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Applied Microbiology and Biotechnology (v.90, #3)

Peptide-based treatment of sepsis by Klaus Brandenburg; Jörg Andrä; Patrick Garidel; Thomas Gutsmann (pp. 799-808).

Sepsis (blood poisoning) is a severe infectious disease with high mortality, and no effective therapy is actually known. In the case of Gram-negative bacteria, endotoxins (lipopolysaccharides) are known to be responsible for the strong inflammation reaction leading to the systemic infection. Peptides based on endotoxin-binding domains of human or animal proteins represent a promising approach in sepsis research. Although so far no medicament is available, the progress in recent years might lead to a breakthrough in this field. In this review, recent investigations are summarised, which may lead to an understanding of the mechanisms of action of peptides to suppress the inflammation reaction in vitro and in vivo (animal models) and thus may allow the development of effective anti-septic drugs.

Keywords: Sepsis; Endotoxin; Lipopolysaccharide; Antimicrobial peptides; Defensins; Limulus

Molecular mechanisms of yeast tolerance and in situ detoxification of lignocellulose hydrolysates by Z. Lewis Liu (pp. 809-825).

Pretreatment of lignocellulose biomass for biofuel production generates inhibitory compounds that interfere with microbial growth and subsequent fermentation. Remediation of the inhibitors by current physical, chemical, and biological abatement means is economically impractical, and overcoming the inhibitory effects of lignocellulose hydrolysate poses a significant technical challenge for lower-cost cellulosic ethanol production. Development of tolerant ethanologenic yeast strains has demonstrated the potential of in situ detoxification for numerous aldehyde inhibitors derived from lignocellulose biomass pretreatment and conversion. In the last decade, significant progress has been made in understanding mechanisms of yeast tolerance for tolerant strain development. Enriched genetic backgrounds, enhanced expression, interplays, and global integration of many key genes enable yeast tolerance. Reprogrammed pathways support yeast functions to withstand the inhibitor stress, detoxify the toxic compounds, maintain energy and redox balance, and complete active metabolism for ethanol fermentation. Complex gene interactions and regulatory networks as well as co-regulation are well recognized as involved in yeast adaptation and tolerance. This review presents our current knowledge on mechanisms of the inhibitor detoxification based on molecular studies and genomic-based approaches. Our improved understanding of yeast tolerance and in situ detoxification provide insight into phenotype-genotype relationships, dissection of tolerance mechanisms, and strategies for more tolerant strain development for biofuels applications.

Keywords: Aldehyde inhibitors; Gene regulatory networks; Genomic adaptation; Lignocellulose-to-ethanol conversion; Reprogrammed pathways; Stress tolerance

Electricity generation from carbon monoxide and syngas in a microbial fuel cell by Abid Hussain; Serge R. Guiot; Punita Mehta; Vijaya Raghavan; Boris Tartakovsky (pp. 827-836).

Electricity generation in microbial fuel cells (MFCs) has been a subject of significant research efforts. MFCs employ the ability of electricigenic bacteria to oxidize organic substrates using an electrode as an electron acceptor. While MFC application for electricity production from a variety of organic sources has been demonstrated, very little research on electricity production from carbon monoxide and synthesis gas (syngas) in an MFC has been reported. Although most of the syngas today is produced from non-renewable sources, syngas production from renewable biomass or poorly degradable organic matter makes energy generation from syngas a sustainable process, which combines energy production with the reprocessing of solid wastes. An MFC-based process of syngas conversion to electricity might offer a number of advantages such as high Coulombic efficiency and biocatalytic activity in the presence of carbon monoxide and sulfur components. This paper presents a discussion on microorganisms and reactor designs that can be used for operating an MFC on syngas.

Keywords: Microbial fuel cell; Syngas; Carbon monoxide

Integrating microbial ecology in bioprocess understanding: the case of gas biofiltration by Léa Cabrol; Luc Malhautier (pp. 837-849).

Biofilters are packed-bed bioreactors where contaminants, once transferred from the gas phase to the biofilm, are oxidized by diverse and complex communities of attached microorganisms. Over the last decade, more and more studies aimed at opening the back box of biofiltration by unraveling the biodiversity-ecosystem function relationship. In this review, we report the insights provided by the microbial ecology approach in biofilters and we emphasize the parallels existing with other engineered ecosystems used for wastewater treatment, as they all constitute relevant model ecosystems to explore ecological issues. We considered three characteristic ecological indicators: the density, the diversity, and the structure of the microbial community. Special attention was paid to the temporal and spatial dynamics of each indicator, insofar as it can disclose the potential relationship, or absence of relation, with any operating or functional parameter. We also focused on the impact of disturbance regime on the microbial community structure, in terms of resistance, resilience, and memory. This literature review led to mitigated conclusions in terms of biodiversity–ecosystem function relationship. Depending on the environmental system itself and the way it is investigated, the spatial and temporal dynamics of the microbial community can be either correlated (e.g., spatial stratification) or uncoupled (e.g., temporal instability) to the ecosystem function. This lack of generality shows the limits of current 16S approach in complex ecosystems, where a functional approach may be more suitable.

Keywords: Microbial ecology; Biofiltration; Diversity; Community structure; Resilience; Stability

Bacteriophages as twenty-first century antibacterial tools for food and medicine by Damien Maura; Laurent Debarbieux (pp. 851-859).

Antibiotic-resistant bacteria are an increasing source of concern in all environments in which these drugs have been used. More stringent regulations have led to a slow but sure decrease in antibiotic use in the food industry worldwide, but have also stimulated the search for alternative antibacterial agents. In medicine, the number of people infected with pan-resistant bacteria is driving research to develop new treatments. Within these contexts, studies on the use of bacteriophages in both medicine and the food industry have recently flourished. This renewed interest has coincided with the demonstration that these viruses are involved in geochemical cycles, revolutionizing our vision of their ecological role on our planet. Bacteriophages have co-evolved with bacteria for billions of years and retain the ability to infect bacteria efficiently. They are undoubtedly one of the best potential sources of new solutions for the management of undesirable bacteria.

Keywords: Antibacterial tools; Bacteriophage; Phage therapy; Antibiotic resistance; Bacterial infections

Microbial Resource Management revisited: successful parameters and new concepts by Suzanne Read; Massimo Marzorati; Beatriz C. M. Guimarães; Nico Boon (pp. 861-871).

In the twenty-first century, scientists will want to steer the microbial black box in (engineered) ecosystems, rather than only study and describe them. This strategy led to a new way of thinking: Microbial Resource Management (MRM). For the last few years, MRM has been utilized to consolidate and communicate our acquired knowledge of the microbiome to many areas of the scientific community. This shared knowledge has brought us closer to formulating a plan toward the analysis, and at a later stage, the management of our varied microbial communities and to look at ways of harnessing their unique abilities for future practices. We require this acquired knowledge for a more sustainable solution to our ongoing global challenges such as our diminishing energy and water supply. Like any successful concept, MRM must be updated to adapt to new molecular technologies, and thus, in this review, MRM has been reengineered to encompass these changes. This review reports how MRM has been used successfully over the last few years within various environments and how we can broaden its capabilities to increase its compliance in the face of state of the art ever changing technologies. Not only have we reengineered and improved MRM, but also we have discussed how newly formed relationships between technologies can provide the full picture of these complex microbial communities and their interactions for future opportunities.

