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Applied Microbiology and Biotechnology (v.97, #5)

Silica sol-gel encapsulation of cyanobacteria: lessons for academic and applied research by David J. Dickson; Roger L. Ely (pp. 1809-1819).

Cyanobacteria inhabit nearly every ecosystem on earth, play a vital role in nutrient cycling, and are useful as model organisms for fundamental research in photosynthesis and carbon and nitrogen fixation. In addition, they are important for several established biotechnologies for producing food additives, nutritional and pharmaceutical compounds, and pigments, as well as emerging biotechnologies for biofuels and other products. Encapsulation of living cyanobacteria into a porous silica gel matrix is a recent approach that may dramatically improve the efficiency of certain production processes by retaining the biomass within the reactor and modifying cellular metabolism in helpful ways. Although encapsulation has been explored empirically in the last two decades for a variety of cell types, many challenges remain to achieving optimal encapsulation of cyanobacteria in silica gel. Recent evidence with Synechocystis sp. PCC 6803, for example, suggests that several unknown or uncharacterized proteins are dramatically upregulated as a result of encapsulation. Also, additives commonly used to ease stresses of encapsulating living cells, such as glycerol, have detrimental impacts on photosynthesis in cyanobacteria. This mini-review is intended to address the current status of research on silica sol-gel encapsulation of cyanobacteria and research areas that may further the development of this approach for biotechnology applications.

Keywords: Cyanobacteria; Encapsulation; Silica gel; Sol-gel; Stress; Bioproducts; Bioenergy

Recent advances on physiological functions and biotechnological production of epilactose by Wanmeng Mu; Qiuxi Li; Chen Fan; Chen Zhou; Bo Jiang (pp. 1821-1827).

Epilactose (4-O-β-d-galactopyranosyl-d-mannose), an epimer of lactose, is a rare disaccharide existing extremely small quantities in heat-treated milk, in which epilactose is produced by non-enzymatic catalysis from lactose. This disaccharide is a kind of non-digestible carbohydrate, has a good prebiotic effect, and promotes intestinal mineral absorption. This article presents a review of recent studies on epilactose formation in food system, qualitative and quantitative analysis, and its physiological functions. In addition, the biochemical properties and kinetic parameters of the epilactose-producing enzyme, cellobiose 2-epimerase, are compared, and the biotechnological production of epilactose from lactose is reviewed.

Keywords: Epilactose; Cellobiose 2-epimerase; Lactose; Physiological function; Biotechnological production

An overview of transducers as platform for the rapid detection of foodborne pathogens by Pooja Arora; Annu Sindhu; Harmanmeet Kaur; Neeraj Dilbaghi; Ashok Chaudhury (pp. 1829-1840).

The driving advent of portable, integrated biosensing ways for pathogen detection methods offers increased sensitivity and specificity over traditional microbiological techniques. The miniaturization and automation of integrated detection systems present a significant advantage for rapid, portable detection of foodborne microbes. In this review, we have highlighted current developments and directions in foodborne pathogen detection systems. Recent progress in the biosensor protocols toward the detection of specific microbes has been elaborated in detail. It also includes strategies and challenges for the implementation of a portable platform toward rapid foodborne sensing systems.

Keywords: Biosensor; Food; Detection; Pathogen; Transducer

Ecological characteristics of anaerobic ammonia oxidizing bacteria by Shuang Ding; Ping Zheng; Huifeng Lu; Jianwei Chen; Qaisar Mahmood; Ghulam Abbas (pp. 1841-1849).

Anaerobic ammonium oxidation (anammox) is the microbial conversion of ammonium and nitrite to dinitrogen gas. The functional microbes of anammox reaction are anammox bacteria, which were discovered in a wastewater treatment system for nitrogen removal. Anammox bacteria are prevalent in anoxic ecosystems and play an important role in both biological nitrogen cycle and nitrogen pollution control. In this paper, we reviewed the investigation on ecological characteristics of anammox bacteria, and tried to figure out their complicated intraspecies and interspecies relationships. As for intraspecies relationship, we focused on the quorum sensing system, a cell density-dependent phenomenon. As for interspecies relationship, we focused on the synergism and competition of anammox bacteria with other microorganisms for substrate and space. Finally, we discussed the great influence of environmental factors (e.g., dissolved oxygen, organic matters) on the constitution, structure and function of anammox bacteria community.

Keywords: Anaerobic ammonium-oxidizing bacteria; Quorum sensing; Synergism; Competition; Nitrogen removal

Pseudomonas: a promising biocatalyst for the bioconversion of terpenes by Gustavo Molina; Mariana R. Pimentel; Gláucia M. Pastore (pp. 1851-1864).

The Pseudomonas genus is one of the most diverse and ecologically significant groups of known bacteria, and it includes species that have been isolated worldwide in all types of environments. The bacteria from this genus are characterized by an elevated metabolic versatility, which is due to the presence of a complex enzymatic system. Investigations since the early 1960s have demonstrated their potential as biocatalysts for the production of industrially relevant and value-added flavor compounds from terpenes. Although terpenes are often removed from essential oils as undesirable components, its synthetic oxy-functionalized derivatives have broad applications in flavors/fragrances and pharmaceutical industries. Hence, biotransformation appears to be an effective tool for the structural modification of terpene hydrocarbons and terpenoids to synthesize novel and high-valued compounds. This review highlights the potential of Pseudomonas spp. as biocatalysts for the bioconversion of terpenes and summarizes the presently known bioflavors that are obtained from these processes.

Keywords: Pseudomonas ; Terpenes; Biotransformation; Bioconversion; Bioflavors

Metabolic reconstruction and flux analysis of industrial Pichia yeasts by Bevan Kai-Sheng Chung; Meiyappan Lakshmanan; Maximilian Klement; Chi Bun Ching; Dong-Yup Lee (pp. 1865-1873).

Pichia yeasts have been recognized as important microbial cell factories in the biotechnological industry. Notably, the Pichia pastoris and Pichia stipitis species have attracted much research interest due to their unique cellular physiology and metabolic capability: P. pastoris has the ability to utilize methanol for cell growth and recombinant protein production, while P. stipitis is capable of assimilating xylose to produce ethanol under oxygen-limited conditions. To harness these characteristics for biotechnological applications, it is highly required to characterize their metabolic behavior. Recently, following the genome sequencing of these two Pichia species, genome-scale metabolic networks have been reconstructed to model the yeasts’ metabolism from a systems perspective. To date, there are three genome-scale models available for each of P. pastoris and P. stipitis. In this mini-review, we provide an overview of the models, discuss certain limitations of previous studies, and propose potential future works that can be conducted to better understand and engineer Pichia yeasts for industrial applications.

Keywords: Genome-scale metabolic modeling; Constraints-based flux analysis; Pichia pastoris ; Pichia stipitis ; Microbial cell factories; Systems biotechnology

Sophorolipids: improvement of the selective production by Starmerella bombicola through the design of nutritional requirements by Isabel A. Ribeiro; M. Rosário Bronze; Matilde F. Castro; Maria H. L. Ribeiro (pp. 1875-1887).

Microtiter plates were used as minireactors to study Starmerella bombicola growth and sophorolipid (SL) production. Compositional analysis of SL mixtures by liquid chromatography with electrospray ionization tandem mass spectrometry showed similar results on SLs produced using the laboratory scale (shake flask) and the microscale (24-well microtiter plates (MTP)) approach. MTP suitability on SL production was proven, being this approach, especially advantageous on SL screening. Several hydrophilic carbon sources, hydrophobic co-substrates and nitrogen sources were supplied to culture media, and their influence on SL production was evaluated. The selection of specific hydrophobic co-substrate and nitrogen sources influenced the ratio acidic/lactonic SLs. In fact, it was observed that the production of acidic C18:1 diacetylated hydroxy fatty acid SLs was favoured when culture media was supplied with avocado, argan, sweet almond and jojoba oil or when NaNO₃ was supplied instead of urea. This last case was observed after 144 h of cultivation. A new SL, lactonic C18:3 hydroxy fatty acid diacetylated SL, was detected when borage and onagra oils were used individually as co-substrates. Overall results indicated the potential of the selective production of different and new sophorolipids by Starmerella bombicola based on the selection of carbon and nitrogen sources to culture media.

Keywords: Sophorolipids; Carbon and nitrogen sources; Microscale approach; HPLC–ELSD; LC–MS/MS

Production of 3-O-xylosyl quercetin in Escherichia coli by Ramesh Prasad Pandey; Sailesh Malla; Dinesh Simkhada; Byung-Gee Kim; Jae Kyung Sohng (pp. 1889-1901).

