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Applied Microbiology and Biotechnology (v.89, #2)

Proteomics of industrial fungi: trends and insights for biotechnology by José Miguel P. Ferreira de Oliveira; Leo H. de Graaff (pp. 225-237).

Filamentous fungi are widely known for their industrial applications, namely, the production of food-processing enzymes and metabolites such as antibiotics and organic acids. In the past decade, the full genome sequencing of filamentous fungi increased the potential to predict encoded proteins enormously, namely, hydrolytic enzymes or proteins involved in the biosynthesis of metabolites of interest. The integration of genome sequence information with possible phenotypes requires, however, the knowledge of all the proteins in the cell in a system-wise manner, given by proteomics. This review summarises the progress of proteomics and its importance for the study of biotechnological processes in filamentous fungi. A major step forward in proteomics was to couple protein separation with high-resolution mass spectrometry, allowing accurate protein quantification. Despite the fact that most fungal proteomic studies have been focused on proteins from mycelial extracts, many proteins are related to processes which are compartmentalised in the fungal cell, e.g. β-lactam antibiotic production in the microbody. For the study of such processes, a targeted approach is required, e.g. by organelle proteomics. Typical workflows for sample preparation in fungal organelle proteomics are discussed, including homogenisation and sub-cellular fractionation. Finally, examples are presented of fungal organelle proteomic studies, which have enlarged the knowledge on areas of interest to biotechnology, such as protein secretion, energy production or antibiotic biosynthesis.

Keywords: Aspergillus niger ; Trichoderma; Cell-organelle proteomics; Protein secretion; Metabolites; Antibiotics

Proteomics of industrial fungi: trends and insights for biotechnology by José Miguel P. Ferreira de Oliveira; Leo H. de Graaff (pp. 225-237).

Keywords: Aspergillus niger ; Trichoderma; Cell-organelle proteomics; Protein secretion; Metabolites; Antibiotics

Engineering of nitrogen metabolism and its regulation in Corynebacterium glutamicum: influence on amino acid pools and production by Nadine Rehm; Andreas Burkovski (pp. 239-248).

Nitrogen is one of the macronutrients necessary for living cells, and consequently, assimilation of nitrogen is a crucial step for metabolism. To satisfy their nitrogen demand and to ensure a sufficient nitrogen supply even in situations of nitrogen limitation, microorganisms have evolved sophisticated uptake and assimilation mechanisms for different nitrogen sources. This mini-review focuses on nitrogen metabolism and its control in the biotechnology workhorse Corynebacterium glutamicum, which is used for the industrial production of more than 2 million tons of l-amino acids annually. Ammonium assimilation and connected control mechanisms on activity and transcription level are summarized, and the influence of mutations on amino acid pools and production is described with emphasis on l-glutamate, l-glutamine, and l-lysine.

Keywords: Amino acid production; AmtR; Glutamate; Glutamine; Lysine; Nitrogen control; Nitrogen regulation

Engineering of nitrogen metabolism and its regulation in Corynebacterium glutamicum: influence on amino acid pools and production by Nadine Rehm; Andreas Burkovski (pp. 239-248).

Keywords: Amino acid production; AmtR; Glutamate; Glutamine; Lysine; Nitrogen control; Nitrogen regulation

YqhD: a broad-substrate range aldehyde reductase with various applications in production of biorenewable fuels and chemicals by Laura R. Jarboe (pp. 249-257).

