastiaan E. Van Mulders; Maarten Ghequire; Luk Daenen; Pieter J. Verbelen; Kevin J. Verstrepen; Freddy R. Delvaux (pp. 1321-1331).

The brewer’s yeast genome encodes a ‘Flo’ flocculin family responsible for flocculation. Controlled floc formation or flocculation at the end of fermentation is of great importance in the brewing industry since it is a cost-effective and environmental-friendly technique to separate yeast cells from the final beer. FLO genes have the notable capacity to evolve and diverge many times faster than other genes. In actual practice, this genetic variability may directly alter the flocculin structure, which in turn may affect the flocculation onset and/or strength in an uncontrolled manner. Here, 16 ale and lager yeast strains from different breweries, one laboratory Saccharomyces cerevisiae and one reference Saccharomyces pastorianus strain, with divergent flocculation strengths, were selected and screened for characteristic FLO gene sequences. Most of the strains could be distinguished by a typical pattern of these FLO gene markers. The FLO1 and FLO10 markers were only present in five out of the 18 yeast strains, while the FLO9 marker was ubiquitous in all the tested strains. Surprisingly, three strongly flocculating ale yeast strains in this screening also share a typical ‘lager’ yeast FLO gene marker. Further analysis revealed that a complete Lg-FLO1 allele was present in these ale yeasts. Taken together, this explicit genetic variation between flocculation genes hampers attempts to understand and control the flocculation behavior in industrial brewer’s yeasts.

Keywords: Flocculation; Saccharomyces cerevisiae ; Adhesin; Tandem repeats; Brewery fermentations

Laser capture microdissection and metagenomic analysis of intact mucosa-associated microbial communities of human colon by Yunwei Wang; Dionysios A. Antonopoulos; Xiaorong Zhu; Laura Harrell; Ira Hanan; John C. Alverdy; Folker Meyer; Mark W. Musch; Vincent B. Young; Eugene B. Chang (pp. 1333-1342).

Metagenomic analysis of colonic mucosa-associated microbes has been complicated by technical challenges that disrupt or alter community structure and function. In the present study, we determined the feasibility of laser capture microdissection (LCM) of intact regional human colonic mucosa-associated microbes followed by phi29 multiple displacement amplification (MDA) and massively parallel sequencing for metagenomic analysis. Samples were obtained from the healthy human subject without bowel preparation and frozen sections immediately prepared. Regional mucosa-associated microbes were successfully dissected using LCM with minimal contamination by host cells, their DNA extracted and subjected to phi29 MDA with a high fidelity, prior to shotgun sequencing using the GS-FLX DNA sequencer. Metagenomic analysis of approximately 67 million base pairs of DNA sequences from two samples revealed that the metabolic functional profiles in mucosa-associated microbes were as diverse as those reported in feces, specifically the representation of functional genes associated with carbohydrate, protein, and nucleic acid utilization. In summary, these studies demonstrate the feasibility of the approach to study the structure and metagenomic profiles of human intestinal mucosa-associated microbial communities at small spatial scales.

Keywords: Laser capture microdissection; Metagenomics; Mucosa-associated microbes; Multiple displacement amplification; Pyrosequencing; Host-microbe interactions

Proteome analysis of Azotobacter vinelandii ∆arrF mutant that overproduces poly-β-hydroxybutyrate polymer by Rajkumar Pyla; Tae-Jo Kim; Juan L. Silva; Yean-Sung Jung (pp. 1343-1354).

Azotobacter vinelandii ArrF is an iron-responsive small RNA that is under negative control of Ferric uptake regulator protein. A. vinelandii ∆arrF mutant that had a deletion of the entire arrF gene was known to overproduce poly-β-hydroxybutyrate (PHB). Proteins differentially expressed in the mutant were identified by gel-based proteomics and confirmed by real-time RT-PCR. 6-Phosphogluconolactonase and E₁ component of pyruvate dehydrogenase complex, which leads to the production of NADPH and acetyl-CoA, were upregulated, while proteins in the tricarboxylic acid cycle that consumes acetyl-CoA were downregulated. Heat-shock proteins such as HSP20 and GroEL were highly overexpressed in the mutant. Antioxidant proteins such as Fe-containing superoxide dismutase (FeSOD), a putative oxidoreductase, alkyl hydroperoxide reductase, flavorprotein WrbA, and cysteine synthase were also overexpressed in the ∆arrF mutant, indicating that the PHB accumulation is stressful to the cells. Upregulated in the ∆arrF mutant were acetyl-CoA carboxylase, flagellin, and adenylate kinase, though the reasons for their overexpression are unclear. Among genes upregulated in the mutant, sodB coding for FeSOD and phbF encoding PHB synthesis regulator PhbF were negatively regulated by small RNA ArrF probably in an antisense mechanism. The deletion of arrF gene, therefore, would increase PhbF and FeSOD levels, which favors PHB synthesis in the mutant. On the other hand, glutamate synthetase, elongation factor-Tu, iron ABC transporter, and major outer membrane porin OprF were downregulated in the ∆arrF mutant. Based on the results, it is concluded that multiple factors including the direct effect of small RNA ArrF might be responsible for the PHB overproduction in the mutant.