Quercetin, a flavonol aglycone, is one of the most abundant flavonoids with high medicinal value. The bioavailability and pharmacokinetic properties of quercetin are influenced by the type of sugars attached to the molecule. To efficiently diversify the therapeutic uses of quercetin, Escherichia coli was harnessed as a production factory by the installation of various plant and bacterial UDP-xylose sugar biosynthetic genes. The genes encoding for the UDP-xylose pathway enzymes phosphoglucomutase (nfa44530), glucose-1-phosphate uridylyltransferase (galU), UDP-glucose dehydrogenase (calS8), and UDP-glucuronic acid decarboxylase (calS9) were overexpressed in E. coli BL21 (DE3) along with a glycosyltransferase (arGt-3) from Arabidopsis thaliana. Furthermore, E. coli BL21(DE3)/∆pgi, E. coli BL21(DE3)/∆zwf, E. coli BL21(DE3)/∆pgi∆zwf, and E. coli BL21(DE3)/∆pgi∆zwf∆ushA mutants carrying the aforementioned UDP-xylose sugar biosynthetic genes and glycosyltransferase and the galU-integrated E. coli BL21(DE3)/∆pgi host harboring only calS8, calS9, and arGt-3 were constructed to enhance whole-cell bioconversion of exogeneously supplied quercetin into 3-O-xylosyl quercetin. Here, we report the highest production of 3-O-xylosyl quercetin with E. coli BL21 (DE3)/∆pgi∆zwf∆ushA carrying UDP-xylose sugar biosynthetic genes and glycosyltransferase. The maximum concentration of 3-O-xylosyl quercetin achieved was 23.78 mg/L (54.75 μM), representing 54.75 % bioconversion, which was an ~4.8-fold higher bioconversion than that shown by E. coli BL21 (DE3) with the same set of genes when the reaction was carried out in 5-mL culture tubes with 100 μM quercetin under optimized conditions. Bioconversion was further improved by 98 % when the reaction was scaled up in a 3-L fermentor at 36 h.

Keywords: Biotransformation; E. coli ; Glycosylation; Metabolic engineering; Quercetin

High cell density cultivation of Pseudomonas putida strain HKT554 and its application for optically active sulfoxide production by Said Hamad Ramadhan; Toru Matsui; Kazuma Nakano; Hirofumi Minami (pp. 1903-1907).

Culture conditions with Pseudomonas putida strain HKT554, expressing naphthalene dioxygenase, known as the biocatalyst showing wide substrate specificity, were optimized for high cell density cultivation (HCDC). Culture in a medium TK-B modified from that for HCDC of Escherichia coli with glucose fed-batch and dissolved oxygen stat resulted in a high cell density growth of 114 g dry cell/l at 40 h of cultivation. This system was further applied for S-(+)-methyl phenyl sulfoxide (MPSO) production from methyl phenyl sulfide. Addition of nonpolar organic solvent, such as n-hexadecane, greatly enhanced the MPSO production due to the prevention of substrate evaporation, resulting in a MPSO production up to 39 mM in 30 h with a conversion rate of 95.7 mol%.

Keywords: Pseudomonas putida ; High cell density cultivation; Naphthalene dioxygenase; Sulfoxide

Characterization of rhamnolipids produced by wild-type and engineered Burkholderia kururiensis by Luiz F. D. Tavares; Patrícia M. Silva; Magno Junqueira; Danielly C. O. Mariano; Fábio C. S. Nogueira; Gilberto B. Domont; Denise M. G. Freire; Bianca C. Neves (pp. 1909-1921).

Biosurfactants are a class of functional molecules produced and secreted by microorganisms, which play important roles in cell physiology such as flagellum-dependent or -independent bacterial spreading, cell signaling, and biofilm formation. They are amphipathic compounds and comprise a variety of chemical structures, including rhamnolipids, typically produced by Pseudomonas spp. and also reported within other bacterial genera. The present study is focused on Burkholderia kururiensis KP23^T, a trichloroethylene (TCE)-degrading, N-fixing, and plant growth-promoting bacterium. Herein, we describe the production of rhamnolipids by B. kururiensis, and its characterization by LTQ-Orbitrap Hybrid Mass Spectrometry, a powerful tool that allowed efficient identification of molecular subpopulations, due to its high selectivity, mass accuracy, and resolving power. The population of rhamnolipids produced by B. kururiensis revealed molecular species commonly observed in Pseudomonas spp. and/or Burkholderia spp. In addition, this strain was used as a platform for expression of two Pseudomonas aeruginosa biosynthetic enzymes: RhlA, which directly utilizes β-hydroxydecanoyl-ACP intermediates in fatty acid synthesis to generate the HAA, and RhlB, the rhamnosyltransferase 1, which catalyzes the transfer of dTDP-L-rhamnose to β-hydroxy fatty acids in the biosynthesis of rhamnolipids. We show that rhamnolipid production by the engineered B. kururiensis was increased over 600 % when compared to the wild type. Structural analyses demonstrated a molecular population composed mainly of monorhamnolipids, as opposed to wild-type B. kururiensis and P. aeruginosa in which dirhamnolipids are predominant. We conclude that B. kururiensis is a promising biosurfactant-producing organism, with great potential for environmental and biotechnological applications due to its non-pathogenic characteristics and efficiency as a platform for metabolic engineering and production of tailor-made biosurfactants.

Keywords: Burkholderia kururiensis ; Pseudomonas aeruginosa ; Biosurfactants; Rhamnolipids; Heterologous expression; LTQ Orbitrap Hybrid Mass Spectrometer

Integrated process of starch ethanol and cellulosic lactic acid for ethanol and lactic acid production by Yong Tang; Liwei Zhu; Weiming Zhang; Xinhui Shang; Jianxin Jiang (pp. 1923-1932).

The sequential production of bioethanol and lactic acid from starch materials and lignocellulosic materials was investigated as ethanol fermentation broth (EFB) can provide nutrients for lactic acid bacteria. A complete process was developed, and all major operations are discussed, including ethanol fermentation, broth treatment, lactic acid fermentation, and product separation. The effect of process parameters, including ethanol fermentation conditions, treatment methods, and the amount of EFB used in simultaneous saccharification and fermentation (SSF), is investigated. Under the selected process conditions, the integrated process without additional chemical consumption provides a 1.08 acid/alcohol ratio (the broth containing 22.4 g/L ethanol and 47.6 g/L lactic acid), which corresponds to a polysaccharide utilization ratio of 86.9 %. Starch ethanol can thus promote cellulosic lactic acid by providing important nutrients for lactic acid bacteria, and in turn, cellulosic lactic acid could promote starch ethanol by improving the profit of the ethanol production process. Two process alternatives for the integration of starch ethanol and cellulosic lactic acid are compared, and some suggestions are given regarding the reuse of yeast following the cellulosic SSF step for lactic acid production.

Keywords: Ethanol; Lactic acid; Simultaneous saccharification and fermentation; Yeast; Integrated process

Overexpression of acetyl-CoA synthetase increased the biomass and fatty acid proportion in microalga Schizochytrium by Jinfei Yan; Rubin Cheng; Xiangzhi Lin; Song You; Ke Li; Hui Rong; Yong Ma (pp. 1933-1939).

High acetate accumulation was produced during glucose fermentation in high cell density cultures, which is harmful to cell growth. In order to reduce the negative impact of acetate accumulation on the fermentation products, we introduced the Escherichia coli acetyl-CoA synthetase (ACS) gene into the marine microalga Schizochytrium sp. TIO1101, generating genetically modified ACS transformants. The results of PCR and blotting analyses showed that the exogenous ACS gene was incorporated into the genome and successfully expressed. The engineered Schizochytrium increased the pH value and reduced the acetate concentration in the final fermentation medium significantly. Furthermore, the ACS transformants exhibited faster growth and glucose consumption rates than the wild-type strain. The biomass and fatty acid proportion of ACS transformants increased by 29.9 and 11.3 %, respectively. Taken together, the data suggest that ACS overexpression in Schizochytrium might improve the utilization of carbon resource and decrease the production of acetate byproduct. These results demonstrate that application of ACS in metabolic genetic engineering could improve the properties of Schizochytrium significantly.

Keywords: Acetate; Acetyl-CoA synthetase; Schizochytrium ; Transgene; 18S rDNA

Characterization of a S-layer protein from Lactobacillus crispatus K313 and the domains responsible for binding to cell wall and adherence to collagen by Zhilan Sun; Jian Kong; Shumin Hu; Wentao Kong; Wenwei Lu; Wei Liu (pp. 1941-1952).

