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Applied Microbiology and Biotechnology (v.90, #4)

An overview of lipid metabolism in yeasts and its impact on biotechnological processes by Athanasios Beopoulos; Jean-Marc Nicaud; Claude Gaillardin (pp. 1193-1206).

High energy prices, depletion of crude oil supplies, and price imbalance created by the increasing demand of plant oils or animal fat for biodiesel and specific lipid derivatives such as lubricants, adhesives, and plastics have given rise to heated debates on land-use practices and to environmental concerns about oil production strategies. However, commercialization of microbial oils with similar composition and energy value to plant and animal oils could have many advantages, such as being non-competitive with food, having shorter process cycle and being independent of season and climate factors. This review focuses on the ongoing research on different oleaginous yeasts producing high added value lipids and on the prospects of such microbial oils to be used in different biotechnological processes and applications. It covers the basic biochemical mechanisms of lipid synthesis and accumulation in these organisms, along with the latest insights on the metabolic processes involved. The key elements of lipid accumulation, the mechanisms suspected to confer the oleaginous character of the cell, and the potential metabolic routes enhancing lipid production are also extensively discussed.

Keywords: Yeast; Lipid metabolism; Bio-oils; Biotechnology; Genetic engineering

Beneficial effects of Monascus purpureus NTU 568-fermented products: a review by Yeu-Ching Shi; Tzu-Ming Pan (pp. 1207-1217).

Monascus-fermented products have been used in food, medicine, and industry dating back over a thousand years in Asian countries. Monascus-fermented products contained several bioactive metabolites such as pigments, polyketide monacolins, dimerumic acid, and γ-aminobutyric acid. Scientific reports showed that Monascus-fermented products proved to be effective for the management of blood cholesterol, diabetes, blood pressure, obesity, Alzheimer’s disease, and prevention of cancer development. This review article describes the beneficial effects about using Monascus-fermented products in human beings and animals.

Keywords: Monascus ; Red mold rice; Red mold dioscorea; Hyperlipidemia; Metabolite

Oily yeasts as oleaginous cell factories by Jose Manuel Ageitos; Juan Andres Vallejo; Patricia Veiga-Crespo; Tomas G. Villa (pp. 1219-1227).

Oily yeasts have been described to be able to accumulate lipids up to 20% of their cellular dry weight. These yeasts represent a minor proportion of the total yeast population, and only 5% of them have been reported as able to accumulate more than 25% of lipids. The oily yeast genera include Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon, and Lipomyces. More specifically, examples of oleaginous yeasts include the species: Lipomyces starkeyi, Rhodosporidium toruloides, Rhodotorula glutinis, and Yarrowia lipolytica. Yeast do exhibit advantages for lipid production over other microbial sources, namely, their duplication times are usually lower than 1 h, are much less affected than plants by season or climate conditions, and their cultures are more easily scaled up than those of microalgae. Additionally, some oily yeasts have been reported to accumulate oil up to 80% of their dry weight and can indeed generate different lipids from different carbon sources or from lipids present in the culture media. Thus, they can vary their lipid composition by replacing the fatty acids present in their triglycerides. Due to the diversity of microorganisms and growth conditions, oily yeasts can be useful for the production of triglycerides, surfactants, or polyunsaturated fatty acids.

Keywords: Oleaginous; Yeast; Cryptococcus ; Rhodotorula ; Rodosporidum ; Candida ; Lipids; Triglycerides

Production and metabolic engineering of bioactive substances in plant hairy root culture by Mei-Liang Zhou; Xue-Mei Zhu; Ji-Rong Shao; Yi-Xiong Tang; Yan-Min Wu (pp. 1229-1239).

In the past three decades, hairy roots research for the production of valuable biological active substances has received a lot of attention. The addition of knowledge to enhance the yields of desired substances and the development of novel tools for biomass engineering offer new possibilities for large-scale cultivation of the plant hairy root. Hairy roots can also produce recombinant proteins through the transfer of Agrobacterium T-DNA into the plant genome, and thereby hold immense potential for the pharmaceutical industry. This review highlights some of the significant progress made in the past few years and outlines future prospects for exploiting the potential utility of hairy root cultures as “chemical factories” for producing bioactive substances.

Keywords: Elicitor; Hairy roots; Metabolic engineering; Pharmaceutical protein; Secondary metabolites

Advances in methods for detection of anaerobic ammonium oxidizing (anammox) bacteria by Meng Li; Ji-Dong Gu (pp. 1241-1252).

Anaerobic ammonium oxidation (anammox), the biochemical process oxidizing ammonium into dinitrogen gas using nitrite as an electron acceptor, has only been recognized for its significant role in the global nitrogen cycle not long ago, and its ubiquitous distribution in a wide range of environments has changed our knowledge about the contributors to the global nitrogen cycle. Currently, several groups of methods are used in detection of anammox bacteria based on their physiological and biochemical characteristics, cellular chemical composition, and both 16S rRNA gene and selective functional genes as biomarkers, including hydrazine oxidoreductase and nitrite reductase encoding genes hzo and nirS, respectively. Results from these methods coupling with advances in quantitative PCR, reverse transcription of mRNA genes and stable isotope labeling have improved our understanding on the distribution, diversity, and activity of anammox bacteria in different environments both natural and engineered ones. In this review, we summarize these methods used in detection of anammox bacteria from various environments, highlight the strengths and weakness of these methods, and also discuss the new development potentials on the existing and new techniques in the future.

Keywords: Anammox bacteria; Isotope pairing technique; Lipid analysis; Molecular technique; 16S rRNA gene; Functional biomarkers

Development of recombinant Klebsiella pneumoniae ∆dhaT strain for the co-production of 3-hydroxypropionic acid and 1,3-propanediol from glycerol by Somasundar Ashok; Subramanian Mohan Raj; Chelladurai Rathnasingh; Sunghoon Park (pp. 1253-1265).

Klebsiella pneumoniae converts glycerol to the specialty chemical 1,3-propanediol (1,3-PDO), which is used for the production of polytrimethylene terepthalate (PTT). In this study, an NAD⁺-dependent gamma-glutamyl-gamma-aminobutyraldehyde dehydrogenase (PuuC) of K. pneumoniae DSM 2026, which oxidizes 3-hydroxypropionaldehyde to a platform chemical 3-hydroxypropionic acid (3-HP), was cloned and overexpressed in K. pneumoniae DSM 2026 for the co-production of 3-HP and 1,3-PDO from glycerol. In addition, the gene dhaT, encoding NADH-dependent 1,3-propanediol oxidoreductase (1,3-PDOR), was deleted from the chromosome for the balanced production of 3-HP and 1,3-PDO. The recombinant K. pneumoniae ∆dhaT, expressing puuC, produced 3.6 g 3-HP and 3.0 g 1,3-PDO per liter with an average yield of 81% on glycerol carbon in shake flask culture under microaerobic conditions. When a fed-batch culture was carried out under microaerobic conditions at pH 7.0 in a 5-l bioreactor, the recombinant K. pneumoniae ∆dhaT (puuC) strain produced 16.0 g 3-HP and 16.8 g 1,3-PDO per liter with a cumulative yield of 51% on glycerol carbon in 24 h. The production of 1,3-PDO in the dhaT-deletion mutant was attributed to the expression of NAD(P)H-dependent hypothetical oxidoreductase. This study demonstrates the feasibility of obtaining two commercially valuable chemicals, 3-HP and 1,3-PDO, at a significant scale.