Keywords: Poly-β-hydroxybutyrate; Azotobacter vinelandii ; Small RNA ArrF; Proteomics; Real-time reverse transcription PCR

Lead toxicity in Saccharomyces cerevisiae by Maarten Van der Heggen; Sara Martins; Gisela Flores; Eduardo V. Soares (pp. 1355-1361).

The effect of Pb on Saccharomyces cerevisiae cell structure and function was examined. Membrane integrity was assessed by the release of UV-absorbing compounds and by the intracellular K⁺ efflux. No leakage of UV₂₆₀-absorbing compounds or loss of K⁺ were observed in Pb (until 1,000 μmol/l) treated cells up to 30 min; these results suggest that plasma membrane seems not to be the immediate and primary target of Pb toxicity. The effect of Pb on yeast metabolism was examined using the fluorescent probe FUN-1 and compared with the ability to reproduce, evaluated by colony-forming units counting. The exposition of yeast cells, during 60 min to 1,000 μmol/l Pb, induces a decrease in the ability to process FUN-1 although the cells retain its proliferation capacity. A more prolonged contact time (120 min) of yeast cells with Pb induces a marked (> 50%) loss of yeast cells metabolic activity and replication competence through a mechanism which most likely requires protein synthesis.

Keywords: Cell membrane; FUN-1; Lead; Metal toxicity; Yeast viability

Hydrogen formation by an arsenate-reducing Pseudomonas putida, isolated from arsenic-contaminated groundwater in West Bengal, India by Dominik Freikowski; Josef Winter; Claudia Gallert (pp. 1363-1371).

Anaerobic growth of a newly isolated Pseudomonas putida strain WB from an arsenic-contaminated soil in West Bengal, India on glucose, l-lactate, and acetate required the presence of arsenate, which was reduced to arsenite. During aerobic growth in the presence of arsenite arsenate was formed. Anaerobic growth of P. putida WB on glucose was made possible presumably by the non-energy-conserving arsenate reductase ArsC with energy derived only from substrate level phosphorylation. Two moles of acetate were generated intermediarily and the reducing equivalents of glycolysis and pyruvate decarboxylation served for arsenate reduction or were released as H₂. Anaerobic growth on acetate and lactate was apparently made possible by arsenate reductase ArrA coupled to respiratory electron chain energy conservation. In the presence of arsenate, both substrates were totally oxidized to CO₂ and H₂ with part of the H₂ serving for respiratory arsenate reduction to deliver energy for growth. The growth yield for anaerobic glucose degradation to acetate was Y _Glucose = 20 g/mol, leading to an energy coefficient of Y _ATP = 10 g/mol adenosine-5'-triphosphate (ATP), if the Emden–Meyerhof–Parnas pathway with generation of 2 mol ATP/mol glucose was used. During growth on lactate and acetate no substrate chain phosphorylation was possible. The energy gain by reduction of arsenate was Y _Arsenate = 6.9 g/mol, which would be little less than one ATP/mol of arsenate.

Keywords: Pseudomonas putida ; Arsenate; Hydrogen; Glucose; Acetate; Lactate

Quantitative identification of fecal water pollution sources by TaqMan real-time PCR assays using Bacteroidales 16S rRNA genetic markers by Dae-Young Lee; Susan C. Weir; Hung Lee; Jack T. Trevors (pp. 1373-1383).

PCR-based analysis of Bacteroidales 16S rRNA genes has emerged as a promising tool to identify sources of fecal water pollution. In this study, three TaqMan real-time PCR assays (BacGeneral, BacHuman, and BacBovine) were developed and evaluated for their ability to quantitatively detect general (total), human-specific, and bovine-specific Bacteroidales 16S rRNA genetic markers. The detection sensitivity was determined to be 6.5 copies of 16S rRNA gene for the BacGeneral and BacHuman assays and 10 copies for the BacBovine assay. The assays were capable of detecting approximately one to two cells per PCR. When tested with 70 fecal samples from various sources (human, cattle, pig, deer, dog, cat, goose, gull, horse, and raccoon), the three assays positively identified the target markers in all samples without any false-negative results. The BacHuman and BacBovine assays exhibited false-positive reactions with non-target samples in a few cases. However, the level of the false-positive reactions was about 50 times smaller than that of the true-positive ones, and therefore, these cross-reactions were unlikely to cause misidentifications of the fecal pollution sources. Microbial source-tracking capability was tested at two freshwater streams of which water quality was influenced by human and cattle feces, respectively. The assays accurately detected the presence of the corresponding host-specific markers upon fecal pollution and the persistence of the markers in downstream areas. The assays are expected to reliably determine human and bovine fecal pollution sources in environmental water samples.

Keywords: Water pollution; Fecal source tracking; Bacteroidales ; 16S rRNA genetic marker; TaqMan real-time PCR

Enrichment of Escherichia coli spheroplasts displaying scFv antibodies specific for antigens expressed on the human cell surface by Jun-Kang Qiu; Sang-Taek Jung; George Georgiou; Hai-Ying Hang (pp. 1385-1391).