It was previously shown that the surface (S)-layer proteins covering the cell surface of Lactobacillus crispatus K313 were involved in the adherence of this strain to human intestinal cell line HT-29. To further elucidate the structures and functions of S-layers, three putative S-layer protein genes (slpA, slpB, and slpC) of L. crispatus K313 were amplified by PCR, sequenced, and characterized in detail. Quantitative real-time PCR analysis reveals that slpA was silent under the tested conditions; whereas slpB and slpC, the putative amino acid sequences which exhibited minor similarities to the previously reported S-layer proteins in L. crispatus, were actively expressed. slpB, which was predominantly expressed in L. crispatus K313, was further investigated for its functional domains. Genetic truncation of the untranslated leader sequence (UTLS) of slpB results in a reduction in protein production, indicating that the UTLS contributed to the efficient S-layer protein expression. By producing a set of N- and C-terminally truncated recombinant SlpB proteins in Escherichia coli, the cell wall-binding region was mapped to the C terminus, where rSlpB_380–501 was sufficient for binding to isolated cell wall fragments. Moreover, the binding ability of the C terminus was variable among the Lactobacillus species (S-layer- and non-S-layer-producing strains), and teichoic acid may be acting as the receptor of SlpB. To determine the adhesion region of SlpB to extracellular matrix proteins, ELISA was performed. Binding to immobilized types I and IV collagen was observed with the His-SlpB_1–379 peptides, suggesting that the extracellular matrix protein-binding domain was located in the N terminus.

Keywords: Lactobacillus crispatus ; S-layer·cell wall fragments; Extracellular matrix protein; Enzyme-linked immunosorbent assay

Changes in tyrosinase specificity by ionic liquids and sodium dodecyl sulfate by Mor Goldfeder; Mor Egozy; Vered Shuster Ben-Yosef; Noam Adir; Ayelet Fishman (pp. 1953-1961).

Tyrosinase is a member of the type 3 copper enzyme family involved in the production of melanin in a wide range of organisms. The ability of tyrosinases to convert monophenols into diphenols has stimulated studies regarding the production of substituted catechols, important intermediates for the synthesis of pharmaceuticals, agrochemicals, polymerization inhibitors, and antioxidants. Despite its enormous potential, the use of tyrosinases for catechol synthesis has been limited due to the low monophenolase/diphenolase activity ratio. In the presence of two water miscible ionic liquids, [BMIM][BF₄] and ethylammonium nitrate, the selectivity of a tyrosinase from Bacillus megaterium (TyrBm) was altered, and the ratio of monophenolase/diphenolase activity increased by up to 5-fold. Furthermore, the addition of sodium dodecyl sulphate (SDS) at levels of 2–50 mM increased the activity of TyrBm by 2-fold towards the natural substrates l-tyrosine and l-Dopa and 15- to 20-fold towards the non-native phenol and catechol. The R209H tyrosinase variant we previously identified as having a preferential ratio of monophenolase/diphenolase activity was shown to have a 45-fold increase in activity towards phenol in the presence of SDS. We propose that the effect of SDS on the ability of tyrosinase to convert non-natural substrates is due to the interaction of surfactant molecules with residues located at the entrance to the active site, as visualized by the newly determined crystal structure of TyrBm in the presence of SDS. The effect of SDS on R209 may enable less polar substrates such as phenol and catechol, to penetrate more efficiently into the enzyme catalytic pocket.

Keywords: Tyrosinase; Bacillus megaterium ; Ionic liquids; Sodium dodecyl sulphate; Diphenols

Exploring the allosteric mechanism of dihydrodipicolinate synthase by reverse engineering of the allosteric inhibitor binding sites and its application for lysine production by Feng Geng; Zhen Chen; Ping Zheng; Jibin Sun; An-Ping Zeng (pp. 1963-1971).

Dihydrodipicolinate synthase (DHDPS, EC 4.2.1.52) catalyzes the first committed reaction of l-lysine biosynthesis in bacteria and plants and is allosterically regulated by l-lysine. In previous studies, DHDPSs from different species were proved to have different sensitivity to l-lysine inhibition. In this study, we investigated the key determinants of feedback regulation between two industrially important DHDPSs, the l-lysine-sensitive DHDPS from Escherichia coli and l-lysine-insensitive DHDPS from Corynebacterium glutamicum, by sequence and structure comparisons and site-directed mutation. Feedback inhibition of E. coli DHDPS was successfully alleviated after substitution of the residues around the inhibitor’s binding sites with those of C. glutamicum DHDPS. Interestingly, mutagenesis of the lysine binding sites of C. glutamicum DHDPS according to E. coli DHDPS did not recover the expected feedback inhibition but an activation of DHDPS by l-lysine, probably due to differences in the allosteic signal transduction in the DHDPS of these two organisms. Overexpression of l-lysine-insensitive E. coli DHDPS mutants in E. coli MG1655 resulted in an improvement of l-lysine production yield by 46 %.

Keywords: Dihydrodipicolinate synthase (DHDPS); Allosteric binding site; Escherichia coli ; Corynebacterium glutamicum ; l-Lysine overproduction

Identification and characterization of new Δ-17 fatty acid desaturases by Zhixiong Xue; Hongxian He; Dieter Hollerbach; Daniel J. Macool; Narendra S. Yadav; Hongxiang Zhang; Bogdan Szostek; Quinn Zhu (pp. 1973-1985).

ω-3 fatty acid desaturase is a key enzyme for the biosynthesis of ω-3 polyunsaturated fatty acids via the oxidative desaturase/elongase pathways. Here we report the identification of three ω-3 desaturases from oomycetes, Pythium aphanidermatum, Phytophthora sojae, and Phytophthora ramorum. These new ω-3 desaturases share 55 % identity at the amino acid level with the known Δ-17 desaturase of Saprolegnia diclina, and about 31 % identity with the bifunctional Δ-12/Δ-15 desaturase of Fusarium monoliforme. The three enzymes were expressed in either wild-type or codon optimized form in an engineered arachidonic acid producing strain of Yarrowia lipolytica to study their activity and substrate specificity. All three were able to convert the ω-6 arachidonic acid to the ω-3 eicosapentanoic acid, with a substrate conversion efficiency of 54–65 %. These enzymes have a broad ω-6 fatty acid substrate spectrum, including both C18 and C20 ω-6 fatty acids although they prefer the C20 substrates, and have strong Δ-17 desaturase activity but weaker Δ-15 desaturase activity. Thus, they belong to the Δ-17 desaturase class. Unlike the previously identified bifunctional Δ-12/Δ-15 desaturase from F. monoliforme, they lack Δ-12 desaturase activity. The newly identified Δ-17 desaturases could use fatty acids in both acyl-CoA and phospholipid fraction as substrates. The identification of these Δ-17 desaturases provides a set of powerful new tools for genetic engineering of microbes and plants to produce ω-3 fatty acids, such as eicosapentanoic acid and docosahexanoic acid, at high levels.

Keywords: ω-3 Desaturase; Δ-17 Desaturase; Long-chain polyunsaturated fatty acids; ω-3 Fatty acids; Yarrowia lipolytica ; Metabolic engineering

Heterologous expression of the C-terminal antigenic domain of the malaria vaccine candidate Pfs48/45 in the green algae Chlamydomonas reinhardtii by Carla S. Jones; Tiffany Luong; Michael Hannon; Miller Tran; James A. Gregory; Zhouxin Shen; Steven P. Briggs; Stephen P. Mayfield (pp. 1987-1995).

Malaria is a widespread and infectious disease that is a leading cause of death in many parts of the world. Eradication of malaria has been a major world health goal for decades, but one that still remains elusive. Other diseases have been eradicated using vaccination, but traditional vaccination methods have thus far been unsuccessful for malaria. Infection by Plasmodium species, the causative agent of malaria, is currently treated with drug-based therapies, but an increase in drug resistance has led to the need for new methods of treatment. A promising strategy for malaria treatment is to combine transmission blocking vaccines (TBVs) that prevent spread of disease with drug-based therapies to treat infected individuals. TBVs can be developed against surface protein antigens that are expressed during parasite reproduction in the mosquito. When the mosquito ingests blood from a vaccinated individual harboring the Plasmodium parasite, the antibodies generated by vaccination prevent completion of the parasites life-cycle. Animal studies have shown that immunization with Pfs48/45 results in the production of malaria transmission blocking antibodies; however, the development of this vaccine candidate has been hindered by poor expression in both prokaryotic and eukaryotic hosts. Recently, the chloroplast of Chlamydomonas reinhardtii has been used to express complex recombinant proteins. In this study, we show that the C-terminal antigenic region of the Pfs48/45 antigen can be expressed in the chloroplast of the green algae C. reinhardtii and that this recombinant protein has a conformation recognized by known transmission blocking antibodies. Production of this protein in algae has the potential to scale to the very large volumes required to meet the needs of millions at risk for contracting malaria.