Keywords: Microbial Resource Management; 454 pyrosequencing; Bioreactors; Engineering; Steering

Maximal release of highly bifidogenic soluble dietary fibers from industrial potato pulp by minimal enzymatic treatment by Lise V. Thomassen; Louise K. Vigsnæs; Tine R. Licht; Jørn D. Mikkelsen; Anne S. Meyer (pp. 873-884).

Potato pulp is a poorly utilized, high-volume co-processing product resulting from industrial potato starch manufacturing. Potato pulp mainly consists of the tuber plant cell wall material and is particularly rich in pectin, notably galactan branched rhamnogalacturonan I type pectin which has previously been shown to exhibit promising properties as dietary fiber. The objective of this study was to solubilize dietary fibers from potato pulp by a one-step minimal treatment procedure and evaluate the prebiotic potential of the fibers. Statistically designed experiments were conducted to investigate the influence of enzyme type, dosage, substrate level, incubation time, and temperature on the enzyme catalyzed solubilization to define the optimal minimal enzyme treatment for maximal fiber solubilization. The result was a method that within 1 min released 75% [weight/weight (w/w)] dry matter from 1% (w/w) potato pulp treated with 1.0% (w/w) [enzyme/substrate (E/S)] pectin lyase from Aspergillus nidulans and 1.0% (w/w) E/S polygalacturonase from Aspergillus aculeatus at pH 6.0 and 60 °C. Molecular size fractionation of the solubilized fibers revealed two major fractions: one fraction rich in galacturonic acid of 10–100 kDa indicating mainly homogalacturonan, and a fraction >100 kDa rich in galactose, presumably mainly made up of β-1,4-galactan chains of rhamnogalacturonan I. When fermented in vitro by microbial communities derived from fecal samples from three healthy human volunteers, both of the solubilized fiber fractions were more bifidogenic than fructo-oligosaccharides (FOS). Notably the fibers having molecular masses of >100 kDa selectively increased the densities of Bifidobacterium spp. and Lactobacillus spp. 2–3 times more than FOS.

Keywords: Potato pulp; Dietary fiber; Pectin lyase; Polygalacturonase; Galactan; Bifidobacterium

Improved p-hydroxybenzoate production by engineered Pseudomonas putida S12 by using a mixed-substrate feeding strategy by Jean-Paul Meijnen; Suzanne Verhoef; Ashwin A. Briedjlal; Johannes H. de Winde; Harald J. Ruijssenaars (pp. 885-893).

The key precursors for p-hydroxybenzoate production by engineered Pseudomonas putida S12 are phosphoenolpyruvate (PEP) and erythrose-4-phosphate (E4P), for which the pentose phosphate (PP) pathway is an important source. Since PP pathway fluxes are typically low in pseudomonads, E4P and PEP availability is a likely bottleneck for aromatics production which may be alleviated by stimulating PP pathway fluxes via co-feeding of pentoses in addition to glucose or glycerol. As P. putida S12 lacks the natural ability to utilize xylose, the xylose isomerase pathway from E. coli was introduced into the p-hydroxybenzoate producing strain P. putida S12palB2. The initially inefficient xylose utilization was improved by evolutionary selection after which the p-hydroxybenzoate production was evaluated. Even without xylose-co-feeding, p-hydroxybenzoate production was improved in the evolved xylose-utilizing strain, which may indicate an intrinsically elevated PP pathway activity. Xylose co-feeding further improved the p-hydroxybenzoate yield when co-fed with either glucose or glycerol, up to 16.3 Cmol% (0.1 g p-hydroxybenzoate/g substrate). The yield improvements were most pronounced with glycerol, which probably related to the availability of the PEP precursor glyceraldehyde-3-phosphate (GAP). Thus, it was demonstrated that the production of aromatics such as p-hydroxybenzoate can be improved by co-feeding different carbon sources via different and partially artificial pathways. Moreover, this approach opens new perspectives for the efficient production of (fine) chemicals from renewable feedstocks such as lignocellulose that typically has a high content of both glucose and xylose and (crude) glycerol.

Keywords: Pseudomonas putida S12; Aromatics; Mixed substrate feeding; Glucose; Xylose

Glutamate production from β-glucan using endoglucanase-secreting Corynebacterium glutamicum by Takeyuki Tsuchidate; Toshihiro Tateno; Naoko Okai; Tsutomu Tanaka; Chiaki Ogino; Akihiko Kondo (pp. 895-901).

We demonstrate glutamate production from β-glucan using endoglucanase (EG)-expressing Corynebacterium glutamicum. The signal sequence torA derived from Escherichia coli K12, which belongs to the Tat pathway, was suitable for secreting EG of Clostridium thermocellum using C. glutamicum as a host. Using the torA signal sequence, endoglucanase from Clostridium cellulovorans 743B was successfully expressed, and the secreted EG produced 123 mg of reducing sugar from 5 g of β-glucan at 30 °C for 72 h, which is the optimal condition for C. glutamicum growth. Subsequently, glutamate fermentation from β-glucan was carried out with the addition of Aspergillus aculeatus β-glucosidase produced by recombinant Aspergillus oryzae. Using EG-secreting C. glutamicum, 178 mg/l of glutamate was produced from 15 g of β-glucan. This is the first report of glutamate fermentation from β-glucan using endoglucanase-secreting C. glutamicum.

Keywords: Corynebacterium glutamicum ; Endoglucanase; Signal sequence; Glutamate fermentation; β-Glucan

Removal of l-alanine from the production of l-2-aminobutyric acid by introduction of alanine racemase and d-amino acid oxidase by Li Zhu; Rongsheng Tao; Yi Wang; Yu Jiang; Xin Lin; Yunliu Yang; Huabao Zheng; Weihong Jiang; Sheng Yang (pp. 903-910).

l-2-Aminobutyric acid can be synthesized in a transamination reaction from l-threonine and l-aspartic acid as substrates by the action of threonine deaminase and aromatic aminotransferase, but the by-product l-alanine was produced simultaneously. A small amount of l-alanine increased the complexity of the l-2-aminobutyric acid recovery process because of their extreme similarity in physical and chemical properties. Acetolactate synthase has been introduced to remove the pyruvate intermediate for reducing the l-alanine concentration partially. To eliminate the remnant l-alanine, alanine racemase of Bacillus subtilis in combination with d-amino acid oxidase of Rhodotorula gracilis or Trigonopsis variabilis respectively was introduced into the reaction system for the l-2-aminobutyric acid synthesis. l-Alanine could be completely removed by the action of alanine racemase of B. subtilis and d-amino acid oxidase of R. gracilis; thereby, high-purity l-2-aminobutyric acid was achieved. The results revealed that alanine racemase could discriminate effectively between l-alanine and l-2-aminobutyric acid, and selectively catalyzed l-alanine to d-alanine reversibly. d-Amino acid oxidase then catalyzed d-alanine to pyruvate stereoselectively. Furthermore, this method was also successfully used to remove the by-product l-alanine in the production of other neutral amino acids such as l-tertiary leucine and l-valine, suggesting that multienzymatic whole-cell catalysis can be employed to provide high purity products.