Keywords: 3-hydroxypropionic acid; 1,3-propanediol; Glycerol; Co-production; Gamma-glutamyl-gamma-aminobutyraldehyde dehydrogenase; Klebsiella pneumoniae

A novel downstream process for 1,3-propanediol from glycerol-based fermentation by Pinki Anand; Rajendra Kumar Saxena; Ruchi G. Marwah (pp. 1267-1276).

In this paper, a downstream process for purification of 1,3-propanediol from glycerol-based fermentation broth was investigated. The purification of 1,3-propanediol from fermentation broth was achieved by a process combining microfiltration, charcoal treatment, vacuum distillation, and silica gel chromatography. The broth was first filtered through hollow fiber cartridge, wherein 98.7% of biomass was removed. Soluble proteins and other color impurities in the broth were removed by the use of activated charcoal at optimal concentration of 30 g l⁻¹ where the soluble proteins in the broth decreased to 0.1 g l⁻¹ (96.0% protein loss). The obtained broth when concentrated by vacuum distillation resulted in the crystallization of inorganic salts. Subsequently, 1,3-propanediol was purified by gradient chromatography using silica gel as a stationary phase and mixture of chloroform and methanol as a mobile phase. Finally, with the optimal flow rate of 10 ml min⁻¹ and loading amount of 80 ml, the yield of 1,3-propanediol achieved was 89%. The overall yield of 1,3-propanediol using the proposed procedure was 75.47%. The developed method was found to be a simple, rapid, and efficient procedure for the purification of 1,3-propanediol from fermentation broth.

Keywords: 1,3-Propanediol; Glycerol; Downstream processing; Silica gel; Adsorption chromatography

A novel rapid and continuous procedure for large-scale purification of magnetosomes from Magnetospirillum gryphiswaldense by Fangfang Guo; Yang Liu; Yanping Chen; Tao Tang; Wei Jiang; Ying Li; Jilun Li (pp. 1277-1283).

A new rapid and continuous procedure was developed for purifying magnetosomes from Magnetospirillum gryphiswaldense MSR-1 cells on a large scale. The procedure included these steps: disruption of cells with a high-pressure homogeniser, isolation of magnetosomes with a continuous magnetism isolation system accompanied by low-power ultrasonication and urea treatment, removal of adsorbed and surface proteins with proteinase K, removal of nucleic acids with electro-elution, and replacement of the PBS buffer with distilled water by a magnetically stirred system. The purified magnetosomes were stored at −20 °C after lyophilized and treated with γ-rays. The time required for purification was reduced from 20–30 to 2–5 days. Evaluation of the purity of the resulting magnetosomes was carried out with SDS-PAGE, PCR, and Fourier-transform infrared spectroscopy. The overall data suggest that the method presented here is a simple, rapid, continuous, and highly efficient procedure for large-scale purification of magnetosomes.

Keywords: Magnetospirillum gryphiswaldense ; Magnetosomes; Large-scale purification

Properties of recombinant Strep-tagged and untagged hyperthermophilic D-arabitol dehydrogenase from Thermotoga maritima by Verena Kallnik; Christian Schultz; Paul Schweiger; Uwe Deppenmeier (pp. 1285-1293).

The first hyperthermophilic d-arabitol dehydrogenase from Thermotoga maritima was heterologously purified from Escherichia coli. The protein was purified with and without a Strep-tag. The enzyme exclusively catalyzed the NAD(H)-dependent oxidoreduction of d-arabitol, d-xylitol, d-ribulose, or d-xylulose. A twofold increase of catalytic rates was observed upon addition of Mg²⁺ or K⁺. Interestingly, only the tag-less protein was thermostable, retaining 90% of its activity after 90 min at 85 °C. However, the tag-less form of d-arabitol dehydrogenase had similar kinetic parameters compared to the tagged enzyme, demonstrating that the Strep-tag was not deleterious to protein function but decreased protein stability. A single band at 27.6 kDa was observed on SDS-PAGE and native PAGE revealed that the protein formed a homohexamer and a homododecamer. The enzyme catalyzed oxidation of d-arabitol to d-ribulose and therefore belongs to the class of d-arabitol 2-dehydrogenases, which are typically observed in yeast and not bacteria. The product d-ribulose is a rare ketopentose sugar that has numerous industrially applications. Given its thermostability and specificity, d-arabitol 2-dehydrogenase is a desirable biocatalyst for the production of rare sugar precursors.

Keywords: Thermotoga ; Thermozymes; Thermophile; Biotechnology; Polyol dehydrogenase; Short-chain dehydrogenase/reductase

Catalytic efficiency of HAP phytases is determined by a key residue in close proximity to the active site by Dawei Fu; Zhongyuan Li; Huoqing Huang; Tiezheng Yuan; Pengjun Shi; Huiying Luo; Kun Meng; Peilong Yang; Bin Yao (pp. 1295-1302).

The maximum activity of Yersinia enterocolitica phytase (YeAPPA) occurs at pH 5.0 and 45 °C, and notably, its specific activity (3.28 ± 0.24 U mg⁻¹) is 800-fold less than that of its Yersinia kristeensenii homolog (YkAPPA; 88% amino acid sequence identity). Sequence alignment and molecular modeling show that the arginine at position 79 (Arg79) in YeAPPA corresponding to Gly in YkAPPA as well as other histidine acid phosphatase (HAP) phytases is the only non-conserved residue near the catalytic site. To characterize the effects of the corresponding residue on the specific activities of HAP phytases, Escherichia coli EcAPPA, a well-characterized phytase with a known crystal structure, was selected for mutagenesis—its Gly73 was replaced with Arg, Asp, Glu, Ser, Thr, Leu, or Tyr. The results show that the specific activities of all of the corresponding EcAPPA mutants (17–2,400 U mg⁻¹) were less than that of the wild-type phytase (3,524 U mg⁻¹), and the activity levels were approximately proportional to the molecular volumes of the substituted residues’ side chains. Site-directed replacement of Arg79 in YeAPPA (corresponding to Gly73 of EcAPPA) with Ser, Leu, and Gly largely increased the specific activity, which further verified the key role of the residue at position 79 for determining phytase activity. Thus, a new determinant that influences the catalytic efficiency of HAP phytases has been identified.

Keywords: Specific activity; Phytase; Site-directed mutagenesis; Yersinia enterocolitica ; Escherichia coli

Functional characterization of the recombinant antimicrobial peptide Trx-Ace-AMP1 and its application on the control of tomato early blight disease by Yin Wu; Yue He; Xiaochun Ge (pp. 1303-1310).

Ace-AMP1 is a potent antifungal peptide found in onion (Allium cepa) seeds with sequence similarity to plant lipid transfer proteins. Transgenic plants over-expressing Ace-AMP1 gene have enhanced disease resistance to some fungal pathogens. However, mass production in heterologous systems and in vitro application of this peptide have not been reported. In this study, Ace-AMP1 was highly expressed in a prokaryotic Escherichia coli system as a fusion protein. The purified protein inhibited the growth of many plant fungal pathogens, especially Alternaria solani, Fusarium oxysporum f. sp. vasinfectum, and Verticillium dahliae. The inhibitory effect was accompanied by hyphal hyperbranching for V. dahliae but not for F. oxysporum f. sp. vasinfectum and A. solani, suggesting that the mode of action of Ace-AMP1 on different fungi might be different. Application of Ace-AMP1 on tomato leaves showed that the recombinant protein conferred strong resistance to the tomato pathogen A. solani and could be used as an effective fungicide.

