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Applied Microbiology and Biotechnology (v.79, #6)

Occurrence, production, and applications of gellan: current state and perspectives by Arsenio M. Fialho; Leonilde M. Moreira; Ana Teresa Granja; Alma O. Popescu; Karen Hoffmann; Isabel Sá-Correia (pp. 889-900).

Bacterial exopolysaccharides (EPS) are products of biotechnology that are of high interest due to their rheological properties. This is the case of sphingans, a group of structurally related EPS secreted by members of the genus Sphingomonas. Among these, gellan is a multifunctional gelling agent produced in high yields by the non-pathogenic strain Sphingomonas elodea ATCC 31461. In its native form, gellan is a linear anionic EPS based on a tetrasaccharide repeat unit composed of two molecules of d-glucose, one of l-rhamnose and one of d-glucuronic acid. The native gellan is partially esterified with acyl substituents (1 mol of glycerate and 0.5 mol of acetate) per repeat unit. Gellan has unique characteristics and has many applications, particularly in the food, pharmaceutical, and biomedical fields. This review summarizes current knowledge on the structure and properties of gellan and provides details about the biosynthesis of this exopolysaccharide. In addition, a highlight of the importance of gellan in industrial and medicinal applications is given.

Keywords: Bacterial exopolysaccharides; Sphingans; Gellan; Sphingomonas elodea

Minimizing losses in bio-electrochemical systems: the road to applications by Peter Clauwaert; Peter Aelterman; The Hai Pham; Liesje De Schamphelaire; Marta Carballa; Korneel Rabaey; Willy Verstraete (pp. 901-913).

Bio-electrochemical systems (BESs) enable microbial catalysis of electrochemical reactions. Plain electrical power production combined with wastewater treatment by microbial fuel cells (MFCs) has been the primary application purpose for BESs. However, large-scale power production and a high chemical oxygen demand conversion rates must be achieved at a benchmark cost to make MFCs economical competitive in this context. Recently, a number of valuable oxidation or reduction reactions demonstrating the versatility of BESs have been described. Indeed, BESs can produce hydrogen, bring about denitrification, or reductive dehalogenation. Moreover, BESs also appear to be promising in the field of online biosensors. To effectively apply BESs in practice, both biological and electrochemical losses need to be further minimized. At present, the costs of reactor materials have to be decreased, and the volumetric biocatalyst activity in the systems has to be increased substantially. Furthermore, both the ohmic cell resistance and the pH gradients need to be minimized. In this review, these losses and constraints are discussed from an electrochemical viewpoint. Finally, an overview of potential applications and innovative research lines is given for BESs.

Keywords: Biofuel cell; Bioenergy; Biocatalyzed electrolysis; Overpotentials; Biocatalysts; Ohmic resistance

Optimization of alkaline protease production by Aspergillus clavatus ES1 in Mirabilis jalapa tuber powder using statistical experimental design by Mohamed Hajji; Ahmed Rebai; Néji Gharsallah; Moncef Nasri (pp. 915-923).

Medium composition and culture conditions for the bleaching stable alkaline protease production by Aspergillus clavatus ES1 were optimized. Two statistical methods were used. Plackett–Burman design was applied to find the key ingredients and conditions for the best yield. Response surface methodology (RSM) including full factorial design was used to determine the optimal concentrations and conditions. Results indicated that Mirabilis jalapa tubers powder (MJTP), culture temperature, and initial medium pH had significant effects on the production. Under the proposed optimized conditions, the protease experimental yield (770.66 U/ml) closely matched the yield predicted by the statistical model (749.94 U/ml) with R ² = 0.98. The optimum operating conditions obtained from the RSM were MJTP concentration of 10 g/l, pH 8.0, and temperature of 30 °C, Sardinella heads and viscera flour (SHVF) and other salts were used at low level. The medium optimization contributed an about 14.0-fold higher yield than that of the unoptimized medium (starch 5 g/l, yeast extract 2 g/l, temperature 30 °C, and pH 6.0; 56 U/ml). More interestingly, the optimization was carried out with the by-product sources, which may result in cost–effective production of alkaline protease by the strain.