The Escherichia coli NADPH-dependent aldehyde reductase YqhD has contributed to a variety of metabolic engineering projects for production of biorenewable fuels and chemicals. As a scavenger of toxic aldehydes produced by lipid peroxidation, YqhD has reductase activity for a broad range of short-chain aldehydes, including butyraldehyde, glyceraldehyde, malondialdehyde, isobutyraldehyde, methylglyoxal, propanealdehyde, acrolein, furfural, glyoxal, 3-hydroxypropionaldehyde, glycolaldehyde, acetaldehyde, and acetol. This reductase activity has proven useful for the production of biorenewable fuels and chemicals, such as isobutanol and 1,3- and 1,2-propanediol; additional capability exists for production of 1-butanol, 1-propanol, and allyl alcohol. A drawback of this reductase activity is the diversion of valuable NADPH away from biosynthesis. This YqhD-mediated NADPH depletion provides sufficient burden to contribute to growth inhibition by furfural and 5-hydroxymethyl furfural, inhibitory contaminants of biomass hydrolysate. The structure of YqhD has been characterized, with identification of a Zn atom in the active site. Directed engineering efforts have improved utilization of 3-hydroxypropionaldehyde and NADPH. Most recently, two independent projects have demonstrated regulation of yqhD by YqhC, where YqhC appears to function as an aldehyde sensor.

Keywords: Aldo–keto; Promiscuous; Glutathione; Tolerance; Reverse engineering

YqhD: a broad-substrate range aldehyde reductase with various applications in production of biorenewable fuels and chemicals by Laura R. Jarboe (pp. 249-257).

Keywords: Aldo–keto; Promiscuous; Glutathione; Tolerance; Reverse engineering

Microbial fuel cells for energy self-sufficient domestic wastewater treatment—a review and discussion from energetic consideration by Olivier Lefebvre; Arnaud Uzabiaga; In Seop Chang; Byung-Hong Kim; How Yong Ng (pp. 259-270).

As the microbial fuel cell (MFC) technology is getting nearer to practical applications such as wastewater treatment, it is crucial to consider the different aspects that will make this technology viable in the future. In this paper, we provide information about the specifications of an energy self-sufficient MFC system as a basis to extrapolate on the potential benefits and limits of a future MFC-based wastewater treatment plant. We particularly emphasize on the importance of two crucial parameters that characterize an MFC: its electromotive force (E _emf) and its internal resistance (R _int). A numerical projection using state-of-art values (E _emf = 0.8 V and R _int = 5 Ω) emphasized on the difficulty at this moment to reach self-sufficiency using a reasonable number of MFCs at the laboratory scale. We found that a realistic number of MFCs to provide enough voltage (=5 V) at a sufficient current (=0.8 A) to power a pump requiring 4 W would be of 13 MFCs in series and 10 stacks of MFCs in parallel, resulting in a total number of 130 MFCs. That would result in a treatment capacity of 144 L of domestic wastewater (0.5 g-COD L⁻¹) per day. The total MFC system would be characterized by an internal resistance of 6.5 Ω.

Keywords: Electromotive force; Internal resistance; Microbial fuel cells; Scalability; Stack configuration

The impact of acetate metabolism on yeast fermentative performance and wine quality: reduction of volatile acidity of grape musts and wines by Alice Vilela-Moura; Dorit Schuller; Arlete Mendes-Faia; Rui D. Silva; Susana R. Chaves; Maria João Sousa; Manuela Côrte-Real (pp. 271-280).

Acetic acid is the main component of the volatile acidity of grape musts and wines. It can be formed as a by-product of alcoholic fermentation or as a product of the metabolism of acetic and lactic acid bacteria, which can metabolize residual sugars to increase volatile acidity. Acetic acid has a negative impact on yeast fermentative performance and affects the quality of certain types of wine when present above a given concentration. In this mini-review, we present an overview of fermentation conditions and grape-must composition favoring acetic acid formation, as well the metabolic pathways leading to its formation and degradation by yeast. The negative effect of acetic acid on the fermentative performance of Saccharomyces cerevisiae will also be covered, including its role as a physiological inducer of apoptosis. Finally, currently available wine deacidification processes and new proposed solutions based on zymological deacidification by select S. cerevisiae strains will be discussed.