Keywords: Chlamydomonas reinhardtii ; Recombinant protein; Pfs48/45; Malaria vaccine; Transmission blocking vaccine

Removal of pathogenic factors from 2,3-butanediol-producing Klebsiella species by inactivating virulence-related wabG gene by Sung-Geun Jung; Jun-Ho Jang; Ah-Young Kim; Min-Cheol Lim; Borim Kim; Jinwon Lee; Young-Rok Kim (pp. 1997-2007).

Klebsiella species are the most extensively studied among a number of 2,3-butanediol (2,3-BDO)-producing microorganisms. The ability to metabolize a wide variety of substrates together with the ease of cultivation made this microorganisms particularly promising for the application in industrial-scale production of 2,3-BDO. However, the pathogenic characteristics of encapsulated Klebsiella species are considered to be an obstacle hindering their industrial applications. Here, we removed the virulence factors from three 2,3-BDO-producing strains, Klebsiella pneumoniae KCTC 2242, Klebsiella oxytoca KCTC1686, and K. oxytoca ATCC 43863 through site-specific recombination technique. We generated deletion mutation in wabG gene encoding glucosyltransferase which plays a key role in the synthesis of outer core lipopolysaccharides (LPS) by attaching the first outer core residue d-GalAp to the O-3 position of the l,d-HeppII residue. The morphologies and adhesion properties against epithelial cells were investigated, and the results indicated that the wabG mutant strains were devoid of the outer core LPS and lost the ability to retain capsular structure. The time profile of growth and 2,3-BDO production from K. pneumoniae KCTC 2242 and K. pneumoniae KCTC 2242 ΔwabG were analyzed in batch culture with initial glucose concentration of 70 g/l. The growth was not affected by disrupting wabG gene, but the production of 2,3-BDO decreased from 31.27 to 22.44 g/l in mutant compared with that of parental strain. However, the productions of acetoin and lactate from wabG mutant strain were negligible, whereas that from parental strain reached to ~5 g/l.

Keywords: Klebsiella pneumoniae ; Klebsiella oxytoca ; 2,3-Butanediol; Virulence; wabG ; Lipopolysaccharides; Capsule

Increasing oxidative stress tolerance and subculturing stability of Cordyceps militaris by overexpression of a glutathione peroxidase gene by Chenghui Xiong; Yongliang Xia; Peng Zheng; Chengshu Wang (pp. 2009-2015).

Like other filamentous fungi, the medicinal ascomycete Cordyceps militaris frequently degenerates during continuous maintenance in culture by showing loss of the ability to reproduce sexually or asexually. Degeneration of fungal cultures has been related with cellular accumulation of reactive oxygen species (ROS). In this study, an antioxidant glutathione peroxidase (Gpx) gene from Aspergillus nidulans was engineered into two C. militaris strains, i.e., the Cm01 strain which can fruit normally and the Cm04 strain which has lost the ability to form fruiting bodies on different media through subculturing. The results showed that the mitotically stable mutants had higher Gpx activities and stronger capacity to scavenge cellular ROS than their parental strains. Most significantly, the fruiting ability of Cm04 strain was restored by overexpression of the antioxidant enzyme. However, after being successively transferred for up to ten generations, two of three Cm04 mutants again lost the ability to fruit on insect pupae while Cm01 transformants remained fertile. This study confirms the relationship between fungal culture degeneration and cellular ROS accumulation. Our results indicate that genetic engineering with an antioxidant gene can be an effective way to reverse fungal degeneration during subculturing.

Keywords: Cordyceps militaris ; Culture degeneration; Oxidative stress; Glutathione peroxidase; Antioxidation; Fruiting body

A novel transcriptional regulator, ClbR, controls the cellobiose- and cellulose-responsive induction of cellulase and xylanase genes regulated by two distinct signaling pathways in Aspergillus aculeatus by Emi Kunitake; Shuji Tani; Jun-ichi Sumitani; Takashi Kawaguchi (pp. 2017-2028).

The cellobiose- and cellulose-responsive induction of the FIII-avicelase (cbhI), FII-carboxymethyl cellulase (cmc2), and FIa-xylanase (xynIa) genes is not regulated by XlnR in Aspergillus aculeatus, which suggests that this fungus possesses an unknown cellulase gene-activating pathway. To identify the regulatory factors involved in this pathway, we constructed a random insertional mutagenesis library using Agrobacterium tumefaciens-mediated transformation of A. aculeatus NCP2, which harbors a transcriptional fusion between the cbhI promoter (P CBHI) and the orotidine 5′-phosphate decarboxylase gene (pyrG). Of the ~6,000 transformants screened, one 5-FOA-resistant transformant, S4-22, grew poorly on cellulose-containing media and exhibited reduced cellobiose-induced expression of cbhI. Southern blot analysis and nucleotide sequencing of the flanking regions of the T-DNA inserted in S4-22 indicated that the T-DNA was inserted within the coding region of a previously unreported Zn(II)₂Cys₆-transcription factor, which we designated the cellobiose response regulator (ClbR). The disruption of the clbR gene resulted in a significant reduction in the expression of cbhI and cmc2 in response to cellobiose and cellulose. Interestingly, the cellulose-responsive induction of FI-carboxymethyl cellulase (cmc1) and FIb-xylanase (xynIb) genes that are under the control of XlnR, was also reduced in the clbR-deficient mutant, but there was no effect on the induction of these genes in response to d-xylose or l-arabinose. These data demonstrate that ClbR participates in both XlnR-dependent and XlnR-independent cellobiose- and cellulose-responsive induction signaling pathways in A. aculeatus.

Keywords: XlnR-independent signaling pathway; XlnR-dependent signaling pathway; Cellulase induction; Xylanase induction; Cellobiose response regulator ClbR; Aspergillus aculeatus

Characterization of Saccharomyces cerevisiae promoters for heterologous gene expression in Kluyveromyces marxianus by Ki-Sung Lee; Jun-Seob Kim; Paul Heo; Tae-Jun Yang; Young-Je Sung; Yuna Cheon; Hyun Min Koo; Byung Jo Yu; Jin-Ho Seo; Yong-Su Jin; Jae Chan Park; Dae-Hyuk Kweon (pp. 2029-2041).

Kluyveromyces marxianus is now considered one of the best choices of option for industrial applications of yeast because the strain is able to grow at high temperature, utilizes various carbon sources, and grows fast. However, the use of K. marxianus as a host for industrial applications is still limited. This limitation is largely due to a lack of knowledge on the characteristics of the promoters since the time and amount of protein expression is strongly dependent on the promoter employed. In this study, four well-known constitutive promoters (P CYC, P TEF, P GPD, and P ADH) of Saccharomyces cerevisiae were characterized in K. marxianus in terms of protein expression level and their stochastic behavior. After constructing five URA3-auxotrophic K. marxianus strains and a plasmid vector, four cassettes each comprising one of the promoters—the gene for the green fluorescence protein (GFP)—CYC1 terminator (T CYC) were inserted into the vector. GFP expression under the control of each one of the promoters was analyzed by reverse transcription PCR, fluorescence microscopy, and flow cytometer. Using these combined methods, the promoter strength was determined to be in the order of P GPD > P ADH ∼ P TEF >> P CYC. All promoters except for the P CYC exhibited three distinctive populations, including non-expressing cells, weakly expressing cells, and strongly expressing cells. The relative ratios between populations were strongly dependent on the promoter and culture time. Forward scattering was independent of GFP fluorescence intensity, indicating that the different fluorescence intensities were not just due to different cell sizes derived from budding. It also excluded the possibility that the non-expressing cells resulted from plasmid loss because plasmid stability was maintained at almost 100 % over the culture time. The same cassettes, cloned into a single copy plasmid pRS416 and transformed into S. cerevisiae, showed only one population. When the cassettes were integrated into the chromosome, the stochastic behavior was markedly reduced. These combined results imply that the gene expression stochasticity should be overcome in order to use this strain for delicate metabolic engineering, which would require the co-expression of several genes.

Keywords: Kluyveromyces marxianus ; Promoter; Strength; Stochasticity; Flow cytometer

A non-cyclic baboon θ-defensin derivative exhibiting antimicrobial activity against the phytopathogen Verticillium dahliae by Mi Ni; Yijing Zhao; Noreen Bibi; Mingyan Shao; Shuna Yuan; Kai Fan; Gaixia Zhang; Feng Li; Xuede Wang (pp. 2043-2052).