Keywords: Aspergillus clavatus ES1; Bleaching stable alkaline protease; Plackett–Burman design; Full factorial design; Mirabilis jalapa tuber powder; Economy

The macromolecule with antimicrobial activity synthesized by Pseudoalteromonas luteoviolacea strains is an l-amino acid oxidase by Daniel Gómez; Elena Espinosa; Marcelo Bertazzo; Patricia Lucas-Elío; Francisco Solano; Antonio Sanchez-Amat (pp. 925-930).

Two purple pigmented bacterial strains, CPMOR-1 and CPMOR-2, have been newly isolated from the Mediterranean Sea. 16S RNA sequencing and phenotypic characteristics indicate that they belong to the species Pseudoalteromonas luteoviolacea. The synthesis of macromolecules with antimicrobial activity is a capacity described in many strains of this species although the nature of those macromolecules has not been reported up to now. The search for antimicrobial compounds in the two new strains described in this work shows that they synthesize a macromolecule with antimicrobial activity that can be inhibited by catalase, as it had been described in the type strain P. luteoviolacea NCIMB 1893^T. This work elucidates the nature of such macromolecule as a novel l-amino acid oxidase (LAO) with broad substrate specificity. The enzyme is most active with Met, Gln, Leu, Phe, Glu, and Trp. In growth media containing those amino acids, the hydrogen peroxide generated by the reaction catalyzed by the LAO mediates its antimicrobial activity.

Keywords: l-Amino acid oxidase; Antimicrobial macromolecule; Hydrogen peroxide; Pseudoalteromonas luteoviolacea

Cloning, expression and characterisation of CYP102A7, a self-sufficient P450 monooxygenase from Bacillus licheniformis by Matthias Dietrich; Sabine Eiben; Chimene Asta; Tuan Anh Do; Juergen Pleiss; Vlada B. Urlacher (pp. 931-940).

Cytochrome P450 monooxygenases of the CYP102A subfamily are single-component natural fusion proteins consisting of a heme domain and a diflavin reductase. The characterised CYP102A enzymes are fatty acid hydroxylases with turnover rates of several thousands per minute. In search of new P450s with similar activities, but with a broader substrate spectrum, we cloned, expressed and characterised CYP102A7 from Bacillus licheniformis. As expected, CYP102A7 was active towards medium-chain fatty acids but showed a strong preference for saturated over unsaturated fatty acids, which could not be observed for either of the CYP102A members so far. Besides fatty acids, CYP102A7 was able to catalyse the oxidation of cyclic and acyclic terpenes with high activity and coupling efficiency. For example, (R)-(+)-limonene was converted with activity of 220 nmol nmol P450⁻¹ min⁻¹ and 80% coupling. Unusual for enzymes of the CYP102A subfamily was the deethylation activity of CYP102A7 towards 7-ethoxycoumarin. Furthermore, this monooxygenase, though having a moderate thermal stability, exhibited 50% of its initial activity in the presence of 26% DMSO. Comparison of the homology models of CYP102A7 and other members of the CYP102A subfamily revealed distinct differences in the shape of the substrate access channel and the active site, which might explain differences in catalytic properties of these homologous enzymes.

Keywords: Cytochrome P450 monooxygenases; CYP102A; Bacillus licheniformis ; Self-sufficient

Cloning and characterization of a novel exo-α-1,5-L-arabinanase gene and the enzyme by Dominic W. S. Wong; Victor J. Chan; Sarah B. Batt (pp. 941-949).

A novel exo-α-1,5-l-arabinanase gene (arn3) was isolated, cloned, and expressed in E. coli. The recombinant enzyme (ARN3) had a pH optimum of 6.0–7.0 and a pH 3.0–7.0 stability range. The temperature optimum was 50°C with a stability less than or equal to 45°C. The recombinant ARN3 cleaved carboxymethyl (CM)-arabinan, debranched arabinan, and linear arabinan at a decreasing rate and is inactive on sugar beet arabinan, wheat arabinoxylan, and p-nitrophenyl-α-l-arabinofuranoside. The enzyme hydrolyzed debranched arabinan and synthetic arabino-oligosaccharides entirely to arabinose. The apparent K _m and V _max values were determined to be 6.2 ± 0.3 mg/ml and 0.86 ± 0.01 mg ml⁻¹ min⁻¹, respectively (pH 7.0, 37°C, CM-arabinan). Multiple sequence alignment and homology modeling revealed unique short sequences of amino acids extending the loop involved in partial blocking of one end of the substrate-binding site on the surface of the molecule.