Keywords: Ace-AMP1; Antifungal activity; Fungicide; Plant pathogens

Protein disulphide isomerase-assisted functionalization of keratin-based matrices by Margarida M. Fernandes; Andreia C. Gomes; Andreia Vasconcelos; Florentina-Daniela Munteanu; Tzanko Tzanov; Maria Sameiro T. Gonçalves; Nicole End; Kai-Uwe Schoening; Georg M. Guebitz; Artur Cavaco-Paulo (pp. 1311-1321).

In living systems, protein disulphide isomerase (PDI, EC 5.3.4.1) regulates the formation of new disulphide bonds in proteins (oxidase activity) and catalyzes the rearrangement of non-native disulphide bonds (isomerase activity), leading proteins towards their native configuration. In this study, PDI was used to attach cysteine-containing compounds (CCCs) onto hair, to enhance compound migration within hair fibre and to trigger protein release. A fluorescent (5(6)-TAMRA)-labelled keratin peptide was incorporated into hair by using PDI. Similarly, PDI promoted the grafting of a cysteine-functionalized dye onto wool, as suggested by matrix-assisted laser desorption and ionization time-of-flight results. These reactions were thought to involve oxidation of disulphide bonds between CCCs and wool or hair cysteine residues, catalyzed by the oxidized PDI active site. On the other hand, PDI was demonstrated to enhance the migration of a disulphide bond-functionalized dye within the keratin matrix and trigger the release of RNase A from wool fibres’ surface. These observations may indicate that an isomerisation reaction occurred, catalyzed by the reduced PDI active site, to achieve the thiol-disulphide exchange, i.e. the rearrangement of disulphide bonds between CCCs and keratin. The present communication aims to highlight promising biotechnological applications of PDI, derived from its almost unique properties within the isomerase family.

Keywords: Cysteine-containing compounds (CCCs); Disulphide bonds; Keratin fibres; Protein disulphide isomerase (PDI); Protein release

The α-glucuronidase Agu1 from Schizophyllum commune is a member of a novel glycoside hydrolase family (GH115) by Sun-Li Chong; Evy Battaglia; Pedro M. Coutinho; Bernard Henrissat; Maija Tenkanen; Ronald P. de Vries (pp. 1323-1332).

Schizophyllum commune produces an α-glucuronidase that is active on polymeric xylan, while the ascomycete α-glucuronidases are only active on xylan oligomers. In this study, we have identified the gene (agu1) encoding this enzyme and confirmed the functionality by overexpression of the gene in S. commune and degradation of aldopentauronic acids, (MeGlcA)³-Xyl₄, in the cultivation medium of the transformants. Expression analysis demonstrated that agu1 is not co-regulated with the predominant xylanase-encoding gene (xynA) of S. commune. The detailed sequence analysis of Agu1 demonstrated that this gene belongs to a novel glycoside hydrolase family (GH115) that also contains candidate genes from ascomycete fungi and bacteria. Phylogenetic analysis showed that the fungal GH115 α-glucuronidases are distinctly separate from the prokaryotic clade and distributed over three branches. The identification of putative genes encoding this enzyme in industrial fungi, such as Aspergillus oryzae and Hypocrea jecorina, will provide a starting point for further analysis of the importance of this enzyme for the hydrolysis of plant biomass.

Keywords: α-Glucuronidase; Schizophyllum commune ; GH115; Gene expression; Xylan degradation

Isolation and characterisation of KP34—a novel φKMV-like bacteriophage for Klebsiella pneumoniae by Zuzanna Drulis-Kawa; Paweł Mackiewicz; Agata Kęsik-Szeloch; Ewa Maciaszczyk-Dziubinska; Beata Weber-Dąbrowska; Agata Dorotkiewicz-Jach; Daria Augustyniak; Grażyna Majkowska-Skrobek; Tomasz Bocer; Joanna Empel; Andrew M. Kropinski (pp. 1333-1345).

Bacteriophage KP34 is a novel virus belonging to the subfamily Autographivirinae lytic for extended-spectrum β-lactamase-producing Klebsiella pneumoniae strains. Its biological features, morphology, susceptibility to chemical and physical agents, burst size, host specificity and activity spectrum were determined. As a potential antibacterial agent used in therapy, KP34 molecular features including genome sequence and protein composition were examined. Phylogenetic analyses and clustering of KP34 phage genome sequences revealed its clear relationships with “phiKMV-like viruses”. Simultaneously, whole-genome analyses permitted clustering and classification of all phages, with completely sequenced genomes, belonging to the Podoviridae.

Keywords: φKMV-like bacteriophage; Genome; Klebsiella pneumoniae ; Phage therapy; Podoviridae ; phiKMV-like viruses

Distribution of dTDP-glucose-4,6-dehydratase gene and diversity of potential glycosylated natural products in marine sediment-derived bacteria by Feifei Chen; Ling Lin; Lu Wang; Yi Tan; Hongxia Zhou; Yiguang Wang; Yong Wang; Weiqing He (pp. 1347-1359).

To investigate the distribution of dTDP-glucose-4,6-dehydratase (dTGD) gene and diversity of the potential 6-deoxyhexose (6DOH) glycosylated compounds in marine microorganisms, a total of 91 marine sediment-derived bacteria, representing 48 operational taxonomic units and belonging to 25 genera, were screened by polymerase chain reaction. In total, 84% of the strains were dTGD gene positive, suggesting 6DOH biosynthetic pathway is widespread in these marine sediment-derived bacteria. BLASTp results of dTGD gene fragments indicate a high chemical diversity of the potential 6DOH glycosylated compounds. Close phylogenetic relationship occurred between dTGDs involved in the production of same or similar 6DOH glycosylated compounds, suggesting dTGD can be used to predict the structure of potential 6DOH glycosylated compounds produced by new strains. In two cases, where dTGD shared ≥85% amino acid identity and close phylogenetic relationship with their counterparts, 6DOH glycosylated compounds were accurately predicted. Our results demonstrate that phylogenetic analysis of dTGD gene is useful for structure prediction of glycosylated compounds from newly isolated strains and can therefore guide the chemical purification and structure identification process. The rapid identification of strains that possess dTGD gene provides a bioinformatics assessment of the greatest potential to produce glycosylated compounds despite the absence of fully biosynthetic pathways or genome sequences.

Keywords: dTDP-glucose-4,6-dehydratase (dTGD); Glycosylated compounds; 6-Deoxyhexose (6DOH); Marine sediment-derived bacteria

Gradually accumulating beneficial mutations to improve the thermostability of N-carbamoyl-d-amino acid amidohydrolase by step-wise evolution by Dalong Zhang; Fuyun Zhu; Wenchao Fan; Rongsheng Tao; Hong Yu; Yunliu Yang; Weihong Jiang; Sheng Yang (pp. 1361-1371).