θ-Defensins are the only natural cyclic proteins found in primates. They have strong antimicrobial activity related to their trisulfide ladders and macrocyclic conformation. A non-cyclic baboon θ-defensin (BTD) was synthesized by substituting valine with phenylalanine at position 17, at the C-terminal end of the BTD; this was termed “BTD-S.” The antimicrobial activities of this synthetic peptide were investigated against Escherichia coli and two cotton phytopathogens: Verticillium dahliae and Fusarium oxysporum. The minimum inhibitory concentration (MIC) of BTD-S for E. coli was 10 μg/mL and for V. dahliae was 5 μg/mL, significantly lower than that for F. oxysporum (40.0 μg/mL). A time course analysis of fungal cultures indicated that the growth of V. dahliae was completely inhibited after 96 h of BTD-S treatment. Furthermore, hemolysis assays revealed that BTD-S was not toxic to mammalian cells as it could not induce lysis of sheep red blood cells even at ten times the MIC (50 μg/mL). Scanning electron microscopy and double-stained (calcofluor white and propidium iodide binding) fluorescence microscopy showed that exposure of spores of V. dahliae to BTD-S either disabled normal germination or disintegrated the spores. The size of cells exposed to BTD-S was significantly reduced compared with controls, and their number increased in a dose-dependent curve when measured by flow cytometry. These findings suggest that BTD-S has great potential to inhibit the growth of V. dahliae and can be utilized as an effective remedy to control economic losses caused by Verticillium wilt in the development of wilt-resistant cotton.

Keywords: θ-Defensin; Antifungal property; Verticillium dahliae ; Membrane permeabilization; Germination

The impact of Fusarium culmorum infection on the protein fractions of raw barley and malted grains by Pedro M. Oliveira; Deborah M. Waters; Elke K. Arendt (pp. 2053-2065).

Contaminating fungi, such as Fusarium species, produce metabolites that may interfere with normal barley grain proteolysis pattern and consequently, affect malt and beer quality. Protein compositional changes of an initial mixture of 20 % Fusarium culmorum infected and 80 % noninfected mature barley grains and respective malt are reported here. Proteolytic activity of infected barley grains (IBG) and respective malt, with controls (uninfected grains), were characterized using protease inhibitors from each class of this enzyme, including metallo-, cysteine, serine, and aspartic proteases, as well as uninhibited protease fractions. The proteins were extracted according to the Osborne fractionation and separated by size exclusion chromatography. Additionally, two-dimensional (2D) gel electrophoresis (GE) was used to analyze hydrophobic storage proteins isolated from the control and IBG. Analyses revealed that F. culmorum IBG had a twofold increase of proteolytic activity compared to the control sample, which showed an increase in all protease classes with aspartic proteases dominating. Infected and control malt grains were comparable with cysteine proteases representing almost 50 % of all proteolytic enzymes detected. Protein extractability was 31 % higher in IBG compared to the control barley. The albumin fraction showed that several metabolic proteins decreased and increased at different rates during infection and malting, thus showing a complex F. culmorum infection interdependence. Prolamin storage proteins were more hydrophobic during barley fungal infection. F. culmorum interfered with the grain hydrolytic protein profile, thereby altering the grain's protein content and quality.

Keywords: Fusarium culmorum ; Osborne protein fractions; Protease activity; Capillary electrophoresis; Infected barley; Malt

Comparative functional genomics to reveal the molecular basis of phenotypic diversities and guide the genetic breeding of industrial yeast strains by Dao-Qiong Zheng; Tian-Zhe Liu; Jie Chen; Ke Zhang; Ou Li; Liang Zhu; Yu-Hua Zhao; Xue-Chang Wu; Pin-Mei Wang (pp. 2067-2076).

An understanding of the genetic basis underlying the phenotypic variations of yeast strains would guide the breeding of this useful microorganism. Here, comparative functional genomics (CFG) of two bioethanol Saccharomyces cerevisiae strains (YJS329 and ZK2) with different stress tolerances and ethanol fermentation performances were performed. Our analysis indicated that different patterns of gene expression in the central carbon metabolism, antioxidative factors, and membrane compositions of these two strains are the main contributors to their various traits. Some of the differently expressed genes were directly caused by the genomic structural variations between YJS329 and ZK2. Moreover, CFG of these two strains also led to novel insights into the mechanism of stress tolerance in yeast. For example, it was found that more oleic acid in the plasma membrane contributes to the acetic acid tolerance of yeast. Based on the genetic information particular to each strain, strategies to improve their adaptability and ethanol fermentation performances were designed and confirmed. Thus, CFG could not only help reveal basis of phenotypic diversities but also guide the genetic breeding of industrial microorganisms.

Keywords: Saccharomyces cerevisiae ; Bioethanol; Stress; RNA-Seq; Genetic breeding

Immunoproteomic identification of immunogenic proteins in Cronobacter sakazakii strain BAA-894 by Jian Wang; Xin-Jun Du; Xiao-Nan Lu; Shuo Wang (pp. 2077-2091).

Cronobacter spp. are emerging opportunistic pathogens. Cronobacter sakazakii is considered as the predominant species in all infections. So far, our understanding of the species’ immunogens and potential virulence factors of Cronobacter spp. remains limited. In this study, an immunoproteomic approach was used to investigate soluble and insoluble proteins from the genome-sequenced strain C. sakazakii ATCC BAA-894. Proteins were separated using two-dimensional electrophoresis, detected by Western blotting with polyclonal antibodies of C. sakazakii BAA-894, and identified using tandem mass spectrometry (MALDI-MS and MALDI-MS/MS, MS/MSMS). A total of 11 immunoreactive proteins were initially identified in C. sakazakii BAA-894, including two outer membrane proteins, four periplasmic proteins, and five cytoplasmic proteins. In silico functional analysis of the 11 identified proteins indicated three proteins that were initially described as immunogens of pathogenic bacteria. For the remaining eight proteins, one protein was categorized as a potential virulence factor involved in protection against reactive oxygen species, and seven proteins were considered to play potential roles in adhesion, invasion, and biofilm formation. To our knowledge, this is the first time that immunogenic proteins of C. sakazakii BAA-894 have been identified as immunogens and potential virulence factors by an immunoproteomics approach. Future studies should investigate the roles of these proteins in bacterial pathogenesis and modulation of host immune responses during infection to identify their potential as molecular therapeutic targets.

Keywords: C. sakazakii BAA-894; MS/MSMS; Immunoproteomics; Immunogen; Virulence factor

High hydrostatic pressure activates gene expression that leads to ethanol production enhancement in a Saccharomyces cerevisiae distillery strain by Fernanda Bravim; Soyeon I. Lippman; Lucas F. da Silva; Diego T. Souza; A. Alberto R. Fernandes; Claudio A. Masuda; James R. Broach; Patricia M. B. Fernandes (pp. 2093-2107).

High hydrostatic pressure (HHP) is a stress that exerts broad effects on microorganisms with characteristics similar to those of common environmental stresses. In this study, we aimed to identify genetic mechanisms that can enhance alcoholic fermentation of wild Saccharomyces cerevisiae isolated from Brazilian spirit fermentation vats. Accordingly, we performed a time course microarray analysis on a S. cerevisiae strain submitted to mild sublethal pressure treatment of 50 MPa for 30 min at room temperature, followed by incubation for 5, 10 and 15 min without pressure treatment. The obtained transcriptional profiles demonstrate the importance of post-pressurisation period on the activation of several genes related to cell recovery and stress tolerance. Based on these results, we over-expressed genes strongly induced by HHP in the same wild yeast strain and identified genes, particularly SYM1, whose over-expression results in enhanced ethanol production and stress tolerance upon fermentation. The present study validates the use of HHP as a biotechnological tool for the fermentative industries.

Keywords: Saccharomyces cerevisiae ; High hydrostatic pressure; Fermentative process; Ethanol production; Microarray; Stress response

Effects of Lactobacillus salivarius, Lactobacillus reuteri, and Pediococcus acidilactici on the nematode Caenorhabditis elegans include possible antitumor activity by Michael K. Fasseas; Costas Fasseas; Konstantinos C. Mountzouris; Popi Syntichaki (pp. 2109-2118).

This study examined the effects of three lactic acid bacteria (LAB) strains on the nematode Caenorhabditis elegans. Lactobacillus salivarius, Lactobacillus reuteri, and Pediococcus acidilactici were found to inhibit the development and growth of the worm. Compared to Escherichia coli used as the control, L. reuteri and P. acidilactici reduced the lifespan of wild-type and short-lived daf-16 worms. On the contrary, L. salivarius extended the lifespan of daf-16 worms when used live, but reduced it as UV-killed bacteria. The three LAB induced the expression of genes involved in pathogen response and inhibited the growth of tumor-like germ cells, without affecting DAF16 localization or increasing corpse cells. Our results suggest the possible use of C. elegans as a model for studying the antitumor attributes of LAB. The negative effects of these LAB strains on the nematode also indicate their potential use against parasitic nematodes.