Keywords: Arabinanase; Exo-1,5-α-l-arabinanase; Pectinase; Arabinan-degrading enzyme

Potential of a 7-dimethylallyltryptophan synthase as a tool for production of prenylated indole derivatives by Anika Kremer; Shu-Ming Li (pp. 951-961).

Recently, a gene for a 7-dimethylallyltryptophan synthase (7-DMATS) was identified in Aspergillus fumigatus and its enzymatic function was proven biochemically. In this study, the behaviour of 7-DMATS towards aromatic substrates was investigated and compared with that of the 4-dimethylallyltryptophan synthase FgaPT2 from the same fungus. In total, 24 simple indole derivatives were tested as potential substrates for 7-DMATS. With an exception of 7-methyltryptophan, all of the substances were accepted by 7-DMATS and converted to their prenylated derivatives, indicating a more flexible substrate specificity of 7-DMATS in comparison to that of FgaPT2. The relative activities of 7-DMATS towards these substrates were from 4% to 89% of that of l-tryptophan, much higher than that of FgaPT2. Structural elucidation of the isolated enzymatic products by nuclear magnetic resonance and mass spectrometry analysis proved unequivocally the prenylation at position C7 of the indole ring. Overnight incubation with eight substances showed that the conversion ratios were in the range of 55.9% to 99.7%. This study provided an additional example that prenylated indole derivatives can be effectively produced by using the overproduced and purified 7-DMATS.

Keywords: Aspergillus fumigatus ; Chemoenzymatic synthesis; 7-dimethylallyltryptophan synthase; Prenylated indole derivatives; Prenyltransferase; Prenylation

Pasteurella multocida sialic acid aldolase: a promising biocatalyst by Yanhong Li; Hai Yu; Hongzhi Cao; Kam Lau; Saddam Muthana; Vinod Kumar Tiwari; Bryan Son; Xi Chen (pp. 963-970).

Sialic acid aldolases or N-acetylneuraminate lyases (NanAs) catalyze the reversible aldol cleavage of N-acetylneuraminic acid (Neu5Ac) to form pyruvate and N-acetyl-d-mannosamine (ManNAc). A capillary electrophoresis assay was developed to directly characterize the activities of NanAs in both Neu5Ac cleavage and Neu5Ac synthesis directions. The assay was used to obtain the pH profile and the kinetic data of a NanA cloned from Pasteurella multocida P-1059 (PmNanA) and a previously reported recombinant Escherichia coli K12 NanA (EcNanA). Both enzymes are active in a broad pH range of 6.0–9.0 in both reaction directions and have similar kinetic parameters. Substrates specificity studies showed that 5-O-methyl-ManNAc, a ManNAc derivative, can be used efficiently as a substrate by PmNanA, but not efficiently by EcNanA, for the synthesis of 8-O-methyl Neu5Ac. In addition, PmNanA (250 mg l⁻¹ culture) has a higher expression level (2.5-fold) than EcNanA (94 mg l⁻¹ culture). The higher expression level and a broader substrate tolerance make PmNanA a better catalyst than EcNanA for the chemoenzymatic synthesis of sialic acids and their derivatives.

Keywords: Aldolase; Capillary electrophoresis; Escherichia coli ; Lyase; NanA; Pasteurella multocida

Heterologous expression of lcc1 from Lentinula edodes in tobacco BY-2 cells results in the production an active, secreted form of fungal laccase by Yuichi Sakamoto; Keiko Nakade; Akira Yano; Yuko Nakagawa; Tatsuya Hirano; Toshikazu Irie; Hisayuki Watanabe; Masaru Nagai; Toshitsugu Sato (pp. 971-980).

Laccase (Lcc) is a lignin-degrading enzyme produced by white-rot fungi and has been the subject of much interest in the field of bioremediation due to its ability to oxidize phenolic compounds. In this report, we describe the isolation and characterization of lcc1, a novel gene of Lentinula edodes that encodes Lcc1, and demonstrate that recombinant Lcc1 is expressed in an active, secreted form in tobacco BY-2 cells in culture. The open reading frame of lcc1 was 1,557 base pairs in length and encoded a putative protein of 518 amino acids. We introduced a chimeric form of lcc1 (CaMV35Sp:clcc1) into tobacco BY-2 cells and obtained several stable clcc1 transformants that expressed active Lcc1. Lcc1 activity in BY-2 culture media was higher than in cellular extracts, which indicated that recombinant Lcc1 was produced in a secreted form. Recombinant Lcc1 had a smaller apparent molecular weight and exhibited a different pattern of posttranslational modification than Lcc1 purified from L. edodes. The substrate specificity of purified recombinant Lcc1 was similar to L. edodes Lcc1, and both enzymes were able to decolorize the same set of dyes. These results suggest that heterologous expression of fungal Lcc1 in BY-2 cells will be a valuable tool for the production of sufficient quantities of active laccase for bioremediation.