To further enhance repeated batch reactions with immobilized N-carbamoyl-d-amino acid amidohydrolase (DCase), which can be used for the industrial production of d-amino acids, the stability of high soluble mutant DCase-M3 from Ralstonia pickettii CGMCC1596 was improved by step-wise evolution. In our previous report, six thermostability-related sites were identified by error-prone PCR. Based on the above result, an improved mutant B5 (Q12L/Q23L/H248Q/T262A/T263S) was obtained through two rounds of DNA shuffling, showing a 10°C increase in the T ₅₀ (defined as the temperature at which heat treatment for 15 min reduced the initial activity by 50%) compared with the parental enzyme DCase-M3. Furthermore, several thermostability-related sites (Met³¹, Asn⁹³, Gln²⁰⁷, Asn²⁴², Glu²⁶⁶, Thr²⁷¹, Ala²⁷³) on B5 were identified using amino acid consensus approach based on sequence alignment of homologous DCases. These sites were further investigated by iterative saturation mutagenesis (ISM), and a combinational mutant D1 (Q12L/Q23L/Q207E/N242G/H248Q/T262A/T263S/E266D/T271I/A273P) that enhanced the T ₅₀ by about 16°C over DCase-M3 was obtained. Oxidative stability assay showed that the most heat-resisting mutant displayed only a slight increase in resistance to hydrogen peroxide. Comparative characterization showed that D1 not only maintained its characteristic high solubility but also shared similar k _cat and K _m values and optimum reaction pHs with the parental enzyme. The significantly improved mutants in the immobilized form are expected to be applied in the industrial production of d-p-hydroxyphenylglycine.

Keywords: N-carbamoyl-d-amino acid amidohydrolase; Directed evolution; Thermostability; Consensus concept; d-p-Hydroxyphenylglycine

Co-expression of a cellobiose phosphorylase and lactose permease enables intracellular cellobiose utilisation by Saccharomyces cerevisiae by Christa J. Sadie; Shaunita H. Rose; Riaan den Haan; Willem H. van Zyl (pp. 1373-1380).

The cellobiose phosphorylase (cepA) gene from Clostridium stercorarium was cloned and successfully expressed under transcriptional control of the phosphoglycerate kinase gene (PGK1) promoter and terminator in Saccharomyces cerevisiae Y294. The recombinant CepA enzyme showed optimal activity at 60 °C and pH 5 and displayed a K _m value of 92.85 mM and 1.69 mM on cellobiose and pNPG, respectively. A codon-optimised synthetic cepA gene was also expressed; however, it did not enhance cellobiose utilisation. Transport of cellobiose was subsequently facilitated through the heterologous expression of the lac12 of Kluyveromyces lactis. Strains co-producing the heterologous CepA and Lac12 were able to grow on cellobiose as sole carbon source. This is the first report of successful intracellular utilisation of cellobiose by S. cerevisiae producing a cellobiose phosphorylase and of cellobiose transport into S. cerevisiae via the K. lactis lac12 encoded permease.

Keywords: Cellobiose phosphorylase; Lactose permease; Disaccharide transport; Cellobiose utilisation

T cell augments the antitumor activity of tumor-targeting Salmonella by Che-Hsin Lee; Jeng-Long Hsieh; Chao-Liang Wu; Pei-Yu Hsu; Ai-Li Shiau (pp. 1381-1388).

Systemic administration of Salmonella to tumor-bearing mice leads to preferential accumulation within tumor sites and retardation of tumor growth. However, the detailed mechanism of Salmonella-induced antitumor immune response via host T cell remains uncertain. Herein, we used wild-type, CD4⁺ T-cell-deficient, and CD8⁺ T-cell-deficient mice to study the role of T cell in the antitumor immune responses induced by Salmonella enterica serovar Choleraesuis (Salmonella Choleraesuis). When systemically administered into mice bearing tumors, Salmonella Choleraesuis significantly inhibited tumor growth by 50%. In contrast, in T-cell-deficient mice, there was only 34–42% inhibition of tumor growth. We found that treatment with Salmonella Choleraesuis significantly upregulates interferon-γ in wild-type and CD8⁺ T-cell-deficient mice, but not in CD4⁺ T-cell-deficient mice. Furthermore, immunohistochemical staining of the tumors revealed more infiltration of macrophages and neutrophils in wild-type mice after Salmonella Choleraesuis treatment compared with those in T-cell-deficient mice. The antitumor therapeutic effect mediated by Salmonella Choleraesuis is associated with an inflammatory immune response at the tumor site and a tumor T helper 1-type immune response. In conclusion, these results suggest that tumor-targeted therapy using Salmonella Choleraesuis, which exerts tumoricidal effects and stimulates T cell activities, represents a potential strategy for the treatment of tumor.

Keywords: T cell; Salmonella enterica serovar Choleraesuis; Interferon-γ; Tumor

Response to saline stress and aquaporin expression in Azospirillum-inoculated barley seedlings by Myriam S. Zawoznik; Mayra Ameneiros; María P. Benavides; Susana Vázquez; María D. Groppa (pp. 1389-1397).

The ability of two strains of Azospirillum brasilense to mitigate NaCl stress in barley plants was evaluated. Barley seedlings were inoculated and subjected to 200 mM NaCl for 18 days. Several days after NaCl treatment, a significant decline in biomass as well as in height was observed in uninoculated plants. However, smaller reductions in biomass and height were detected in plants inoculated with strain Az39. All the stressed plants showed significantly higher Na⁺ but lower K⁺ contents in their shoots. The growth rate of uninoculated plants was adversely affected by saline treatment, which was associated with higher putrescine content and lower levels of HvPIP2;1 transcripts in the roots. Azospirillum inoculation triggered the transcription of this gene. Our results suggest that barley plants inoculated with A. brasilense may be better prepared to thrive under saline conditions. To our knowledge, this is the first report showing an effect of Azospirillum inoculation on the expression of PIP2;1, a gene involved in the synthesis of root water channels.

Keywords: Azospirillum ; Barley; Saline stress; Aquaporins; Polyamines

Increased CO₂ and the effect of pH on growth and calcification of Pleurochrysis carterae and Emiliania huxleyi (Haptophyta) in semicontinuous cultures by Navid R. Moheimani; Michael A. Borowitzka (pp. 1399-1407).

The effects of changes in CO₂ and pH on biomass productivity and carbon uptake of Pleurochrysis carterae and Emiliania huxleyi in open raceway ponds and a plate photobioreactor were studied. The pH of P. carterae cultures increased during day and decreased at night, whereas the pH of E. huxleyi cultures showed no significant diurnal changes. P. carterae coccolith production occurs during the dark period, whereas in E. huxleyi, coccolith production is mainly during the day. Addition of CO₂ at constant pH (pH-stat) resulted in an increase in P. carterae biomass and coccolith productivity, while CO₂ addition lowered E. huxleyi biomass and coccolith production. Neither of these algae could grow at less than pH 7.5. Species-specific diurnal pH and pCO₂ variations could be indicative of significant differences in carbon uptake between these two species. While E. huxleyi has been suggested to be predominantly a bicarbonate user, our results indicate that P. carterae may be using CO₂ as the main C source for photosynthesis and calcification.

Keywords: Pleurochrysis carterae ; Emiliania huxleyi ; Raceway pond; Plate photobioreactor; pH; CO₂

Effect of sporulation conditions on the resistance of Bacillus subtilis spores to heat and high pressure by Hue Nguyen Thi Minh; Alain Durand; Pauline Loison; Jean-Marie Perrier-Cornet; Patrick Gervais (pp. 1409-1417).