Keywords: l-Alanine; Alanine racemase; d-Amino acid oxidase; l-2-Aminobutyric acid

Characterisation of HFBII biosurfactant production and foam fractionation with and without antifoaming agents by James B. Winterburn; Andrew B. Russell; Peter J. Martin (pp. 911-920).

The effects of foaming on the production of the hydrophobin protein HFBII by fermentation have been investigated at two different scales. The foaming behaviour was characterised in standard terms of the product enrichment and recovery achieved. Additional specific attention was given to the rate at which foam, product and biomass overflowed from the fermentation system in order to assess the utility of foam fractionation for HFBII recovery. HFBII was expressed as an extracellular product during fed-batch fermentations with a genetically modified strain of Saccharomyces cerevisiae, which were carried out with and without the antifoam Struktol J647. In the presence of antifoam, HFBII production is shown to be largely unaffected by process scale, with similar yields of HFBII on dry matter obtained. More variation in HFBII yield was observed between fermentations without antifoam. In fermentations without antifoam, a maximum HFBII enrichment in the foam phase of 94.7 was measured with an overall enrichment, averaged over all overflowed material throughout the whole fermentation, of 54.6 at a recovery of 98.1%, leaving a residual HFBII concentration of 5.3 mg L⁻¹ in the fermenter. It is also shown that uncontrolled foaming resulted in reduced concentration of biomass in the fermenter vessel, affecting total production. This study illustrates the potential of foam fractionation for efficient recovery of HFBII through simultaneous high enrichment and recovery which are greater than those reported for similar systems.

Keywords: Biosurfactant; Hydrophobin; Foam; Foam fractionation; Fermentation; Antifoam

Continuous pullulan fermentation in a biofilm reactor by Kuan-Chen Cheng; Ali Demirci; Jeffrey M. Catchmark (pp. 921-927).

Biofilm is a natural form of cell immobilization in which microorganisms attach onto solid support. In this study, a pigment-reduced pullulan-producing strain, Aureobasidium pullulans (ATCC 201253), was used for continuous pullulan fermentation in a plastic composite support (PCS) biofilm reactor. Optimal conditions for the continuous pullulan production were determined by evaluating the effects of the feeding medium with various concentrations of ammonium sulfate and sucrose and dilution rate. Pullulan concentration and production rate reached maximum (8.3 g/l and 1.33 g/l/h) when 15 g/l of sucrose, 0.9 g/l of ammonium sulfate, and 0.4 g/l of yeast extract were applied in the medium, and the dilution rate was at 0.16 h⁻¹. The purity of produced pullulan was 93.0%. The ratio of hyphal cells of A. pullulans increased when it was grown on the PCS shaft. Overall, the increased pullulan productivity can be achieved through biomass retention by using PCS biofilm reactor.

Keywords: Pullulan production; PCS biofilm reactor; Nitrogen limitation; Continuous fermentation; Cell morphology

Comparative analysis of tertiary alcohol esterase activity in bacterial strains isolated from enrichment cultures and from screening strain libraries by Susanne Herter; Giang-Son Nguyen; Mark L. Thompson; Fabian Steffen-Munsberg; Frieder Schauer; Uwe T. Bornscheuer; Robert Kourist (pp. 929-939).

The preparation of enantiopure tertiary alcohols is of great contemporary interest due to the application of these versatile building blocks in organic synthesis and as precursors towards high value pharmaceutical compounds. Herein, we describe two approaches taken towards the discovery of novel biocatalysts for the synthesis of these valuable compounds. The first approach was initiated with screening of 47 bacterial strains for hydrolytic activity towards the simple tertiary alcohol ester tert-butyl acetate. In conjunction, a second method focussed on the isolation of strains competent for growth on tert-butyl acetate as the sole source of carbon and energy. From functional screening, 10 Gram-positive Actinomycetes showed hydrolytic activity, whilst enrichment selection resulted in the identification of 14 active strains, of which five belong to the Gram-negative cell-wall type. Bacterial strains obtained from both approaches were viable for enantioselective hydrolysis of pyridine substituted tertiary alcohol esters in addition to bulky aliphatic and keto-derived substrates from the same class. Activity towards each of the test substrates was uncovered, with promising enantioselectivities of up to E = 71 in the hydrolysis of a para-substituted pyridine tertiary alcohol ester using a strain of Rhodococcus ruber. Interestingly strains of Microbacterium and Alcaligenes sp. gave opposite enantiopreference in the hydrolysis of a meta-substituted pyridine tertiary alcohol ester with E values of 17 and 54. These approaches show that via both possibilities, screening established strain collections and performing enrichment selection, it is possible to identify novel species which show activity towards sterically challenging substrates.

Keywords: Biotransformation; Tertiary alcohol; Esterase; Enrichment; Screening

Production and characterisation of AoSOX2 from Aspergillus oryzae, a novel flavin-dependent sulfhydryl oxidase with good pH and temperature stability by Greta Faccio; Kristiina Kruus; Johanna Buchert; Markku Saloheimo (pp. 941-949).

Sulfhydryl oxidases have found application in the improvement of both dairy and baking products due to their ability to oxidise thiol groups in small molecules and cysteine residues in proteins. A genome mining study of the available fungal genomes had previously been performed by our group in order to identify novel sulfhydryl oxidases suitable for industrial applications and a representative enzyme was produced, AoSOX1 from Aspergillus oryzae (Faccio et al. BMC Biochem 11:31, 2010). As a result of the study, a second gene coding for a potentially secreted sulfhydryl oxidase, AoSOX2, was identified in the genome of A. oryzae. The protein AoSOX2 was heterologously expressed in Trichoderma reesei and characterised with regard to both biochemical properties as well as preliminary structural analysis. AoSOX2 showed activity on dithiothreitol and glutathione, and to a lesser extent on D/L-cysteine and beta-mercaptoethanol. AoSOX2 was a homodimeric flavin-dependent protein of approximately 78 kDa (monomer 42412 Da) and its secondary structure presents alpha-helical elements. A. oryzae AoSOX2 showed a significant stability to pH and temperature.

Keywords: Sulfhydryl oxidase; Secreted; Fungal; Flavoenzyme; Production; Characterization

Expression and characterization of (R)-specific enoyl coenzyme A hydratases making a channeling route to polyhydroxyalkanoate biosynthesis in Pseudomonas putida by Shun Sato; Hiromi Kanazawa; Takeharu Tsuge (pp. 951-959).