Keywords: Bio-remediation; BY-2; Heterologous expression; Laccase; Lentinula edodes

Production of hydroxy-fatty acid derivatives from waste oil by Escherichia coli cells producing fungal cytochrome P450foxy by Tatsuya Kitazume; Yuya Yamazaki; Shigeru Matsuyama; Hirofumi Shoun; Naoki Takaya (pp. 981-988).

Cytochrome P450foxy (P450foxy) is a fatty acid (FA) monooxygenase that is characterized by self-sufficient catalysis and high turnover numbers due to the fused structure of cytochrome P450 and its reductase. Here we found that resting recombinant Escherichia coli cells producing P450foxy converted saturated FA with a chain length of 7–16 carbon atoms to their ω − 1 to ω − 3 hydroxy derivatives. Most products were recovered from the culture supernatant. Decanoic acid was most efficiently converted to ω − 1 to ω − 3 hydroxy decanoic acids in the order of ω − 1 > ω − 2 > ω − 3, with a total product yield of 47%. We also found that P450foxy was more active against physiological fatty acyl esters such as monopalmitoyl glycerol, monopalmitoyl phospholipid, and palmitoyl CoA than free palmitic acid. The bacteria producing P450foxy were applicable as biocatalysts in the production of ω − 1 hydroxy palmitic acid from lard, vegetable, and soy sauce oil wastes from the food industry.

Keywords: Cytochrome P450; Hydroxyl fatty acid; Bio-conversion; Fusarium oxysporum

Purification, biochemical and molecular characterization of a metalloprotease from Pseudomonas aeruginosa MN7 grown on shrimp wastes by Kemel Jellouli; Ahmed Bayoudh; Laila Manni; Rym Agrebi; Moncef Nasri (pp. 989-999).

A protease-producing bacterium was isolated and identified as Pseudomonas aeruginosa MN7. The strain was found to produce proteases when it was grown in media containing only shrimp waste powder (SWP), indicating that it can obtain its carbon, nitrogen, and salts requirements directly from shrimp waste. The use of 60 g/l SWP resulted in a high protease production. Elastase, the major protease produced by P. aeruginosa MN7, was purified from the culture supernatant to homogeneity using acetone precipitation, Sephadex G-75 gel filtration, and ultrafiltration using a 10-kDa cut-off membrane, with a 5.2-fold increase in specific activity and 38.4% recovery. The molecular weight of the purified elastase was estimated to be 34 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration. The optimum temperature and pH for protease activity were 60°C and 8.0, respectively. The activity of the enzyme was totally lost in the presence of ethylene glycol tetraacetic acid, suggesting that the purified enzyme is a metalloprotease. The purified enzyme was highly stable in the presence of organic solvents, retaining 100% of its initial activity after 60 days of incubation at 30°C in the presence of dimethyl sulfoxide and methanol. The lasB gene, encoding the MN7 elastase, was isolated and its DNA sequence was determined.

Keywords: Pseudomonas aeruginosa ; Organic solvent-stable; Elastase; Metalloprotease; DNA sequence; Shrimp waste

High-level overproduction of Thermus enzymes in Streptomyces lividans by Margarita Díaz; Eloy Ferreras; Renata Moreno; Ana Yepes; José Berenguer; Ramón Santamaría (pp. 1001-1008).