Bacillus subtilis(B. subtilis) cells were placed in various environmental conditions to study the effects of aeration, water activity of the medium, temperature, pH, and calcium content on spore formation and the resulting properties. Modification of the sporulation conditions lengthened the growth period of B. subtilis and its sporulation. In some cases, it reduced the final spore concentration. The sporulation conditions significantly affected the spore properties, including germination capacity and resistance to heat treatment in water (30 min at 97°C) or to high pressure (60 min at 350 MPa and 40°C). The relationship between the modifications of these spore properties and the change in the spore structure induced by different sporulation conditions is also considered. According to this study, sporulation conditions must be carefully taken into account during settling sterilization processes applied in the food industry.

Keywords: Bacillus subtilis ; Spore resistance; Sporulation; Germination; Heat; High pressure

From shake flasks to bioreactors: survival of E. coli cells harboring pGST–hPTH through auto-induction by controlling initial content of yeast extract by Lianghui Jia; Hairong Cheng; Hengwei Wang; Huairong Luo; Hua Yan (pp. 1419-1428).

A high content of yeast extract in complex media can cause auto-induction of phage T7 RNA polymerase and the consequent expression of recombinant protein in Escherichia coli BL21(DE3) during long-term cultivation. Our study demonstrated that the auto-induction of recombinant protein varied in different vectors harboring heterologous genes. Trx, GST, and their fusion proteins such as GST–human parathyroid hormone (hPTH), expressed by pET32a (+), were easily auto-induced by media containing a high content of yeast extract; however, rtPA was not easily auto-induced when using pET22b (+), although both pET systems were under the control of T7lac promoter. Furthermore, the auto-induction of GST–hPTH may start within 1–2 h after inoculation in bioreactors, which is a deficiency in the scale-up from shake flasks to bioreactors. Our results indicated that too much yeast extract in bioreactor cultivations may be responsible for the early auto-induction of target proteins and consequent loss of cell viability and plasmid instability. To achieve a satisfactory yield, host cells with both high cell viability and plasmid stability were necessary for the starter cultures in shake flasks and pre-induction cultures in bioreactors. This could be achieved simply by controlling the initial content of yeast extract and its subsequent supplementation.

Keywords: Auto-induction; E. coli ; pET systems; Yeast extract; Cell viability; Plasmid stability

The effect of light, salinity, and nitrogen availability on lipid production by Nannochloropsis sp. by Dipasmita Pal; Inna Khozin-Goldberg; Zvi Cohen; Sammy Boussiba (pp. 1429-1441).

We examined responses of batch cultures of the marine microalga Nannochloropsis sp. to combined alterations in salinity (13, 27, and 40 g/l NaCl) and light intensity (170 and 700 μmol photons/m²·s). Major growth parameters and lipid productivity (based on total fatty acid determination) were determined in nitrogen-replete and nitrogen-depleted cultures of an initial biomass of 0.8 and 1.4 g/l, respectively. On the nitrogen-replete medium, increases in light intensity and salinity increased the cellular content of dry weight and lipids due to enhanced formation of triacylglycerols (TAG). Maximum average productivity of ca. 410 mg TFA/l/d were obtained at 700 μmol photons/m²·s and 40 g/l NaCl within 7 days. Under stressful conditions, content of the major LC-PUFA, eicosapentaenoic acid (EPA), was significantly reduced while TAG reached 25% of biomass. In contrast, lower salinity tended to improve major growth parameters, consistent with less variation in EPA contents. Combined higher salinity and light intensity was detrimental to lipid productivity under nitrogen starvation; biomass TFA content, and lipid productivity amounted for only 33% of DW and ca. 200 mg TFA/l/day, respectively. The highest biomass TFA content (ca. 47% DW) and average lipid productivity of ca. 360 mg TFA/l/day were achieved at 13 g/l NaCl and 700 μmol photons/m²·s. Our data further support selecting Nannochloropsis as promising microalgae for biodiesel production. Moreover, appropriate cultivation regimes may render Nannochloropsis microalgae to produce simultaneously major valuable components, EPA, and TAG, while sustaining relatively high biomass growth rates.

Keywords: Biodiesel; EPA; Microalga; Nannochloropsis sp.; Salinity; Triacylglycerol

Identification and application of a different glucose uptake system that functions as an alternative to the phosphotransferase system in Corynebacterium glutamicum by Masato Ikeda; Yuta Mizuno; Shin-ichi Awane; Masahiro Hayashi; Satoshi Mitsuhashi; Seiki Takeno (pp. 1443-1451).

Corynebacterium glutamicum uses the phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS) to uptake and phosphorylate glucose; no other route has yet been identified. Disruption of the ptsH gene in wild-type C. glutamicum resulted, as expected, in a phenotype exhibiting little growth on any of the PTS sugars: glucose, fructose, and sucrose. However, a suppressor mutant that grew on glucose but not on the other two sugars was spontaneously isolated from the PTS-negative strain WTΔptsH. The suppressor strain SPH2, unlike the wild-type strain, exhibited a phenotype of resistance to 2-deoxyglucose which is known to be a toxic substrate for the glucose-PTS of this microbe, suggesting that strain SPH2 utilizes glucose via a different system involving a permease and native glucokinases. Analysis of the C. glutamicum genome sequence using Escherichia coli galactose permease, which can transport glucose, led to the identification of two candidate genes, iolT1 and iolT2, both of which have been reported as myo-inositol transporters. When cultured on glucose medium supplemented with myo-inositol, strain WTΔptsH was able to consume glucose, suggesting that glucose uptake was mediated by one or more myo-inositol-induced transporters. Overexpression of iolT1 alone and that of iolT2 alone under the gapA promoter in strain WTΔptsH rendered the strain capable of growing on glucose, proving that each transporter played a role in glucose uptake. Disruption of iolT1 in strain SPH2 abolished growth on glucose, whereas disruption of iolT2 did not, revealing that iolT1 was responsible for glucose uptake in strain SPH2. Sequence analysis of the iol gene cluster and its surrounding region identified a single-base deletion in the putative transcriptional regulator gene Cgl0157 of strain SPH2. Introduction of the frameshift mutation allowed strain WTΔptsH to grow on glucose, and further deletion of iolT1 abolished the growth again, indicating that inactivation of Cgl0157 under a PTS-negative background can be a means by which to express the iolT1-specified glucose uptake bypass instead of the native PTS. When this strategy was applied to a defined lysine producer, the engineered strain displayed increased lysine production from glucose.

Keywords: Glucose uptake bypass; Phosphotransferase system; myo-inositol transporter; Corynebacterium glutamicum ; Lysine production

A simple and novel volumetric method to metre low gas flows from laboratory-scale bioreactors and its application on laboratory sludge digesters by Tamás Tauber; Brigitta Berta; Zsolt Szabó; József Kovács; Károly Márialigeti; Erika M. Tóth (pp. 1453-1461).