Keywords: Lactic acid bacteria; Probiotic; C. elegans ; Antitumor; Immune response

The atf2 gene is involved in triacylglycerol biosynthesis and accumulation in the oleaginous Rhodococcus opacus PD630 by Martín A. Hernández; Ana Arabolaza; Eduardo Rodríguez; Hugo Gramajo; Héctor M. Alvarez (pp. 2119-2130).

Rhodococcus opacus PD630 is an oleaginous bacterium able to accumulate large amounts of triacylglycerols (TAG) in different carbon sources. The last reaction for TAG biosynthesis is catalyzed by the bifunctional wax ester synthase/acyl-CoA:diacylglycerol acyltransferase (WS/DGAT) enzymes encoded by atf genes. R. opacus PD630 possesses at least 17 putative atf homologous genes in its genome, but only atf1 and atf2 exhibited a significant DGAT activity when expressed in E. coli, as revealed in a previous study. The contribution of atf1 gene to TAG accumulation by strain PD630 has been demonstrated previously, although additional Atfs may also contribute to lipid accumulation, since the atf1-disrupted mutant is still able to produce significant amounts of TAG (Alvarez et al., Microbiology 154:2327–2335, 2008). In this study, we investigated the in vivo role of atf2 gene in TAG accumulation by R. opacus PD630 by using different genetic strategies. The atf2-disrupted mutant exhibited a decrease in TAG accumulation (up to 25–30 %, w/w) and an approximately tenfold increase in glycogen formation in comparison with the wild-type strain. Surprisingly, in contrast to single mutants, a double mutant generated by the disruption of atf1 and atf2 genes only showed a very low effect in TAG and in glycogen accumulation under lipid storage conditions. Overexpression of atf1 and atf2 genes in strain PD630 promoted an increase of approximately 10 % (w/w) in TAG accumulation, while heterologous expression of atf2 gene in Mycobacterium smegmatis caused an increase in TAG accumulation during cultivation in nitrogen-rich media. This study demonstrated that, in addition to atf1 gene, atf2 is actively involved in TAG accumulation by the oleaginous R. opacus PD630.

Keywords: Rhodococcus ; Triacylglycerols; atf ; DGAT

Distinct and effective biotransformation of hexavalent chromium by a novel isolate under aerobic growth followed by facultative anaerobic incubation by Shimei Ge; Maohong Zhou; Xinjiao Dong; Yang Lu; Shichao Ge (pp. 2131-2137).

A bacterial isolate (G161) with high Cr(VI)-reducing capacity was isolated from Cr(VI)-contaminated soil and identified as Leucobacter sp. on the basis of 16S rRNA gene sequence analysis. The isolate was a Gram-positive, aerobic rod. The hexavalent chromate-reducing capability of the isolate was investigated under three conditions of oxygen stress. The isolate was found to reduce Cr(VI) under all conditions but performed most effectively during aerobic growth followed by facultative anaerobic incubation. Under these conditions, the isolate tolerated K₂Cr₂O₇ concentrations up to 1,000 mg/l and completely reduced 400 mg/l K₂Cr₂O₇ within 96 h. The strain reduced Cr(VI) over a wide range of pH (6.0–11.0) and temperatures (15–45 °C) with optimum performance at pH 8.0 and 35 °C. The presence of other metals, such as Ca²⁺, Co²⁺, Cu²⁺, Mn²⁺, Ni²⁺, and Zn²⁺, induced no effect or else played a stimulatory role on Cr(VI)-reduction activity of the strain. The strain was tested for Cr(VI) removal in wastewaters and proved capable of completely reducing the contained Cr(VI). This is the novel report of a bacterial growth and Cr(VI)-reduction process under sequential aerobic growth and facultative anaerobic conditions. The study suggested that the isolate possesses a distinct capability for Cr(VI) reduction which could be harnessed for the detoxification of chromate-contaminated wastewaters.

Keywords: Leucobacter sp G161; 16S rRNA; Cr(VI) pollution; Aerobic growth; Facultative anaerobic Cr(VI) reduction

Taxonomy and chemically semi-defined media for the analysis of the tacrolimus producer ‘Streptomyces tsukubaensis’ by Miriam Martínez-Castro; Zahra Salehi-Najafabadi; Francisco Romero; Rodrigo Pérez-Sanchiz; Rosa Isabel Fernández-Chimeno; Juan Francisco Martín; Carlos Barreiro (pp. 2139-2152).

‘Streptomyces tsukubaensis’ was the first tacrolimus producer strain identified. Although it has been included in the Streptomyces genus, its taxonomic position has not been rigorously determined. By using a polyphasic approach, we have established that the tacrolimus producer strain ‘S. tsukubaensis’ NRRL 18488 represents a unique species in the Streptomyces genus, which is phylogenetically distant from other subsequently described producers. This fact means a horizontal transference of the tacrolimus-producing gene cluster. Physiology, nutrient requirement, and molecular genetics analyses of tacrolimus biosynthesis in ‘S. tsukubaensis’ necessitate chemically defined or semi-defined media, which work as a jigsaw puzzle and allow for pieces (nutrients) exchange. To date, studies related to ‘S. tsukubaensis’ have been mainly focused in the improvement of tacrolimus production using complex industrial fermentation media, which difficulty allows testing of tacrolimus overproduction enhancers or inhibitors because of the presence of non‐defined substances. In the present work, two semi-defined media were developed in order to study the main factors involved in tacrolimus production in ‘S. tsukubaensis’.

Keywords: ‘Streptomyces tsukubaensis’ ; Tacrolimus; FK506; Taxonomy; Fermentation media

Use of propidium monoazide and increased amplicon length reduce false-positive signals in quantitative PCR for bioburden analysis by Franz Schnetzinger; Youwen Pan; Andreas Nocker (pp. 2153-2162).

Rapid microbiological methods (RMMs) as an alternative to conventional cultivation-based bioburden analysis are receiving increasing attention although no single technology is currently able to satisfy the needs of the health care industry. Among the RMMs, quantitative PCR (qPCR) seems particularly suited. Its implementation is, however, hampered by false-positive signals originating from free DNA in PCR reagents or from dead cells in the samples to be analysed. In this study, we assessed the capability of propidium monoazide (PMA) to inactivate exogenous DNA in PCR reagents and thus to minimise its impact in bioburden analysis. PMA is a membrane-impermeant dye that intercalates into DNA and covalently binds to it upon photoactivation leading to strong inhibition of PCR amplification. PMA is currently used mainly for treatment of microbiological samples to exclude signals from membrane-compromised cells, but is also very useful for suppression of exogenous DNA signals. In addition to testing the effect of different PMA concentrations on non-template controls and target DNA, we demonstrate the effect of amplicon length on the exclusion of background amplification. Targeting a 1,108-bp 16S rRNA gene fragment using universal bacterial primers and PCR reagents treated with 5 μM PMA resulted in complete suppression of signals from exogenous DNA within 50 cycles of amplification, while a limit of detection of 10 copies of Escherichia coli genomic DNA per PCR reaction was achieved. A combined PMA treatment of sample and PCR reagents furthermore improved the selective detection of live cells making this method appear a highly attractive RMM.

Keywords: Propidium monoazide; False-positive signals; Real-time PCR; Bioburden analysis; Rapid microbiological method

An integrated high-throughput strategy for rapid screening of poly(γ-glutamic acid)-producing bacteria by Wei Zeng; Yuanshan Lin; Zongxian Qi; Yangyang He; Dayun Wang; Guiguang Chen; Zhiqun Liang (pp. 2163-2172).

Poly(γ-glutamic acid) (γ-PGA) is a promising biomaterial with a wide range of unique applications. To extensively screen γ-PGA-producing bacteria with high yield and different molecular weight, we developed an integrated high-throughput strategy. Firstly, γ-PGA-producing bacteria were selected in a primary screen plate containing a basic dye (neutral red) based on the concentric zone formed through the electrostatic interaction between the dye and the secreted acidic polymer γ-PGA. Then, the isolates were cultured in 50 ml tubes instead of 250 ml flasks. A good correlation of fermentation results in 50 ml tubes and 250 ml flasks was observed. Thirdly, the γ-PGA yield and weight-average molecular weight (M _w) were simultaneously determined by spectrophotomic assay (UV assay) and neutral red plate assay. The results showed that the diameter of the concentric zone varied among isolates and was negatively correlated with the weight-average molecular weight of γ-PGA. The accuracy of the methods was comparable to that of high-performance liquid chromatography and gel permeation chromatography assay. Lastly, γ-PGA obtained from the target isolates was rapidly identified using thin layer chromatography assay. With this strategy, 13 bacteria with high yield and various molecular weights of γ-PGA from 500 obvious single colonies on the primary screen plate were obtained.