Anchored periplasmic expression (APEx) is a method for isolating high affinity ligand-binding proteins from large combinatorial libraries, and antibodies highly specific for soluble antigens were successfully isolated from APEx antibody libraries in combination with flow cytometric sorting (Harvey et al., Proc Natl Acad Sci USA 101(25):9193–9198, 2004). However, many disease markers and drug targets are localized on the cell surface, and often, unique posttranslational modifications and/or properly folded epitopes are lost when they were expressed and isolated in soluble form. In this study, we demonstrate that Escherichia coli spheroplasts, displaying antibodies and screened by a combination of plate-panning and flow cytometric sorting, can be used for isolating antibodies specific for antigens on the human cell surface. Two rounds of plate-panning followed by one round of flow cytometric sorting resulted in 7,200-fold enrichment of antibodies specific for the protective antigen of Bacillus anthracis from a large excess of spheroplasts expressing a scFv antibody to digoxin (a negative control). There is the potential to use this technique for library screening to find novel antibodies against disease cell surface antigens.

Keywords: Anchored periplasmic expression; Spheroplasts; scFv; Antigens on the human cell surface

Identification and quantification of mycothiol in Actinobacteria by a novel enzymatic method by Ya-Jie Yin; Bao-Jun Wang; Cheng-Ying Jiang; Yuan-Ming Luo; Jing-Hua Jin; Shuang-Jiang Liu (pp. 1393-1401).

Mycothiol (MSH) was reported to be the dominant low molecular weight thiol in members of the Actinobacteria. In this study, a simple, fast, and sensitive method for qualitative and quantitative determination of MSH molecules was developed based on maleylpyruvate isomerase (MPI) from Corynebacterium glutamicum. The principle of this method is that the activity of MPI from C. glutamicum was dependent on MSH molecules. It was found that this MPI activity displayed a linear response (R ² = 0.9928) at MSH amounts ranging from 0.12 to 3.98 pmol in the defined assay system. This observation was applied to calculate the MSH levels, and the newly developed method was compared with thiol-specific fluorescent-labeling high-performance liquid chromatography method. Forty-eight genera of Actinobacteria were screened for MSH and 43 genera were reported for MSH occurrence, and the MSH levels in Actinobacteria were determined to be 0.01 to 9.69 μmol/g of residual dry cell weight.

Keywords: Mycothiol (MSH); Quantitative and qualitative determination; Actinobacteria; MSH-dependent maleylpyruvate isomerase (MDMPI)

Estimation of nitrifier abundances in a partial nitrification reactor treating ammonium-rich saline wastewater using DGGE, T-RFLP and mathematical modeling by Lin Ye; Tong Zhang (pp. 1403-1412).

The bacterial community in a partial nitrification reactor was analyzed on the basis of 16S rRNA gene by cloning–sequencing method, and the percentages of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) in the activated sludge were quantified by three independent methods, namely, denaturing gradient gel electrophoresis (DGGE), terminal restriction fragment length polymorphism (T-RFLP) and Double Monod modeling. The clone library results suggested that there were only a dominant AOB and a dominant NOB species in the reactor, belonging to Nitrosomonas genus and Nitrospira genus, respectively. The percentages of NOB in total bacterial community increased from almost 0% to 30% when dissolved oxygen (DO) levels were changed from 0.15 mg/L to 0.5 mg/L, coinciding with the accumulation and conversion of nitrite, while the percentages of AOB changed little in the two phases. The results confirmed the importance of low DO level for inhibiting NOB to achieve partial nitrification. Furthermore, the percentages of AOB and NOB in the total bacteria community were estimated based on the results of batch experiments using Double Monod model, and the results were comparable with those determined according to profiles of DGGE and T-RFLP.

Keywords: Partial nitrification; DGGE; T-RFLP; Double Monod

Response of microbial community structure to microbial plugging in a mesothermic petroleum reservoir in China by Fan Zhang; Yue Hui She; Sha Sha Ma; Ji Ming Hu; Ibrahim M. Banat; Du Jie Hou (pp. 1413-1422).

Microbial plugging, a microbial enhancement of oil recovery (MEOR) technique, has been applied in a candidate oil reservoir of Daqing Oil Field (China). The goal of this study is to monitor the survival of injected bacteria and reveal the response of microbial communities in field trial of microbial plugging through injection of selected microbial culture broth and nutrients. Culture-dependent enrichment and culture-independent 16S rDNA clone library methods were used. The results show that it was easy to activate targeted biopolymer-producing bacteria in a laboratory environment, and it was difficult for injected exogenous bacteria to survive. In addition, microbial communities in the oil reservoir also changed before and after the field trial. However, microbial communities, activated by fermentative medium for biopolymer-producing bacteria, appeared to show greater differences in the laboratory than in the natural reservoir. It was concluded that microbial populations monitoring was important to MEOR; results of response of microbial communities could provide a guide for the future field trials.

Keywords: Microbial plugging; Microbial enhancement of oil recovery (MEOR); Microbial communities; Biopolymer-producing bacteria; 16S rDNA clone library

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Applied Microbiology and Biotechnology (v.88, #6)