A novel, cheap and easy to use method has been developed to measure low gas yields in bioreactors, based on the principle of bubble counting via digital imaging techniques and pattern recognition. No external hardware control is required for the measurements, and the device can be turned into a multichannel tool without further detector accessories. The method proved to perform outstandingly according to the testing and calibrating measurements against standard gas flow and revealed a short periodicity in the gas yields of two parallel laboratory-scale mesophilic biogas reactors providing well analysable data about them. For exact characterization of the oscillation, the data were converted into Morell wavelet spectra, which showed that every feeding period had a characteristic and similarly shaped wavelet profile, thereby this rhythm must have had an immanent source in the community metabolism, and it was not an artefact.

Keywords: Biogas production; Bubble counting; Laboratory-scale digester; Low gas yield; Pattern recognition

Simultaneous detection of influenza virus type B and influenza A virus subtypes H1N1, H3N2, and H5N1 using multiplex real-time RT-PCR by Fang Shisong; Li Jianxiong; Cheng Xiaowen; Zhao Cunyou; Wang Ting; Lv Xing; Wang Xin; Wu Chunli; Zhang Renli; Cheng Jinquan; Xue Hong; Yu Muhua (pp. 1463-1470).

Use of multiplex real-time reverse transcription polymerase chain reaction (RT-PCR) for the simultaneous detection of influenza type B virus and influenza A virus subtypes H5N1, H3N2, and H1N1 has been described. The method exhibited a high specificity and sensitivity of approximately 10¹–10² copies per microliter or 10⁻³–10⁻² TCID50/L for each subtype, as well as a high reproducibility with coefficient of variation (CV) ranging from 0.27% to 4.20%. The assays can be performed commendably on various models of real-time PCR instruments; including ABI7500, ROCH 2.0, and Mx3005p. In an analysis of 436 clinical samples from patients during the year 2009, this detection method has successfully identified 261 positive samples, as compared to only 189 positive samples using the conventional cell culture systems, and at the same time further differentiated them as 35 type B, 21 subtype H1N1, and 205 subtype H3N2. The results indicate that the multiplex real-time RT-PCR method is a potential tool for rapid screening of influenza virus from a large pool of clinical samples during flu pandemics and facilitates early influenza virus identification in most public health laboratories around the world.

Keywords: Influenza virus; Real-time RT-PCR; Differentiation

Biodegradation of deltamethrin and its hydrolysis product 3-phenoxybenzaldehyde by a newly isolated Streptomyces aureus strain HP-S-01 by Shaohua Chen; Kaiping Lai; Yanan Li; Meiying Hu; Yanbo Zhang; Yong Zeng (pp. 1471-1483).

A newly isolated actinomycete strain HP-S-01 from activated sludge could effectively degrade deltamethrin and its major hydrolysis product 3-phenoxybenzaldehyde. Based on the morphological, cultural, physio-biochemical characteristics, and 16S rDNA sequence analysis, strain HP-S-01 was identified as Streptomyces aureus. Strain HP-S-01 was also found highly efficient in degrading cyfluthrin, bifenthrin, fenvalerate, fenpropathrin, permethrin, and cypermethrin. Strain HP-S-01 rapidly degraded deltamethrin without a lag phase over a wide range of temperature (18~38°C) and pH (5~10), and metabolized to produce α-hydroxy-3-phenoxy-benzeneacetonitrile and 3-phenoxybenzaldehyde by hydrolysis of the carboxylester linkage. The 3-phenoxybenzaldehyde was further oxidized to form 2-hydroxy-4-methoxy benzophenone resulting in its detoxification. No persistent accumulative product was detected by gas chromatography-mass spectrometry (GC/MS) analysis. Response surface methodology was used to optimize degradation conditions. Strain HP-S-01 completely removed 50~300 mg L⁻¹ deltamethrin within 7 days under the optimal degradation conditions. Furthermore, the biodegradation kinetics corresponded with the first-order model. Therefore, strain HP-S-01 is suitable for the efficient and rapid bioremediation of pyrethroid-contaminated environment.

Keywords: Pyrethroids; Deltamethrin; 3-Phenoxybenzaldehyde; Biodegradation; Streptomyces aureus ; Kinetics

Analysis of denitrifier community in a bioaugmented sequencing batch reactor for the treatment of coking wastewater containing pyridine and quinoline by Yaohui Bai; Qinghua Sun; Rui Xing; Donghui Wen; Xiaoyan Tang (pp. 1485-1492).

The denitrifier community and associated nitrate and nitrite reduction in the bioaugmented and general sequencing batch reactors (SBRs) during the treatment of coking wastewater containing pyridine and quinoline were investigated. The efficiency and stability of nitrate and nitrite reduction in SBR was considerably improved after inoculation with four pyridine- or quinoline-degrading bacterial strains (including three denitrifying strains). Terminal restriction fragment length polymorphism (T-RFLP) based on the nosZ gene revealed that the structures of the denitrifier communities in bioaugmented and non-bioaugmented reactors were distinct and varied during the course of the experiment. Bioaugmentation protected indigenous denitrifiers from disruptions caused by pyridine and quinoline. Clone library analysis showed that one of the added denitrifiers comprised approximately 6% of the denitrifier population in the bioaugmented sludge.

Keywords: Bioaugmentation; Denitrifier community; Coking wastewater; SBR

Inactivation of Escherichia coli and bacteriophage T4 by high levels of dissolved CO₂ by Xuehang Cheng; Tsuyoshi Imai; Jantima Teeka; Junki Yamaguchi; Mami Hirose; Takaya Higuchi; Masahiko Sekine (pp. 1493-1500).

Little information is available regarding the effectiveness of water disinfection by CO₂ at low pressure. The aim of this study was to evaluate the use of high levels of dissolved CO₂ at 0.3–0.6 MPa for the inactivation of microorganisms. Bacteriophage T4 was chosen as the model virus and Escherichia coli was selected as the representative bacterium. The results of the study showed a highly effective log inactivation of E. coli and bacteriophage T4 at low and medium initial concentrations by high levels of dissolved CO₂ at 0.3 MPa with a treatment time of 20 min. When the pressure was increased to 0.6 MPa, inactivation of both microorganisms at high initial concentrations was improved to different extents. Neither pressurized air nor O₂ effectively inactivated both E. coli and bacteriophage T4. The pH was not a key factor affecting the inactivation process by this method. The results of scanning electron microscopy of E. coli and transmission electron microscopy of bacteriophage T4 suggested that “CO₂ uptake at high pressure and bursting of cells by depressurization” were the main reasons for lethal effect on microorganisms. This technology has potential for application in the disinfection of water, wastewater, and liquid food in the future.

Keywords: Escherichia coli ; Bacteriophage T4; Inactivation by CO₂ ; Low pressure

Archaeal community dynamics and detection of ammonia-oxidizing archaea during composting of cattle manure using culture-independent DNA analysis by Nozomi Yamamoto; Ryoki Asano; Hiroki Yoshii; Kenichi Otawa; Yutaka Nakai (pp. 1501-1510).

The composting process is carried out under aerobic conditions involving bacteria, archaea, and fungi. Little is known about the diversity of archaeal community in compost, although they may play an important role in methane production and ammonia oxidation. In the present study, archaeal community dynamics during cattle manure composting were analyzed using a clone library of the archaeal 16S rRNA gene. The results indicated that methane-producing archaea (methanogen) and ammonia-oxidizing archaea (AOA) may be the dominant microbes throughout the composting. The community consisted primarily of Methanocorpusculum-like and Methanosarcina-like sequences until day 2, while the number of Candidatus Nitrososphaera-like sequences increased from day 6 to day 30. Methanosarcina thermophila-like sequences were dominant from day 2, suggesting that M. thermophila-like species can adapt to increasing temperature or nutrient loss. A denaturant gradient gel electrophoresis analysis of the archaeal amoA genes revealed that the dominant amoA gene sequence with 99% homology to that of Candidatus Nitrososphaera gargensis was identical to those obtained from a different composting facility. These data suggested that AOA may play a role in ammonia oxidation in several composting practices. Our results provide fundamental information regarding archaeal community dynamics that will help in understanding the collective microbial community in compost.