Keywords: Poly(γ-glutamic acid); Neutral red; High-throughput screening; Bacteria

Iodine from bacterial iodide oxidization by Roseovarius spp. inhibits the growth of other bacteria by Dan Zhao; Choon-Ping Lim; Kazuhiko Miyanaga; Yasunori Tanji (pp. 2173-2182).

Microbial activities in brine, seawater, or estuarine mud are involved in iodine cycle. To investigate the effects of the microbiologically induced iodine on other bacteria in the environment, a total of 13 bacteria that potentially participated in the iodide-oxidizing process were isolated from water or biofilm at a location containing 131 μg ml⁻¹ iodide. Three distinct strains were further identified as Roseovarius spp. based on 16 S rRNA gene sequences after being distinguished by restriction fragment length polymorphism analysis. Morphological characteristics of these three Roseovarius spp. varied considerably across and within strains. Iodine production increased with Roseovarius spp. growth when cultured in Marine Broth with 200 μg ml⁻¹ iodide (I⁻). When 10⁶ CFU/ml Escherichia coli, Pseudomonas aeruginosa, and Bacillus pumilus were exposed to various concentrations of molecular iodine (I₂), the minimum inhibitory concentrations (MICs) were 0.5, 1.0, and 1.0 μg ml⁻¹, respectively. However, fivefold increases in the MICs for Roseovarius spp. were obtained. In co-cultured Roseovarius sp. IOB-7 and E. coli in Marine Broth containing iodide (I⁻), the molecular iodine concentration was estimated to be 0.76 μg ml⁻¹ after 24 h and less than 50 % of E. coli was viable compared to that co-cultured without iodide. The growth inhibition of E. coli was also observed in co-cultures with the two other Roseovarius spp. strains when the molecular iodine concentration was assumed to be 0.52 μg ml⁻¹.

Keywords: Iodide-oxidizing process; Molecular iodine; Roseovarius spp.; Minimum inhibitory concentration; Growth inhibition

Dynamics of specific ammonia-oxidizing bacterial populations and nitrification in response to controlled shifts of ammonium concentrations in wastewater by R. Almstrand; P. Lydmark; P.-E. Lindgren; F. Sörensson; M. Hermansson (pp. 2183-2191).

Ammonia-oxidizing bacteria (AOB) are essential for the nitrification process in wastewater treatment. To retain these slow-growing bacteria in wastewater treatment plants (WWTPs), they are often grown as biofilms, e.g., on nitrifying trickling filters (NTFs) or on carriers in moving bed biofilm reactors (MBBRs). On NTFs, a decreasing ammonium gradient is formed because of the AOB activity, resulting in low ammonium concentrations at the bottom and reduced biomass with depth. To optimize the NTF process, different ammonium feed strategies may be designed. This, however, requires knowledge about AOB population dynamics. Using fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy, we followed biomass changes during 6 months, of three AOB populations on biofilm carriers. These were immersed in aerated MBBR tanks in a pilot plant receiving full-scale wastewater. Tanks were arranged in series, forming a wastewater ammonium gradient mimicking an NTF ammonium gradient. The biomass of one of the dominating Nitrosomonas oligotropha-like populations increased after an ammonium upshift, reaching levels comparable to the high ammonium control in 28 days, whereas a Nitrosomonas europaea-like population increased relatively slowly. The MBBR results, together with competition studies in NTF systems fed with wastewater under controlled ammonium regimes, suggest a differentiation between the two N. oligotropha populations, which may be important for WWTP nitrification.

Keywords: Ammonia-oxidizing bacteria; Nitrification; Nitrosomonas europaea ; Nitrosomonas oligotropha ; Wastewater treatment plant; Biofilm; FISH

Kinetic analysis of biodesulfurization of model oil containing multiple alkyl dibenzothiophenes by Shi-Han Zhang; Han Chen; Wei Li (pp. 2193-2200).

Biodesulfurization is regarded as a promising alternative technology for desulfurization from diesel oil due to its mild operating conditions and its ability to remove sulfur from alky dibenzothiophenes (C_x-DBTs). The diesel oil contains complex mixtures of C_x-DBTs in which individual microbial biodesulfurization may be altered. In this work, interactions among three typical C_x-DBTs such as dibenzothiophenes (DBT), 4-methyldibenzothiophene (4-MDBT), and 4,6-dimethyldibenzothiophene (4,6-DMDBT) were investigated using Mycobacterium sp. ZD-19 in an airlift reactor. The experimental results indicated that the desulfurization rates would decrease in the multiple C_x-DBTs system compared to the single C_x-DBT system. The extent of inhibition depended upon the substrate numbers, concentrations, and affinities of the co-existing substrates. For example, compared to individual desulfurization rate (100 %), DBT desulfurization rate decreased to 75.2 % (DBT + 4,6-DMDBT), 64.8 % (DBT + 4-MDBT), and 54.7 % (DBT + 4,6-DMDBT + 4-MDBT), respectively. This phenomenon was caused by an apparent competitive inhibition of substrates, which was well predicted by a Michaelis–Menten competitive inhibition model.

Keywords: Biodesulfurization kinetics; Competitive inhibition; Multisubstrates; Mycobacterium sp.

The effect of ozone on the biodegradation of 17α-ethinylestradiol and sulfamethoxazole by mixed bacterial cultures by Simone Larcher; Viviane Yargeau (pp. 2201-2210).

The potential development of antibacterial resistance and endocrine disruption has led to increased research investigating the removal of contaminants from wastewater (WW) such as sulfamethoxazole (SMX) and 17α-ethinylestradiol (EE2). These compounds react quickly with ozone (O₃), thus ozonation during WW treatment may result in their complete removal. Also, O₃ has demonstrated the ability to increase the biodegradability of WW and certain pharmaceuticals, suggesting its potential as a pretreatment to activated sludge (AS, biological treatment). The objective of this study was to determine whether ozonation, conducted at doses lower than commonly applied to treated WW, would lead to an increased biodegradability of SMX and EE2. The results show that after ozonation performed at lab-scale the bacterial mixtures removed 5 % to 40 % more SMX; however, 2 % to 23 % less EE2 was removed, which was attributed to the observed preferential degradation of a by-product of EE2 ozonation. These results suggest that although ozonation, used as a pretreatment, was shown in literature to increase the overall biodegradability of AS as well as some specific antibiotic compounds and a blood lipid regulator, the potential for increased removal of pharmaceuticals seems to be compound-dependent and cannot yet be extrapolated to this entire class of compounds.

Keywords: Ozone; Sulfamethoxazole; 17α-ethinylestradiol; Biodegradation; Mixed bacteria

Endurance of methanogenic archaea in anaerobic bioreactors treating oleate-based wastewater by Andreia F. Salvador; Ana J. Cavaleiro; Diana Z. Sousa; M. Madalena Alves; M. Alcina Pereira (pp. 2211-2218).

Methanogenic archaea are reported as very sensitive to lipids and long chain fatty acids (LCFA). Therefore, in conventional anaerobic processes, methane recovery during LCFA-rich wastewater treatment is usually low. By applying a start-up strategy, based on a sequence of step feeding and reaction cycles, an oleate-rich wastewater was efficiently treated at an organic loading rate of 21 kg COD m⁻³ day⁻¹ (50 % as oleate), showing a methane recovery of 72 %. In the present work, the archaeal community developed in that reactor is investigated using a 16S rRNA gene approach. This is the first time that methanogens present in a bioreactor converting efficiently high loads of LCFA to methane are monitored. Denaturing gradient gel electrophoresis profiling showed that major changes on the archaeal community took place during the bioreactor start-up, where phases of continuous feeding were alternated with batch phases. After the start-up, a stable archaeal community (similarity higher than 84 %) was observed and maintained throughout the continuous operation. This community exhibited high LCFA tolerance and high acetoclastic and hydrogenotrophic activity. Cloning and sequencing results showed that Methanobacterium- and Methanosaeta-like microorganisms prevailed in the system and were able to tolerate and endure during prolonged exposure to high LCFA loads, despite the previously reported LCFA sensitivity of methanogens.