We investigated the expression of (R)-specific enoyl coenzyme A hydratase (PhaJ) in Pseudomonas putida KT2440 accumulating polyhydroxyalkanoate (PHA) from sodium octanoate in order to identify biosynthesis pathways of PHAs from fatty acids in pseudomonads. From a database search through the P. putida KT2440 genome, an additional phaJ gene homologous to phaJ4 _Pa from Pseudomonas aeruginosa, termed phaJ4 _Pp, was identified. The gene products of phaJ1 _Pp, which was identified previously, and phaJ4 _Pp were confirmed to be functional in recombinant Escherichia coli on PHA synthesis from sodium dodecanoate. Cytosolic proteins from P. putida grown on sodium octanoate were subjected to anion exchange chromatography and one of the eluted fractions with hydratase activity included PhaJ4_Pp, as revealed by western blot analysis. These results strongly suggest that PhaJ4_Pp forms a channeling route from β-oxidation to PHA biosynthesis in P. putida. Moreover, the substrate specificity of PhaJ1_Pp was suggested to be different from that of PhaJ1_Pa from P. aeruginosa although these two proteins share 67% amino acid sequence identity.

Keywords: Polyhydroxyalkanoate; R-hydratase; Pseudomonas putida ; β-Oxidation

Molecular cloning and characterization of a new cold-active esterase from a deep-sea metagenomic library by Chengzhang Fu; Yongfei Hu; Feng Xie; Hui Guo; Elizabeth Jane Ashforth; Steven W. Polyak; Baoli Zhu; Lixin Zhang (pp. 961-970).

A clone which conferred lipolytic activity at low temperature was identified from a fosmid library constructed from a South China Sea marine sediment sample. The gene responsible, estF, consisted of 1,080 bp that encoded 359 amino acid residues, with a typical N-terminal signal peptide of 28 amino acid residues. A phylogenetic analysis of amino acid sequence with other lipolytic enzymes revealed that EstF and seven closely related putative lipolytic enzymes comprised a unique clade in the phylogenetic tree. Moreover, these hypothetic esterases showed unique conservative sites in the amino acid sequence. The recombinant EstF was overexpressed and purified, and its biochemical properties were partially characterized. The optimal substrate for EstF to hydrolyze among a panel of p-nitrophenyl esters (C2 to C16) was p-nitrophenyl butyrate (C4), with a K _m of 0.46 mM. Activity quickly decreased with substrates containing an acyl chain length longer than 10 carbons. We found that EstF was active in the temperature range of 0–60°C, showed the best activity at 50°C, but was unstable at 60°C. It exhibited a high level of activity in the pH range of 7.0–10.0 showing the highest activity at pH 9.0.

Keywords: Marine sediment; Esterase; Metagenome; Cold active

A novel cold-adapted lipase from Acinetobacter sp. XMZ-26: gene cloning and characterisation by Xiaomei Zheng; Xiaoyu Chu; Wei Zhang; Ningfeng Wu; Yunliu Fan (pp. 971-980).

Acinetobacter sp. XMZ-26 (ACCC 05422) was isolated from soil samples obtained from glaciers in Xinjiang Province, China. The partial nucleotide sequence of a lipase gene was obtained by touchdown PCR using degenerate primers designed based on the conserved domains of cold-adapted lipases. Subsequently, a complete gene sequence encoding a 317 amino acid polypeptide was identified. Our novel lipase gene, lipA, was overexpressed in Escherichia coli. The recombinant protein (LipA) was purified by Ni-affinity chromatography, and then deeply characterised. The LipA resulted to hydrolyse pNP esters of fatty acids with acyl chain length from C2 to C16, and the preferred substrate was pNP octanoate showing a k _cat = 560.52 ± 28.32 s⁻¹, K _m = 0.075 ± 0.008 mM, and a k _cat/K _m = 7,377.29 ± 118.88 s⁻¹ mM⁻¹. Maximal LipA activity was observed at a temperature of 15°C and pH 10.0 using pNP decanoate as substrate. That LipA peaked at such a low temperature and remained most activity between 5°C and 35°C indicated that it was a cold-adapted enzyme. Remarkably, this lipase retained much of its activity in the presence of commercial detergents and organic solvents, including Ninol, Triton X-100, methanol, PEG-600, and DMSO. This cold-adapted lipase may find applications in the detergent industry and organic synthesis.

Keywords: Cold-adapted lipase; Acinetobacter sp.; Detergent additives; Organic synthesis

Isolation and characterization of glucoamylase from a wastewater treatment yeast Hansenula fabianii J640, and construction of expression vector by Miyoshi Kato; Teruhito Kitajima; Haruyuki Iefuji (pp. 981-987).

Hansenula fabianii J640 highly expresses an extracellular glucoamylase (GA). Here, we purified the GA and showed that it has pH and temperature optima of 5.0 and 50 °C, respectively, stable at temperatures up to 50 °C, and is inhibited by Ag²⁺, Hg²⁺, and Cu²⁺. The gene was found in an expression library with anti-GA antibodies. A cDNA was found to encode 491 amino acids, including a putative signal peptide of 21 amino acids. Because of the gene’s high expression, we used its promoter and terminator regions to improve a previously developed H. fabianii J640 expression system.

Keywords: Hansenula; Glucoamylase; Wastewater; Expression vector; Yeast

A recombinant α-dioxygenase from rice to produce fatty aldehydes using E. coli by Fenja Kaehne; Markus Buchhaupt; Jens Schrader (pp. 989-995).

Fatty aldehydes are an important group of fragrance and flavor compounds that are found in different fruits and flowers. A biotechnological synthesis of fatty aldehydes based on Escherichia coli cells expressing an α-dioxygenase (αDOX) from Oryza sativa (rice) is presented. α-Dioxygenases are the initial enzymes of α-oxidation in plants and oxidize long and medium-chain C n fatty acids to 2-hydroperoxy fatty acids. The latter are converted to C n_− 1 fatty aldehydes by spontaneous decarboxylation. Successful expression of αDOX in E. coli was proven by an in vitro luciferase assay. Using resting cells of this recombinant E. coli strain, conversion of different fatty acids to the respective fatty aldehydes shortened by one carbon atom was demonstrated. The usage of Triton X 100 improves the conversion rate up to 1 g aldehyde per liter per hour. Easy reuse of the cells was demonstrated by performing a second biotransformation without any loss of biocatalytic activity.

Keywords: Fatty acid; Fatty aldehyde; Alpha-dioxygenase; Whole-cell biotransformation

Efficient fermentation of xylose to ethanol at high formic acid concentrations by metabolically engineered Saccharomyces cerevisiae by Tomohisa Hasunuma; Kyung-mo Sung; Tomoya Sanda; Kazuya Yoshimura; Fumio Matsuda; Akihiko Kondo (pp. 997-1004).

Recombinant yeast strains highly tolerant to formic acid during xylose fermentation were constructed. Microarray analysis of xylose-fermenting Saccharomyces cerevisiae strain overexpressing endogenous xylulokinase in addition to xylose reductase and xylitol dehydrogenase from Pichia stipitis revealed that upregulation of formate dehydrogenase genes (FDH1 and FDH2) was one of the most prominent transcriptional events against excess formic acid. The quantification of formic acid in medium indicated that the innate activity of FDH was too weak to detoxify formic acid. To reinforce the capability for formic acid breakdown, the FDH1 gene was additionally overexpressed in the xylose-metabolizing recombinant yeast. This modification allowed the yeast to rapidly decompose excess formic acid. The yield and final ethanol concentration in the presence of 20 mM formic acid is as essentially same as that of control. The fermentation profile also indicated that the production of xylitol and glycerol, major by-products in xylose fermentation, was not affected by the upregulation of FDH activity.