Biotechnology needs to explore the capacity of different organisms to overproduce proteins of interest at low cost. In this paper, we show that Streptomyces lividans is a suitable host for the expression of Thermus thermophilus genes and report the overproduction of the corresponding proteins. This capacity was corroborated after cloning the genes corresponding to an alkaline phosphatase (a periplasmic enzyme in T. thermophilus) and that corresponding to a beta-glycosidase (an intracellular enzyme) in Escherichia coli and in S. lividans. Comparison of the production in both hosts revealed that the expression of active protein achieved in S. lividans was much higher than in E. coli, especially in the case of the periplasmic enzyme. In fact, the native signal peptide of the T. thermophilus phosphatase was functional in S. lividans, being processed at the same peptide bond in both organisms, allowing the overproduction and secretion of this protein to the S. lividans culture supernatant. As in E. coli, the thermostability of the expressed proteins allowed a huge purification factor upon thermal denaturation and precipitation of the host proteins. We conclude that S. lividans is a very efficient and industry-friendly host for the expression of thermophilic proteins from Thermus spp.

Role of N-terminal 28-amino-acid region of Rhizopus oryzae lipase in directing proteins to secretory pathway of Aspergillus oryzae by Shinji Hama; Sriappareddy Tamalampudi; Naoki Shindo; Takao Numata; Hideki Yamaji; Hideki Fukuda; Akihiko Kondo (pp. 1009-1018).

To develop a new approach for improving heterologous protein production in Aspergillus oryzae, we focused on the functional role of the N-terminal region of Rhizopus oryzae lipase (ROL). Several N-terminal deletion variants of ROL were expressed in A. oryzae. Interestingly, a segment of 28 amino acids from the C-terminal region of the propeptide (N28) was found to be critical for secretion of ROL into the culture medium. To further investigate the role of N28, the ROL secretory process was visualized in vivo using ROL–green fluorescent protein (GFP) fusion proteins. In cells producing ROL with N28, fluorescence observations showed that the fusion proteins are transported through endoplasmic reticulum (ER), Golgi, and cell wall, which is one of the typical secretory processes in a eukaryotic cell. Because the expression of the mature ROL–GFP fusion protein induced fluorescence accumulation without its translocation into the ER, N28 is considered to play a crucial role in protein transport. When N28 was inserted between the secretion signal and GFP, fluorescence observations showed that GFP, which is originally a cytoplasmic protein, was efficiently translocated into the ER of A. oryzae, resulting in an enhanced secretion of mature GFP after proteolytic cleavage of N28. These findings suggest that N28 facilitates protein translocation into ER and can be a promising candidate for improving heterologous protein production in A. oryzae.

Keywords: Filamentous fungi; Protein secretion; Heterologous protein production; N-terminal peptide; Green fluorescent protein; Visualization

Construction of cell surface-engineered yeasts displaying antigen to detect antibodies by immunofluorescence and yeast-ELISA by Yu Qian Tang; Shuang Yan Han; Hong Zheng; Lin Wu; Mitsuyoshi Ueda; Xiao Ning Wang; Ying Lin (pp. 1019-1026).

In order to detect monoclonal antibodies (MAbs) from insufficient and unavailable human proteins, yeast cells were engineered to display human antigens on their surface and consequently endowed with the ability to specifically bind antibodies. Thus, a fusion gene for the expression of the human proteasome subunit alpha 6 (hPSA6) and human profilin I (hProI) were assembled, respectively, with a His.tag marker at the C-terminal and displayed on yeast surface. With anti-His.tag MAb as the primary antibody and the fluorescein isothiocyanate-conjugated goat anti-mouse Immunogloblin G as the second antibody, the surface display of hPSA6 and hProI were verified by immunofluorescence labeling. The antigen-displayed yeast particles were used for MAbs detection from ascites through both immunofluorescence and yeast-enzyme-linked immunosorbent assay (ELISA) methods. The results were verified by Western blotting and indirect ELISA. By improving the sensitivity, the novel MAbs detection can be applied in the generation and screening of positive hybridoma. It is suggested that by combining the DNA immunization, the present study can evolve into a quick and protein-free way of MAbs production for insufficient and unavailable antigen.

Keywords: Yeast display; Immunofluorescence; Yeast-ELISA; Human proteasome subunit alpha 6; Human profilin I

Secretory delivery of heterologous proteins in attenuated Vibrio anguillarum for potential use in vaccine design by Lingyun Zhou; Qin Liu; Qiyao Wang; Yue Ma; Yuzhou Xu; Zhao Yang; Yan Zhao; Yuanxing Zhang (pp. 1027-1034).