Keywords: Anaerobic treatment; High-rate bioreactor; LCFA; Methanogens; 16S rRNA gene; PCR-DGGE

Responses of bacterial and fungal communities to an elevation gradient in a subtropical montane forest of China by Han Meng; Ke Li; Ming Nie; Jia-Rong Wan; Zhe-Xue Quan; Chang-Ming Fang; Jia-Kuan Chen; Ji-Dong Gu; Bo Li (pp. 2219-2230).

Bacteria and fungi are ecologically important contributors to various functioning of forest ecosystems. In this study, we examined simultaneously the bacterial and fungal distributions in response to elevation changes of a forest. By using clone library analysis from genomic DNA extracted from forest humic clay soils, the composition and diversity of bacterial and fungal communities were determined across an elevation gradient from low via medium to high, in a subtropical forest in the Mountain Lushan, China. Our results showed that soil water content and nutrient availability, specifically total carbon, differed significantly with elevation changes. Although the soil acidity did not differ significantly among the three sites, low pH (around 4) could be an important selection factor selecting for acidophilic Acidobacteria and Alphaproteobacteria, which were the most abundant bacterial clones. As the majority of the fungi recovered, both Basidiomycota and Ascomycota, and their relative abundance were most closely associated with the total carbon. Based on the Shannon–Weaver diversity index and ∫-libshuff analysis, the soil at medium elevation contained the highest diversity of bacteria compared with those at high and low elevations. However, it is difficult to predict overall fungal diversity along elevation. The extreme high soil moisture content which may lead to the formation of anaerobic microhabitats in the forest soils potentially reduces the overall bacterial and fungal diversity.

Keywords: Forest; Fungi; Bacteria; Soil; Elevation gradient

Simultaneous improvement of saccharification and ethanol production from crystalline cellulose by alleviation of irreversible adsorption of cellulase with a cell surface-engineered yeast strain by Yuki Matano; Tomohisa Hasunuma; Akihiko Kondo (pp. 2231-2237).

Enzymatic hydrolysis of cellulosic material is an essential step in the bioethanol production process. However, complete cellulose hydrolysis by cellulase is difficult due to the irreversible adsorption of cellulase onto cellulose. Thus, part of the cellulose remains in crystalline form after hydrolysis. In this study, after 96-h hydrolysis of Avicel crystalline cellulose, 47.1 % of the cellulase was adsorbed on the cellulose surface with 10.8 % crystalline cellulose remaining. In simultaneous saccharification and fermentation of 100 g/L Avicel with 1.0 filter paper unit/mL cellulase, a wild-type yeast strain produced 44.7 g/L ethanol after 96 h. The yield of ethanol was 79.7 % of the theoretical yield. On the other hand, a recombinant yeast strain displaying various cellulases, such as β-glucosidase, cellobiohydrolase, and endoglucanase, produced 48.9 g/L ethanol, which corresponds to 87.3 % of the theoretical yield. Higher ethanol production appears to be attributable to higher efficiency of cellulase displayed on the cell surface. These results suggest that cellulases displayed on the yeast cell surface improve hydrolysis of Avicel crystalline cellulose. Indeed, after the 96-h simultaneous saccharification and fermentation using the cellulase-displaying yeast, the amount of residual cellulose was 1.5 g/L, one quarter of the cellulose remaining using the wild-type strain, a result of the alleviation of irreversible adsorption of cellulases on the crystalline cellulose.

Keywords: Bioethanol; Cellulase; Cell surface display; Protein adsorption; Saccharomyces cerevisiae

Use of secondary-treated wastewater for the production of Muriellopsis sp. by C. Gómez; R. Escudero; M. M Morales; F. L. Figueroa; J. M. Fernández-Sevilla; F. G. Acién (pp. 2239-2249).

In this paper, the use of secondary-treated wastewater as the culture medium for the production of Muriellopsis sp. microalgal biomass is analyzed. Using this wastewater, a maximum biomass productivity of 0.5 g l⁻¹ day⁻¹ was measured, it being only 38 % lower than that achieved using the standard culture medium. Due to the low nitrogen content of secondary-treated wastewater, cultures produced in a medium containing a high percentage of it become nitrate-limited, thus the quantum yield reduces by up to 0.38 g E⁻¹—this compares to 0.67 g E⁻¹ when using a standard culture medium. On the other hand, nitrate limitation enhances the accumulation of lipids and carbohydrates, with values measured at 33 and 66 % dry weight, respectively. It was also demonstrated that secondary-treated wastewater does not have any toxic effect on the growth of Muriellopsis sp. in spite of nitrogen being in the form of ammonium rather than in nitrate. Moreover, the secondary-treated wastewater was depurated when used to produce Muriellopsis sp., with the outlet biological oxygen demand and chemical oxygen demand being lower than at the inlet; the nitrate and phosphate concentrations were zero. Therefore, Muriellopsis sp. production using secondary-treated wastewater allows a reduction in the process cost by decreasing freshwater and fertilizer use, as well as by depurating the water, thus greatly enhancing process sustainability.

Keywords: Microalgae; Wastewater; Nutrient limitation; Productivity; Biochemical composition

Evaluation of stable isotope fingerprinting techniques for the assessment of the predominant methanogenic pathways in anaerobic digesters by M. Nikolausz; R. F. H. Walter; H. Sträuber; J. Liebetrau; T. Schmidt; S. Kleinsteuber; F. Bratfisch; U. Günther; H. H. Richnow (pp. 2251-2262).

Laboratory biogas reactors were operated under various conditions using maize silage, chicken manure, or distillers grains as substrate. In addition to the standard process parameters, the hydrogen and carbon stable isotopic composition of biogas was analyzed to estimate the predominant methanogenic pathways as a potential process control tool. The isotopic fingerprinting technique was evaluated by parallel analysis of mcrA genes and their transcripts to study the diversity and activity of methanogens. The dominant hydrogenotrophs were Methanomicrobiales, while aceticlastic methanogens were represented by Methanosaeta and Methanosarcina at low and high organic loading rates, respectively. Major changes in the relative abundance of mcrA transcripts were observed compared to the results obtained from DNA level. In agreement with the molecular results, the isotope data suggested the predominance of the hydrogenotrophic pathway in one reactor fed with chicken manure, while both pathways were important in the other reactors. Short-term changes in the isotopic composition were followed, and a significant change in isotope values was observed after feeding a reactor digesting maize silage. This ability of stable isotope fingerprinting to follow short-term activity changes shows potential for indicating process failures and makes it a promising technology for process control.

Keywords: Stable isotope fingerprinting; Process control; Biogas; mcrA ; Methanogens; Methanogenic activity

At high temperature lipid production in Ettlia oleoabundans occurs before nitrate depletion by Ying Yang; Benjamin Mininberg; Andrea Tarbet; Pamela Weathers (pp. 2263-2273).

Temperature and light intensity effects on biomass and lipid production were investigated in Ettlia oleoabundans to better understand some fundamental properties of this potentially useful but poorly studied microalgal species. E. oleoabundans entered dormant state at 5 °C, showed growth at 10 °C, and when exposed to light at 70 μmol photons per square meter per second at 10 °C, cells reached a biomass concentration of >2.0 g L⁻¹ with fatty acid methyl esters of 11.5 mg L⁻¹. Highest biomass productivity was at 15 °C and 25 °C regardless of light intensity, and accumulation of intracellular lipids was stimulated by nitrate depletion under these conditions. Although growth was inhibited at 35 °C, at 130 μmol photons per square meter per second lipid content reached 10.37 mg L⁻¹ with fatty acid content more favorable to biodiesel dominating; this occurred without nitrate depletion. In a two-phase temperature shift experiment at two nitrate levels, cells were shifted after 21 days at 15 °C to 35 °C for 8 days. Although after the shift growth continued, lipid productivity per cell was less than that in the 35 °C cultures, again without nitrate depletion. This study showed that E. oleoabundans grows well at low temperature and light intensity, and high temperature can be a useful trigger for lipid accumulation independent of nitrate depletion. This will prove useful for improving our knowledge about lipid production in this and other oleaginous algae for modifying yield and quality of algal lipids being considered for biodiesel production.

Keywords: Neochloris oleoabundans ; Biodiesel; Biomass; Light intensity; Temperature; Nitrate

Erratum to: Cloning of a dibutyl phthalate hydrolase gene from Acinetobacter sp. strain M673 and functional analysis of its expression product in Escherichia coli by Jun Wu; Xuewei Liao; Fangbo Yu; Zhongbo Wei; Liuyan Yang (pp. 2275-2275).

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Applied Microbiology and Biotechnology (v.97, #5)

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Applied Microbiology and Biotechnology (v.97, #5)