Keywords: Saccharomyces cerevisiae ; Bioethanol; Xylose fermentation; Formic acid; Formate dehydrogenase

Overexpression of an apoplastic peroxidase gene CrPrx in transgenic hairy root lines of Catharanthus roseus by Monika Jaggi; Santosh Kumar; Alok Krishna Sinha (pp. 1005-1016).

Peroxidases are a family of isoenzymes found in all higher plants and are known to be involved in a broad range of physiological processes. However, very little information is available concerning their role in Catharanthus roseus. The present study describes the impact of both overexpression and suppression of a peroxidase gene, CrPrx in C. roseus transgenic hairy root lines. Real-time PCR analysis in 35S-CrPrx and CrPrx-RNAi transgenic lines indicated differential transcript profile for peroxidases as well as for genes and regulators involved in MIA (monoterpenoid indole alkaloid) pathway of C. roseus. Comparative analysis revealed that MIA pathway genes showing elevated levels of expression in 35S-CrPrx transgenic lines showed a significant reduction in their transcript level in CrPrx-RNAi transgenic lines. Metabolite analysis detected higher levels of ajmalicine and serpentine accumulation in overexpressed lines. It was observed that all overexpressed transgenic lines produced more amount of H₂O₂. These results indicate a role of CrPrx gene in the regulation of MIA pathway genes and regulators, thus affecting the production of specific alkaloids.

Keywords: Agrobacterium rhizogenes ; Catharanthus roseus ; Monoterpenoid indole alkaloids; Peroxidases; Transgenic hairy roots

Manipulation of quorum sensing regulation in Pseudomonas fluorescens NCIMB 10586 to increase mupirocin production by Joanne Hothersall; Annabel C. Murphy; Zafar Iqbal; Genevieve Campbell; Elton R. Stephens; Ji’en Wu; Helen Cooper; Steve Atkinson; Paul Williams; John Crosby; Christine L. Willis; Russell J. Cox; Thomas J. Simpson; Christopher M. Thomas (pp. 1017-1026).

Transcription of the 74 kb Pseudomonas fluorescens mupirocin [pseudomonic acid (PA)] biosynthesis cluster depends on quorum sensing-dependent regulation via the LuxI/LuxR homologues MupI/MupR. To facilitate analysis of novel PAs from pathway mutants, we investigated factors that affect mup gene expression. First, the signal produced by MupI was identified as N-(3-oxodecanoyl)homoserine lactone, but exogenous addition of this molecule did not activate mupirocin production prematurely nor did expression of mupI in trans increase metabolite production. Second, we confirmed that mupX, encoding an amidase/hydrolase that can degrade N-acylhomoserine lactones, is also required for efficient expression, consistent with its occurrence in a regulatory module linked to unrelated genes in P. fluorescens. Third, and most significantly, mupR expression in trans to wild type and mutants can increase production of antibiotic and novel intermediates up to 17-fold.

Keywords: Antibiotic; Polyketide; Quorum sensing; N-acyl homoserine lactone

Intracellular salicylic acid is involved in signal cascade regulating low ammonium-induced taxoid biosynthesis in suspension cultures of Taxus chinensis by Xin Zhou; Jian-Jiang Zhong (pp. 1027-1036).

It was previously reported that low initial ammonium (2 mM) in medium had significant stimulating effects on the biosynthesis of taxuyunnanine C (Tc) by Taxus chinensis cells. However, the secondary metabolism induction mechanism of the low initial ammonium is yet unknown in plant cells. To provide an insight into the defense signals response to the low initial ammonium, oxidative burst and intracellular salicylic acid (SA) were detected, and their influences on the expression of important genes in taxoid biosynthetic pathway were examined in the cell cultures of T. chinensis. Induced H₂O₂ production, elevated phenylalanine ammonia-lyase (PAL) activity, and enhanced SA biosynthesis were observed. Interestingly, inhibition of SA biosynthesis by paclobutrazol and (BOC-aminooxy) acetic acid significantly depressed the Tc stimulation and up-regulation of Tc biosynthetic genes of geranylgeranyl diphosphate synthase and taxadiene synthase. The role of intracellular SA in regulating Tc biosynthesis was further confirmed by applying exogenous SA in normal ammonium (20 mM) medium. The results indicated that SA acted as a signal in low initial ammonium-induced Tc biosynthesis. A signal transduction cascade from defense signal response to activated transcription of taxoid biosynthetic genes and enhanced Tc production is proposed.

Keywords: Low initial ammonium; H₂O₂ ; Salicylic acid; Plant cell culture; Taxus chinensis ; Secondary metabolite

A new phenol oxidase produced during melanogenesis and encystment stage in the nitrogen-fixing soil bacterium Azotobacter chroococcum by Susanne Herter; Marlen Schmidt; Mark L. Thompson; Annett Mikolasch; Frieder Schauer (pp. 1037-1049).

Laccases are copper-containing phenol oxidases that are commonly found in many types of plant, insect, fungi and bacteria. Whilst phenol oxidases have been well characterized in fungal species, laccase-type enzymes originating from bacteria have been much less well defined. Bacteria belonging to the family Azotobacteraceae share many morphological characteristics with strains already known to exhibit polyphenol and phenol oxidase activity; and hence the aim of this work was to identify and characterize a novel laccase from the isolated strain Azotobacter chroococcum SBUG 1484 in an attempt to provide further understanding of the roles such enzymes play in physiological development. Laccase activity was clearly observed through oxidation of 2,6-dimethoxyphenol, other typical substrates including: methoxy-monophenols, ortho- and para-diphenols, 4-hydroxyindole, and the non-phenolic compound para-phenylenediamine. A. chroococcum SBUG 1484 showed production of a cell-associated phenol oxidase when grown under nitrogen-fixing conditions, and was also observed when cells enter the melanogenic and encystment stages of growth. Catechol which is structurally related to melanin compounds was also released from Azotobacter cells into the surrounding culture medium during nitrogen-fixing growth. From our results we propose that a membrane-bound laccase plays an important role in the formation of melanin, which was monitored to correlate with progression of A. chroococcum SBUG 1484 cells into the encystment stage of growth.

Keywords: Bacterial phenol oxidase; Laccase; Polyphenol oxidase; Melanin; Cyst; Nitrogen fixation

Diversity of metabolic shift in response to oxygen deprivation in Corynebacterium glutamicum and its close relatives by Shogo Yamamoto; Masayuki Sakai; Masayuki Inui; Hideaki Yukawa (pp. 1051-1061).