To synthesize and secrete heterologous proteins in an attenuated Vibrio anguillarum strain for potential multivalent live vaccine development, different antigen-delivery systems based on bacterial-originated secretion signal peptides (SPs) were designed and identified in this work. Four SPs were derived from hemolysin of Escherichia coli, RTX protein of V. cholerae, hemolysin of V. anguillarum, zinc-metalloprotease of V. anguillarum, respectively, and their abilities to support secretion of green fluorescent protein (GFP) in an attenuated V. anguillarum strain MVAV6203 were assayed. Immunodetection of GFP showed that the capability of the tested signal leaders to direct secretion of GFP varied greatly. Although all the four signal peptide-fused GFPs could be expressed correctly and trapped intracellularly in recombinant strains, only the EmpA signal peptide could confer efficient secretion to GFP. For the investigation of its potential application in live bacteria carrier vaccines, a heterologous protein EseB of Edwardsiella tarda was fused to the SP empA antigen-delivery system and introduced into the strain MVAV6203. Further analysis of EseB demonstrated that the constructed SP empA antigen-delivery system could be used to secrete foreign protein in attenuated V. anguillarum and be available for carrier vaccines development.

Keywords: EseB; Green fluorescent protein; Signal peptide; Secretion; Vaccine; Vibrio anguillarum

Effect of folding factors in rescuing unstable heterologous lipase B to enhance its overexpression in the periplasm of Escherichia coli by Yali Xu; Darrell Lewis; C. Perry Chou (pp. 1035-1044).

Functional expression of recombinant Pseudozyma antarctica lipase B (PalB) in Escherichia coli was explored. While PalB was stably expressed in the cytoplasm, most of the expressed gene product aggregated in cells as inactive inclusion bodies. In contrast, PalB was extremely unstable when expressed in the periplasm, also leading to poor expression performance. Such unstable PalB can be rescued by coexpression of several periplasmic folding factors, such as DegP, FkpA, DsbA, and DsbC but not cytoplasmic ones. As a result, the performance for functional PalB expression in the periplasm was significantly improved. To our knowledge, this is the first report demonstrating the use of folding factors to rescue the extremely unstable gene product that is otherwise completely degradable.

Keywords: Chaperone; Escherichia coli ; Folding factor; Gene expression; Lipase B; Pseudozyma antarctica ; Recombinant protein production

Impact of polyunsaturated fatty acid degradation on survival and acidification activity of freeze-dried Weissella paramesenteroides LC11 during storage by Amenan A. Yao; Ibourahema Coulibaly; Georges Lognay; Marie-Laure Fauconnier; Philippe Thonart (pp. 1045-1052).

The impact of polyunsaturated fatty acid (PUFA) degradation on the survival and acidification activity of freeze-dried Weissella paramesenteroides LC11 was investigated over 90-days storage at 4 °C or 20 °C in vacuum-sealed aluminium foil or glass tubes with two water activities (a _w = 0.11 or 0.23). Colony counts, acidification activity (% lactic acid/g), linoleic/palmitic (18:2/16:0) or linolenic/palmitic (18:3/16:0) ratio by gas chromatography and 18:2 or 18:3 oxylipins by reversed phase-high performance liquid chromatography were determined. The viable cells, acidification activity and 18:2/16:0 or 18:3/16:0 ratio decreased as the storage time increased. The survival, acidification activity and 18:2/16:0 or 18:3/16:0 ratio were greatest for the freeze-dried strain held in vacuum-sealed aluminium foil at 4 °C. The 18:2/16:0 or 18:3/16:0 ratio decrease was correlated with the accumulation of 18:2 or 18:3 oxylipins during storage in glass tubes. Hydroperoxy PUFAs, hydroxy PUFAs, divinyl ether PUFAs and oxo PUFAs were the main oxylipins identified. A large decrease in the 18:2/16:0 or 18:3/16:0 ratio and a rapid accumulation of oxylipins during storage might be enough to cause high cell death and loss of metabolic activity. These results provide further experimental support for the hypothesis that lipid oxidation and survival or activity of freeze-dried bacteria might be related.

Keywords: Lactic acid bacteria; Lipid oxidation; Oxylipins; Polyunsaturated fatty acid; Viability

Decolorization of azo dyes by Shewanella sp. under saline conditions by Azeem Khalid; Muhammad Arshad; David E. Crowley (pp. 1053-1059).