Keywords: 16S rRNA gene; Ammonia-oxidizing archaea; Compost; Methanogen

Long-chain acylhomoserine lactones increase the anoxic ammonium oxidation rate in an OLAND biofilm by Haydée De Clippeleir; Tom Defoirdt; Lynn Vanhaecke; Siegfried E. Vlaeminck; Marta Carballa; Willy Verstraete; Nico Boon (pp. 1511-1519).

The oxygen-limited autotrophic nitrification/denitrification (OLAND) process comprises one-stage partial nitritation and anammox, catalyzed by aerobic and anoxic ammonium-oxidizing bacteria (AerAOB and AnAOB), respectively. The goal of this study was to investigate whether quorum sensing influences anoxic ammonium oxidation in an OLAND biofilm, with AnAOB colonizing 13% of the biofilm, as determined with fluorescent in situ hybridization (FISH). At high biomass concentrations, the specific anoxic ammonium oxidation rate of the OLAND biofilm significantly increased with a factor of 1.5 ± 0.2 compared to low biomass concentrations. Supernatant obtained from the biofilm showed no ammonium-oxidizing activity on itself, but its addition to low OLAND biomass concentrations resulted in a significant activity increase of the biomass. In the biofilm supernatant, the presence of long-chain acylhomoserine lactones (AHLs) was shown using the reporter strain Chromobacterium violaceum CV026, and one specific AHL, N-dodecanoyl homoserine lactone (C₁₂-HSL), was identified via LC-MS/MS. Furthermore, C₁₂-HSL was detected in an AnAOB-enriched community, but not in an AerAOB-enriched community. Addition of C₁₂-HSL to low OLAND biomass concentrations resulted in a significantly higher ammonium oxidation rate (p < 0.05). To our knowledge, this is the first report demonstrating that AHLs enhance the anoxic ammonium oxidation process. Future work should confirm which species are responsible for the in situ production of C₁₂-HSL in AnAOB-based applications.

Keywords: Ammonia oxidizing bacteria; Biosensor; Cell-to-cell communication; Cell density; LC-MS/MS; Nitrification

Evaluating the prevalence and genetic diversity of adenovirus and polyomavirus in bovine waste for microbial source tracking by Kelvin Wong; Irene Xagoraraki (pp. 1521-1526).

This study evaluated and compared the occurrence, concentrations, and genetic diversity of bovine polyomavirus (BPyV) and bovine adenovirus (BAdV) in manure and feces samples for the purpose of determining which of these two viruses is more suitable for bovine fecal indication and microbial source tracking. The comparability and correlation between concentrations/prevalence of these viruses and bacterial fecal indicators (cow-associated Bacteroidetes, Escherichia coli and enterococci) in manure and fecal samples was also determined. A total of 26 dairy manure and 18 individual dairy cow feces samples were tested. The results showed the mean concentration of BAdV in all of dairy manure samples was at least 1 log lower than BPyV (p ≤ 0.005). All of the dairy manure samples tested positive for BPyV but not for BAdV. After combining dairy manure measurements, bacterial indicators had 0.3–0.7 log (p ≤ 0.05) and 1.8–2.2 log (p ≤ 0.005) higher concentrations than BPyV and BAdV, respectively. The concentration of BPyV had a significant positive correlation with the concentration of E. coli and enterococci in the manure samples. The partial VP1 genetic sequences of BPyV isolated from three different farms had a 100% homology to each other and to VP1 sequence (D13942.1) reported in previous study. Based on the occurrence, quantitative and genetic diversity results, BPyV may be a better indicator than BAdV for microbial source tracking at manure application sites.

Keywords: Bovine polyomavirus; Bovine adenovirus; Quantitative PCR; Fecal indicator; Microbial source tracking

Assessing the influence of the carbon oxidation-reduction state on organic pollutant biodegradation in algal–bacterial photobioreactors by Melanie Bahr; Alfons J. M. Stams; Francisco De la Rosa; Pedro A. García-Encina; Raul Muñoz (pp. 1527-1536).

The influence of the carbon oxidation–reduction state (CORS) of organic pollutants on their biodegradation in enclosed algal–bacterial photobioreactors was evaluated using a consortium of enriched wild-type methanotrophic bacteria and microalgae. Methane, methanol and glucose (with CORS −4, −2 and 0, respectively) were chosen as model organic pollutants. In the absence of external oxygen supply, microalgal photosynthesis was not capable of supporting a significant methane and methanol biodegradation due to their high oxygen demands per carbon unit, while glucose was fully oxidized by photosynthetic oxygenation. When bicarbonate was added, removal efficiencies of 37 ± 4% (20 days), 65 ± 4% (11 days) and 100% (2 days) were recorded for CH_4, CH₃OH and C₆H₁₂O₆, respectively due to the additional oxygen generated from photosynthetic bicarbonate assimilation. The use of NO ₃ ⁻ instead of NH ₄ ⁺ as nitrogen source (N oxidation–reduction state of +5 vs. −3) resulted in an increase in CH₄ degradation from 0 to 33 ± 3% in the absence of bicarbonate and from 37 ± 4% to 100% in the presence of bicarbonate, likely due to a decrease in the stoichiometric oxygen requirements and the higher photosynthetic oxygen production. Hypothetically, the CORS of the substrates might affect the CORS of the microalgal biomass composition (higher lipid content). However, the total lipid content of the algal–bacterial biomass was 19 ± 7% in the absence and 16 ± 2% in the presence of bicarbonate.

Keywords: Carbon oxidation–reduction state; Microalgal–bacterial symbiosis; Nitrogen source; Photosynthetic oxygenation; Pollutant degradation

OLAND is feasible to treat sewage-like nitrogen concentrations at low hydraulic residence times by Haydée De Clippeleir; Xungang Yan; Willy Verstraete; Siegfried Elias Vlaeminck (pp. 1537-1545).

Energy-positive sewage treatment can, in principle, be obtained by maximizing energy recovery from concentrated organics and by minimizing energy consumption for concentration and residual nitrogen removal in the main stream. To test the feasibility of the latter, sewage-like nitrogen influent concentrations were treated with oxygen-limited autotrophic nitrification/denitrification (OLAND) in a lab-scale rotating biological contactor at 25°C. At influent ammonium concentrations of 66 and 29 mg N L⁻¹ and a volumetric loading rate of 840 mg N L⁻¹ day⁻¹ yielding hydraulic residence times (HRT) of 2.0 and 1.0 h, respectively, relatively high nitrogen removal rates of 444 and 383 mg N L⁻¹ day⁻¹ were obtained, respectively. At low nitrogen levels, adapted nitritation and anammox communities were established. The decrease in nitrogen removal was due to decreased anammox and increased nitratation, with Nitrospira representing 6% of the biofilm. The latter likely occurred given the absence of dissolved oxygen (DO) control, since decreasing the DO concentration from 1.4 to 1.2 mg O₂ L⁻¹ decreased nitratation by 35% and increased anammox by 32%. Provided a sufficient suppression of nitratation, this study showed the feasibility of OLAND to treat low nitrogen levels at low HRT, a prerequisite to energy-positive sewage treatment.