Oxygen-deprived Corynebacterium glutamicum R cells remain metabolically active, producing considerable amounts of organic acids even when not actively growing. We compared the proficiencies of C. glutamicum and close relatives grown under aerobic conditions to metabolize glucose when deprived of oxygen. Eight strains that readily consumed glucose without cell growth subsequently produced organic acids. Among these, the glucose consumption rates of the two C. glutamicum strains (>40 mM/h) and Corynebacterium efficiens (>12 mM/h) were an order of magnitude higher than those of the other five strains. The resultant organic acid yields of these three strains (>86%) consequently exceeded those of the other five (<60%). This difference is probably rooted in the comparatively inferior activities of glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, and malate dehydrogenase observed in the five strains. Moreover, under oxygen deprivation, phosphoenolpyruvate carboxylase (PEPC) activity of C. efficiens was elevated tenfold, but its lack of fumarase activity meant that no succinic acid could be produced. The metabolic shift occasioned by addition of the PEPC substrate sodium bicarbonate resulted in a doubling of the glucose consumption rate of the two C. glutamicum strains but not that of the other six close relatives.

Keywords: Corynebacterium relatives; Oxygen deprivation; Metabolic shift; Glucose consumption; Organic acid production

Extracellular polymeric substances enhanced mass transfer of polycyclic aromatic hydrocarbons in the two-liquid-phase system for biodegradation by Yinping Zhang; Fang Wang; Xinglun Yang; Chenggang Gu; Fredrick Orori Kengara; Qing Hong; Zhengyong Lv; Xin Jiang (pp. 1063-1071).

The objective was to elucidate the role of extracellular polymeric substances (EPS) in biodegradation of polycyclic aromatic hydrocarbons in two-liquid-phase system (TLPs). Therefore, biodegradation of phenanthrene (PHE) was conducted in a typical TLPs—silicone oil–water—with PHE-degrading bacteria capable of producing EPS, Sphingobium sp. PHE3 and Micrococcus sp. PHE9. The results showed that the presence of both strains enhanced mass transfer of PHE from silicone oil to water, and that biodegradation of PHE mainly occurred at the interfaces. The ratios of tightly bound (TB) proteins to TB polysaccharides kept almost constant, whereas the ratios of loosely bound (LB) proteins to LB polysaccharides increased during the biodegradation. Furthermore, polysaccharides led to increased PHE solubility in the bulk water, which resulted in an increased PHE mass transfer. Both LB-EPS and TB-EPS (proteins and polysaccharides) correlated with PHE mass transfer in silicone oil, indicating that both proteins and polysaccharides favored bacterial uptake of PHE at the interfaces. It could be concluded that EPS could facilitate microbial degradation of PHE in the TLPs.

Keywords: Polycyclic aromatic hydrocarbons (PAHs); Extracellular polymeric substances (EPS); The non-aqueous phase liquid (NAPL); Biodegradation; Mass transfer

Nitrate reductase-mediated nitric oxide generation is essential for fungal elicitor-induced camptothecin accumulation of Camptotheca acuminata suspension cell cultures by Dan Lu; Jufang Dong; Haihong Jin; Lina Sun; Xiangbin Xu; Ting Zhou; Yun Zhu; Maojun Xu (pp. 1073-1081).

Secondary metabolite accumulation and nitric oxide (NO) generation are two common responses of plant cells to fungal elicitors, and NO has been reported to play important roles in elicitor-induced secondary metabolite production. However, the source of elicitor-triggered NO generation in plant cells remains largely unknown. To investigate the origin of elicitor-triggered NO, we examined nitrate reductase (NR) activities and the expression levels of NIA1 and NIA2 genes of Camptotheca acuminata cells treated with PB90, a protein elicitor from Phytophthora boehmeriae. The data show that PB90 treatment stimulates NR activity and induces upregulation of NIA1 but does not affect NIA2 expression in the cells. Pretreatment of the cells with NR inhibitors tungstate and Gln abolishes not only the fungal elicitor-triggered NR activities but also the PB90-induced NO generation. Treatment of PB90 enhances camptothecin contents of the cells, suggesting that the fungal elicitor might stimulate camptothecin biosynthesis. Furthermore, application of tungstate and Gln suppresses the fungal elicitor-induced camptothecin accumulation of the cells and the suppression of NR inhibitors on PB90-induced camptothecin production can be reversed by NO via its donor sodium nitroprusside. Together, the results suggest that NIA1 is sensitive to PB90 and the fungal elicitor-induced upregulation of NIA1 may lead to higher NR activity. Furthermore, our data demonstrate that NR is involved in the fungal elicitor-triggered NO generation and the fungal elicitor induces camptothecin production of C. acuminata cells dependently on NR-mediated NO generation.

Keywords: Fungal elicitor; Nitric oxide; Nitrate reductase (NR); Camptothecin; Camptotheca acuminata cells

Antifungal activity of essential oils and their synergy with fluconazole against drug-resistant strains of Aspergillus fumigatus and Trichophyton rubrum by Mohd Sajjad Ahmad Khan; Iqbal Ahmad (pp. 1083-1094).

The aim of this study was to screen certain plant essential oils and active compounds for antifungal activity and their in vitro interaction with fluconazole against drug-resistant pathogenic fungi. The methods employed in this work included disc diffusion, broth macrodilution, time kill methods and checkerboard microtiter tests. Oil compositions were evaluated by gas chromatography-mass spectrometry (GC-MS) analysis. Transmission electron microscopy was used to assess the effect of essential oils on cellular structures of test fungi. Test fungal strains exhibited resistance to at least two drugs (fluconazole and itraconazole). Among the 21 essential oils or active compounds tested, ten showed promising antifungal activity. GC-MS analysis revealed the presence of major active compounds in the essential oils used. Cinnamaldehyde showed the most promising antifungal activity and killing potency against Aspergillus fumigatus MTCC2550 and Trichophyton rubrum IOA-9. Cinnamaldehyde showed strongest synergy with fluconazole against A. fumigatus and T. rubrum by reducing the minimum inhibitory concentration of fluconazole up to 8-fold. Zones of lysis of the cell wall and cell membrane appeared to be where cinnamaldehyde acted on fungi. This study highlights the broad spectrum antifungal activity of essential oils and active compounds and their synergy with fluconazole against drug-resistant fungi.

Keywords: Antifungal drugs; Drug resistance; Essential oils; GC-MS; Synergy; Transmission electron microscopy

Effects of three heavy metals on the bacteria growth kinetics: a bivariate model for toxicological assessment by Diego Rial; José Antonio Vázquez; Miguel Anxo Murado (pp. 1095-1109).