Wastewaters from textile processing and dye-stuff manufacture industries contain substantial amounts of salts in addition to azo dye residues. To examine salinity effects on dye-degrading bacteria, a study was carried out with four azo dyes in the presence of varying concentrations of NaCl (0–100 g l⁻¹) with a previously isolated bacterium, Shewanella putrefaciens strain AS96. Under static, low oxygen conditions, the bacterium decolorized 100 mg dye l⁻¹ at salt concentrations up to 60 g NaCl l⁻¹. There was an inverse relationship between the velocity of the decolorization reaction and salt concentration over the range between 5 and 60 g NaCl l⁻¹ and at dye concentrations between 100 and 500 mg l⁻¹. The addition of either glucose (C source) or NH₄NO₃ (N source) to the medium strongly inhibited the decolorization process, while yeast extract (4 g l⁻¹) and Ca(H₂PO₄)₂·H₂O (1 g l⁻¹) both enhanced decolorization rates. High-performance liquid chromatography analysis demonstrated the presence of 1-amino-2-naphthol, sulfanilic acid and nitroaniline as the major metabolic products of the azo dyes, which could be further degraded by a shift to aerobic conditions. These findings show that Shewanella could be effective for the treatment of dye-containing industrial effluents containing high concentrations of salt.

Keywords: Azo dye; Decolorization; Salinity; Nitroaniline; Wastewater

Ammonia–methane two-stage anaerobic digestion of dehydrated waste-activated sludge by Yutaka Nakashimada; Yasutaka Ohshima; Hisao Minami; Hironori Yabu; Yuzaburo Namba; Naomichi Nishio (pp. 1061-1069).

The study investigated methane production from dehydrated waste-activated sludge (DWAS) with approximately 80% water content under thermophilic conditions. The repeated batch-wise treatment of DWAS using methanogenic sludge unacclimated to high concentrations of ammonia, increased the ammonia production up to 7,600 mg N per kilogram total wet sludge of total ammonia concentration, and stopped the methane production. Investigation revealed that the loading ratio of DWAS for methanogenic sludge influences anaerobic digestion. Methane production significantly decreased and ammonia concentration increased with the increase in loading ratio of DWAS. Since the semicontinuous culture revealed that approximately 50% of organic nitrogen in DWAS converted to ammonia at sludge retention time (SRT) after 4 days at 37°C and 1.33 days at 55°C, the previous stripping of the ammonia produced from DWAS was carried out. The stripping of ammonia increased methane production significantly. This ammonia–methane two-stage anaerobic digestion demonstrated a successful methane production at SRT 20 days in the semicontinuous operation using a laboratory-scale reactor system.

Keywords: Dehydrated waste-activated sludge; Ammonia stripping; Anaerobic digestion

Microbial community of granules in expanded granular sludge bed reactor for simultaneous biological removal of sulfate, nitrate and lactate by Chuan Chen; Nanqi Ren; Aijie Wang; Zhenguo Yu; Duu-Jong Lee (pp. 1071-1077).

This study studied the cultivation of granules from an expanded granular sludge bed reactor that simultaneously transforms sulfates, nitrates, and oxygen to elementary sulfur, nitrogen gas, and carbon dioxides, respectively. The living cells accumulate at the granule outer layers, as revealed by the multicolor staining and confocal laser scanning microscope technique. The microbial community comprises sulfate-reducing bacteria (SRB, Desulfomicrobium sp.), heterotrophic (Pseudomonas aeruginosa and Sulfurospirillum sp.), and autotrophic denitrifiers (Sulfurovum sp. and Paracoccus denitrificans) whose population dynamics at different sulfate and nitrate loading rates are monitored with the single-strand conformation polymorphism and denaturing gradient gel electrophoresis technique. The Desulfomicrobium sp. presents one of the dominating strains following reactor startup. At high sulfate and nitrate loading rates, the heterotrophic denitrifiers overcompete autotrophic denitrifiers to reduce SRB activities. Conversely, suddenly reducing nitrate loading rates completely removes the heterotrophic denitrifier Sulfurospirillum sp. from the granules and activates the autotrophic denitrifiers. The physical fixation of different groups of functional strains in granules fine-tunes the strains’ activities, and hence the reactor performance.

Keywords: Sulfate-reducing bacteria; Denitrifier; Granules; Population dynamics

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Applied Microbiology and Biotechnology (v.79, #6)

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Applied Microbiology and Biotechnology (v.79, #6)