Keywords: Ammonia-oxidizing bacteria; Domestic wastewater; Nitrite-oxidizing bacteria; Nutrient; Sustainable

Evidence for taxonomic and functional drift of an atrazine-degrading culture in response to high atrazine input by Nikolina Udiković-Kolić; Marion Devers-Lamrani; Ines Petrić; Dubravka Hršak; Fabrice Martin-Laurent (pp. 1547-1554).

We evaluated the effects of variations in atrazine input on the evolution of a bacterial culture adapted to a low atrazine concentration. This initial culture (M3-K) was subjected to weekly subculturing in the presence of a high concentration of atrazine as the only N source (100 mg l⁻¹). After four subculturing, M3-K evolved to a new bacterial culture (M3) which exhibited a significant increase in the extent of atrazine mineralization in comparison with the initial culture. Molecular analyses of M3-K and M3 cultures by cloning, restriction analysis, and sequencing of the 16S rRNA genes revealed significant differences in culture structure and composition. M3-K culture comprised mainly Actinobacteria (40%), β-Proteobacteria (26%), and Bacteroidetes (16%). After exposure to a high atrazine concentration, the dominance of Actinobacteria decreased (14%), Bacteroidetes increased (27%), and β-Proteobacteria were replaced by γ-Proteobacteria (32%). Quantitative PCR revealed that the abundance of atzB and atzC genes relative to total bacteria decreased by a factor of 3–4 following the increase in atrazine concentration, while the relative abundance of trzD increased significantly (≈400 times). Presented study shows that variations in atrazine input drive both functional and compositional shifts in the atrazine-degrading bacterial culture.

Keywords: Atrazine; Degradation; Bacterial culture; Evolution; atz genes; trz genes

Decreasing ammonia inhibition in thermophilic methanogenic bioreactors using carbon fiber textiles by Kengo Sasaki; Masahiko Morita; Shin-ichi Hirano; Naoya Ohmura; Yasuo Igarashi (pp. 1555-1561).

Ammonia accumulation is one of the main causes of the loss of methane production observed during fermentation. We investigated the effect of addition of carbon fiber textiles (CFT) to thermophilic methanogenic bioreactors with respect to ammonia tolerance during the process of degradation of artificial garbage slurry, by comparing the performance of the reactors containing CFT with the performance of reactors without CFT. Under total ammonia-N concentrations of 3,000 mg L⁻¹, the reactors containing CFT were found to mediate stable removal of organic compounds and methane production. Under these conditions, high levels of methanogenic archaea were retained at the CFT, as determined by 16S rRNA gene analysis for methanogenic archaea. In addition, Methanobacterium sp. was found to be dominant in the suspended fraction, and Methanosarcina sp. was dominant in the retained fraction of the reactors with CFT. However, the reactors without CFT had lower rates of removal of organic compounds and production of methane under total ammonia-N concentrations of 1,500 mg L⁻¹. Under this ammonia concentration, a significant accumulation of acetate was observed in the reactors without CFT (130.0 mM), relative to the reactors with CFT (4.2 mM). Only Methanobacterium sp. was identified in the reactors without CFT. These results suggest that CFT enables stable proliferation of aceticlastic methanogens by preventing ammonia inhibition. This improves the process of stable garbage degradation and production of methane in thermophilic bioreactors that include high levels of ammonia.

Keywords: Ammonia; Methane fermentation; Supporting material; Methanogenic archaea

Effect of enrichment procedures on performance and microbial diversity of microbial fuel cell for Congo red decolorization and electricity generation by Bin Hou; Jian Sun; Yongyou Hu (pp. 1563-1572).

The purpose of this study was to determine the effect of enrichment procedure on the performance and microbial diversity of an air-cathode microbial fuel cell (MFC) which was explored for simultaneous azo dye decolorization and electricity generation. Two different enrichment procedures in which glucose and Congo red were added into the MFCs sequentially (EP1) or simultaneously (EP2) were tested by operating parallel MFCs independently for more than 6 months. The power density, electrode potential, Congo red decolorization, biofilm morphology, and bacterial diversity of the MFCs under the two enrichment procedures were compared and investigated. The results showed that the enrichment procedures have a negligible effect on the dye decolorization, but significantly affected the electricity generation. More than 90% decolorization at dye concentration of 300 mg/L was achieved within 170 h for the two tested enrichment procedures. However, the MFC with EP2 achieved a maximum power density of 192 mW/m², which was 75% higher than that of the MFC with EP1 (110 mW/m²). The depressed surfaces of the bacteria in the MFC with EP1 indicated the allergic response caused by the subsequent addition of Congo red. 16S rRNA sequencing analysis demonstrated a phylogenetic diversity in the communities of the anode biofilm and showed clear differences between the anode-attached populations in the MFCs with a different enrichment procedure. This study suggests that the enrichment procedure is important for the MFC explored for simultaneous dye decolorization and electricity generation.

Keywords: Microbial fuel cell; Enrichment procedure; Congo red decolorization; Electricity generation; Microbial diversity

Growth and ethanol fermentation ability on hexose and pentose sugars and glucose effect under various conditions in thermotolerant yeast Kluyveromyces marxianus by Nadchanok Rodrussamee; Noppon Lertwattanasakul; Katsushi Hirata; Suprayogi; Savitree Limtong; Tomoyuki Kosaka; Mamoru Yamada (pp. 1573-1586).

Ethanol fermentation ability of the thermotolerant yeast Kluyveromyces marxianus, which is able to utilize various sugars including glucose, mannose, galactose, xylose, and arabinose, was examined under shaking and static conditions at high temperatures. The yeast was found to produce ethanol from all of these sugars except for arabinose under a shaking condition but only from hexose sugars under a static condition. Growth and sugar utilization rate under a static condition were slower than those under a shaking condition, but maximum ethanol yield was slightly higher. Even at 40°C, a level of ethanol production similar to that at 30°C was observed except for galactose under a static condition. Glucose repression on utilization of other sugars was observed, and it was more evident at elevated temperatures. Consistent results were obtained by the addition of 2-deoxyglucose. The glucose effect was further examined at a transcription level, and it was found that KmGAL1 for galactokinase and KmXYL1 for xylose reductase for galactose and xylose/arabinose utilization, respectively, were repressed by glucose at low and high temperatures, but KmHXK2 for hexokinase was not repressed. We discuss the possible mechanism of glucose repression and the potential for utilization of K. marxianus in high-temperature fermentation with mixed sugars containing glucose.

Keywords: Kluyveromyces marxianus ; Ethanol production; Glucose effect; High-temperature fermentation

Erratum to: Properties of recombinant Strep-tagged and untagged hyperthermophilic D-arabitol dehydrogenase from Thermotoga maritima by Verena Kallnik; Christian Schulz; Paul Schweiger; Uwe Deppenmeier (pp. 1587-1587).

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Applied Microbiology and Biotechnology (v.90, #4)

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Applied Microbiology and Biotechnology (v.90, #4)