The effects of three heavy metals (Co, Ni and Cd) on the growth kinetics of five bacterial strains with different characteristics (Pseudomonas sp., Phaeobacter sp. strain 27-4, Listonella anguillarum, Carnobacterium piscicola and Leuconostoc mesenteroides subsp. lysis) were studied in a batch system. A bivariate model, function of time and dose, is proposed to describe simultaneously all the kinetic profiles obtained by incubating a microorganism at increasing concentrations of individual metals. This model combines the logistic equation for describing growth, with a modification of the cumulative Weibull’s function for describing the dose-dependent variations of growth parameters. The comprehensive model thus obtained—which minimizes the effects of the experimental error—was statistically significant in all the studied cases, and it raises doubts about toxicological evaluations that are based on a single growth parameter, especially if it is not obtained from a kinetic equation. In lactic acid bacteria cultures (C. piscicola and L. mesenteroides), Cd induced remarkable differences in yield and time course of characteristic metabolites. A global parameter is defined (ED_50,τ: dose of toxic chemical that reduces the biomass of a culture by 50% compared to that produced by the control at the time corresponding to its semi maximum biomass) that allows comparing toxic effects on growth kinetics using a single value.

Keywords: Bacteria growth kinetics; Logistic and Weibull equations; Heavy metals; Toxicity; Dose–response modelling

Multiplex polymerase chain reaction-based assay for the specific detection of toxin-producing Vibrio cholerae in fish and fishery products by Geevaretnam Jeyasekaran; Kannan Thirumalai Raj; Robinson Jeya Shakila; Albin Jemila Thangarani; Durairaj Sukumar (pp. 1111-1118).

A multiplex polymerase chain reaction (MPCR)-based assay was developed for the simultaneous detection of Vibrios using the genus-specific RNA polymerase subunit A (rpoA) gene and specific detection of toxin-producing Vibrio cholerae strains using two sets of primer based on cholera toxin subunit A (ctxA) and repeat in toxin subunit A (RtxA)-producing genes. The MPCR method developed is applicable to both the simultaneous and the two-step detection of genus Vibrio total and toxigenic V. cholerae species. This assay was specific as no amplification occurred with the other bacterial pathogens tested. The sensitivity of the assay was tested by artificially spiking the shrimp homogenate with the toxigenic strain of V. cholerae (NICED 16582) in different dilutions. The developed MPCR assay could detect three cells of V. cholerae in 12 h pre-enrichment in APW. The proposed method is rapid, sensitive, and specific for the detection of Vibrio genus as well as toxin-producing V. cholerae strains in environmental samples.

Keywords: Multiplex PCR; Vibrio, toxigenic strains of V. cholerae ; rpoA gene; Toxin genes ctxA and RtxA genes

Adsorption and decolorization kinetics of methyl orange by anaerobic sludge by Lei Yu; Wen-Wei Li; Michael Hon-Wah Lam; Han-Qing Yu (pp. 1119-1127).

Adsorption and decolorization kinetics of methyl orange (MO) by anaerobic sludge in anaerobic sequencing batch reactors were investigated. The anaerobic sludge was found to have a saturated adsorption capacity of 36 ± 1 mg g MLSS⁻¹ to MO. UV/visible spectrophotometer and high-performance liquid chromatography analytical results indicated that the MO adsorption and decolorization occurred simultaneously in this system. This process at various substrate concentrations could be well simulated using a modified two-stage model with apparent pseudo first-order kinetics. Furthermore, a noncompetitive inhibition kinetic model was also developed to describe the MO decolorization process at high NaCl concentrations, and an inhibition constant of 3.67 g NaCl l⁻¹ was estimated. This study offers an insight into the adsorption and decolorization processes of azo dyes by anaerobic sludge and provides a better understanding of the anaerobic dye decolorization mechanisms.

Keywords: Adsorption; Anaerobic sludge; Decolorization; Kinetics; Methyl orange (MO); Salt inhibition

Reduction of produced elementary sulfur in denitrifying sulfide removal process by Xu Zhou; Lihong Liu; Chuan Chen; Nanqi Ren; Aijie Wang; Duu-Jong Lee (pp. 1129-1136).

Denitrifying sulfide removal (DSR) processes simultaneously convert sulfide, nitrate, and chemical oxygen demand from industrial wastewater into elemental sulfur, dinitrogen gas, and carbon dioxide, respectively. The failure of a DSR process is signaled by high concentrations of sulfide in reactor effluent. Conventionally, DSR reactor failure is blamed for overcompetition for heterotroph to autotroph communities. This study indicates that the elementary sulfur produced by oxidizing sulfide that is a recoverable resource from sulfide-laden wastewaters can be reduced back to sulfide by sulfur-reducing Methanobacterium sp. The Methanobacterium sp. was stimulated with excess organic carbon (acetate) when nitrite was completely consumed by heterotrophic denitrifiers. Adjusting hydraulic retention time of a DSR reactor when nitrite is completely consumed provides an additional control variable for maximizing DSR performance.

Keywords: Denitrifying sulfide removal; Elementary sulfur; Sulfate-reducing; Hydraulic retention time

Diversity and quantity of ammonia-oxidizing Archaea and Bacteria in sediment of the Pearl River Estuary, China by Tao Jin; Tong Zhang; Lin Ye; On On Lee; Yue Him Wong; Pei Yuan Qian (pp. 1137-1145).

The diversity and abundance of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) in the sediment of the Pearl River Estuary were investigated by cloning and quantitative real-time polymerase chain reaction (qPCR). From one sediment sample S16, 36 AOA OTUs (3% cutoff) were obtained from three clone libraries constructed using three primer sets for amoA gene. Among the 36 OTUs, six were shared by all three clone libraries, two appeared in two clone libraries, and the other 28 were only recovered in one of the libraries. For AOB, only seven OTUs (based on 16S rRNA gene) and eight OTUs (based on amoA gene) were obtained, showing lower diversity than AOA. The qPCR results revealed that AOA amoA gene copy numbers ranged from 9.6 × 10⁶ to 5.1 × 10⁷ copies per gram of sediment and AOB amoA gene ranged from 9.5 × 10⁴ to 6.2 × 10⁵ copies per gram of sediment, indicating that the dominant ammonia-oxidizing microorganisms in the sediment of the Pearl River Estuary were AOA. The terminal restriction fragment length polymorphism results showed that the relative abundance of AOB species in the sediment samples of different salinity were significantly different, indicating that salinity might be a key factor shaping the AOB community composition.

Keywords: Ammonia monooxygenase α-subunit (amoA) gene; Ammonia-oxidizing archaea (AOA); Ammonia-oxidizing bacteria (AOB); T-RFLP; qPCR

Effect of operational conditions on the degradation of organic matter and development of microalgae–bacteria consortia when treating swine slurry by Cristina González-Fernández; Berta Riaño-Irazábal; Beatriz Molinuevo-Salces; Saúl Blanco; Maria Cruz García-González (pp. 1147-1153).

There is great controversy regarding the best substrate (fresh or anaerobically digested swine slurry) for the development of microalgae–bacteria consortia. This study aims to elucidate the best substrate by assessing biomass productivity, microorganism predominance, and their ability for organic matter removal. In addition to the different substrates, different operational conditions and influent strengths were evaluated. Increasing organic matter content when favourable temperature and illumination conditions were present improved biomass production. However, these conditions were not favourable for microalgal growth, but they were favourable for bacteria. Regardless of the operational conditions, reactors fed with fresh slurry not only resulted in the highest biomass productivity, but also the greatest removal of total and soluble chemical oxygen demand (COD). On the other hand, reactors fed with digested slurry showed biomass productivity and COD removal values lower than those obtained for reactors fed with fresh slurry, most probably due to the recalcitrant nature of the former. Nevertheless, digested slurry was the substrate more appropriate for microalgae growth under harsh operational conditions (16 °C and 9-h illumination) at low influent strength and optimum operational conditions (30 °C and 24-h illumination) at higher influent strength.

Keywords: Organic matter; Biomass productivity; Microalgae–bacteria consortium; Swine slurry

Effect of monorhamnolipid on the degradation of n-hexadecane by Candida tropicalis and the association with cell surface properties by Guangming Zeng; Zhifeng Liu; Hua Zhong; Jianbing Li; Xingzhong Yuan; Haiyan Fu; Ying Ding; Jing Wang; Meifang Zhou (pp. 1155-1161).

The effect of monorhamnolipid (monoRL) on the degradation of n-hexadecane by Candida tropicalis was investigated in this study. The concentration of hexadecane, cell growth, cell surface hydrophobicity (CSH), cell surface zeta potential (CSZP), and FT-IR spectra of cellular envelope were tested to determine the mechanisms. MonoRL at the initial concentrations of 11.4, 19, and 38 mg/l improved the degradation of hexadecane, and 19 mg/l was the best concentration. However, 114 mg/l monoRL suppressed the biodegradation probably because of the reduced bioavailability of hexadecane caused by the micelles. The presence of monoRL changed the cell surface properties, which was demonstrated by the increased CSH, the increased CSZP, and the changed FT-IR spectra of cellular envelope at 680 and 620 cm⁻¹. The changes of cell surface properties may be a reason for the enhanced biodegradation of hexadecane by the yeast. The results indicate the potential application of monoRL in the bioremediation of hydrocarbons.

Keywords: Rhamnolipids; Hydrocarbons; Candida tropicalis ; Cell surface hydrophobicity; Cell surface zeta potential

Isolation and characterization of a chromium-resistant bacterium Serratia sp. Cr-10 from a chromate-contaminated site by Kundi Zhang; Fuli Li (pp. 1163-1169).

A novel bacterium, Cr-10, was isolated from a chromium-contaminated site and capable of removing toxic chromium species from solution by reducing hexavalent chromium to an insoluble precipitate. Sequence analysis of 16S rRNA gene of strain Cr-10 showed that it was most closely related to Serratia rubidaea JCM 1240^T (97.68%). Physiological and chemotaxonomic data also supported that strain Cr-10 was identified as Serratia sp., a genus which was never specially reported chromate-resistant before. Serratia sp., Cr-10 was tolerant to a concentration of 1,500 mg Cr(VI) L⁻¹, which was the highest level reported until now. The optimum pH and temperature for reduction of Cr(VI) by Serratia sp. Cr-10 were found to be 7.0 and 37 °C, respectively. The Cr(VI) reduction was significantly influenced by additional carbon sources, and among them fructose and lactose offered maximum reduction, with a rate of 0.28 and 0.25 mg Cr(VI) L⁻¹ h⁻¹, respectively. The cell-free extracts and filtrate of the culture were able to reduce Cr(VI) while concentration of total chromium remained stable in the process, indicating that the enzyme-catalyzed mechanism was applied in Cr(VI) reduction by the isolate. Additionally, it was found that there was hardly any chromium on the cell surface of the strain, further supporting that reduction, rather than bioadsorption, plays a major role in the Cr(VI) removal.

Keywords: Cr(VI) reduction; Serratia sp.; Batch reduction; Bioremediation

Highly efficient biodiesel production by a whole-cell biocatalyst employing a system with high lipase expression in Aspergillus oryzae by Tomohiro Takaya; Risa Koda; Daisuke Adachi; Kazunori Nakashima; Junpei Wada; Takayuki Bogaki; Chiaki Ogino; Akihiko Kondo (pp. 1171-1177).

In the present study, a system with high lipase expression in Aspergillus oryzae was developed using an improved enolase promoter (P-enoA124) and the 5′ untranslated region of a heat-shock protein (Hsp-UTR). P-enoA142 enhanced the transcriptional level of a heterologous lipase gene and Hsp-UTR improved its translational efficiency. Fusarium heterosporum lipase (FHL) was inserted into a pSENSU-FHL expression vector harboring P-enoA142 and Hsp-UTR and was transformed into an A. oryzae NS4 strain. Transformants possessing pSENSU-FHL in single (pSENSU-FHL#1) and double copies (pSENSU-FHL#2) were selected to evaluate the lipase activity of the whole-cell biocatalyst. The two strains, pSENSU-FHL#1 and #2, showed excellent lipase activity in hydrolysis compared with the strain transformed with conventional expression vector pNAN8142-FHL. Furthermore, by using pSENSU-FHL#2, methanolysis could proceed much more effectively without deactivation, which allowed a swift addition of methanol to the reaction mixture, thereby reducing reaction time.

Keywords: Whole-cell biocatalyst; Aspergillus oryzae ; Biodiesel fuel; Transesterification; Fusarium heterosporum lipase

Spatiotemporal development of the bacterial community in a tubular longitudinal microbial fuel cell by Jung Rae Kim; Nelli J. Beecroft; John R. Varcoe; Richard M. Dinsdale; Alan J. Guwy; Robert C. T. Slade; Alfred Thumser; Claudio Avignone-Rossa; Giuliano C. Premier (pp. 1179-1191).

The spatiotemporal development of a bacterial community in an exoelectrogenic biofilm was investigated in sucrose-fed longitudinal tubular microbial fuel cell reactors, consisting of two serially connected modules. The proportional changes in the microbial community composition were assessed by polymerase chain reaction–denaturing gradient gel electrophoresis (DGGE) and DNA sequencing in order to relate them to the performance and stability of the bioelectrochemical system. The reproducibility of duplicated reactors, evaluated by cluster analysis and Jaccard’s coefficient, shows 80–90% similarity in species composition. Biofilm development through fed-batch start-up and subsequent stable continuous operation results in a population shift from γ-Proteobacteria- and Bacteroidetes- to Firmicutes-dominated communities, with other diverse species present at much lower relative proportions. DGGE patterns were analysed by range-weighted richness (Rr) and Pareto–Lorenz evenness distribution curves to investigate the evolution of the bacterial community. The first modules shifted from dominance by species closely related to Bacteroides graminisolvens, Raoultella ornithinolytica and Klebsiella sp. BM21 at the start of continuous-mode operation to a community dominated by Paludibacter propionicigenes-, Lactococcus sp.-, Pantoea agglomerans- and Klebsiella oxytoca-related species with stable power generation (6.0 W/m³) at day 97. Operational strategies that consider the dynamics of the population will provide useful parameters for evaluating system performance in the practical application of microbial fuel cells.

Keywords: Microbial fuel cell (MFC); Electricity generation; DGGE; Identification; Semi-quantitative microbial community analysis

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Applied Microbiology and Biotechnology (v.90, #3)

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Applied Microbiology and Biotechnology (v.90, #3)