Keywords: Yeast; Acetic acid; Metabolism; Wine; Volatile acidity; Deacidification

Recombinant antimicrobial peptide hPAB-β expressed in Pichia pastoris, a potential agent active against methicillin-resistant Staphylococcus aureus by Zhijin Chen; Dongmei Wang; Yanguang Cong; Jing Wang; Junmin Zhu; Jie Yang; Zhen Hu; Xiaomei Hu; Yinling Tan; Fuquan Hu; Xiancai Rao (pp. 281-291).

As a potential therapeutic agent, antimicrobial peptide has received increased attention in recent years. However, high-level expression of a small peptide with antimicrobial activity is still a challenging task. In this study, the coding sequence of antimicrobial peptide hPAB-β, a variant derived from human beta-defensin 2, was cloned into pPIC9K vector and transformed into Pichia pastoris. P. pastoris transformants harbored with multi-copy plasmids were screened by G418 selection. When the transformed cells were induced by methanol, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blot, and matrix-assisted laser desorption ionization-time of flight mass spectrometry revealed recombinant hPAB-β products consisting of three protein species of 4,680.4, 4,485.3, and 4,881.9 Da at proportions of 58%, 36%, and 6%, respectively, which may be due to the incomplete processing of the fusion signal peptide of α-factor by the STE13 protease. Expressed hPAB-β was secreted into the culture medium at a level of 241.2 ± 29.5 mg/L. Purified hPAB-β with 95% homogeneity was obtained by 10 kDa membrane filtration followed by cation ion-exchange chromatography with a SP-Sepharose™ XL column. The two major protein species separated through a SOURCE™ 30RPC reverse phase chromatography column showed definite antimicrobial activities against Staphylococcus aureus. All 22 methicillin-resistant S. aureus (MRSA) isolates with multidrug resistance phenotype were sensitive to the recombinant hPAB-β with minimal inhibitory concentrations of 8–64 μg/ml. Our results show that the methylotrophic yeast-inducible system is suitable for high-level expression of active hPAB-β, and that expressed hPAB-β in P. pastoris may be a potential antimicrobial agent against MRSA infection.

Keywords: Antimicrobial peptide; hPAB-β; Pichia pastoris ; Multidrug resistance; Methicillin-resistant Staphylococcus aureus (MRSA); Antimicrobial activity

Coenzyme Q₁₀ production directly from precursors by free and gel-entrapped Sphingomonas sp. ZUTE03 in a water-organic solvent, two-phase conversion system by Weihong Zhong; Weijian Wang; Zhuoyi Kong; Bin Wu; Li Zhong; Xuanzhen Li; Ji Yu; Fuming Zhang (pp. 293-302).

In a water-organic solvent, two-phase conversion system, CoQ₁₀ could be produced directly from solanesol and para-hydroxybenzoic acid (PHB) by free cells of Sphingomonas sp. ZUTE03 and CoQ₁₀ concentration in the organic solvent phase was significantly higher than that in the cell. CoQ₁₀ yield reached a maximal value of 60.8 mg l⁻¹ in the organic phase and 40.6 mg g⁻¹-DCW after 8 h. CoQ₁₀ also could be produced by gel-entrapped cells in the two-phase conversion system. Soybean oil and hexane were found to be key substances for CoQ₁₀ production by gel-entrapped cells of Sphingomonas sp. ZUTE03. Soybean oil might improve the release of CoQ10 from the gel-entrapped cells while hexane was the suitable solvent to extract CoQ₁₀ from the mixed phase of aqueous and organic. The gel-entrapped cells could be re-used to produce CoQ₁₀ by a repeated-batch culture. After 15 repeats, the yield of CoQ₁₀ kept at a high level of more than 40 mg l⁻¹. After 8 h conversion under optimized precursor’s concentration, CoQ₁₀ yield of gel-trapped cells reached 52.2 mg l⁻¹ with a molar conversion rate of 91% and 89.6% (on PHB and solanesol, respectively). This is the first report on enhanced production of CoQ₁₀ in a two-phase conversion system by gel-entrapped cells of Sphingomonas sp. ZUTE03.

Keywords: Coenzyme Q₁₀ ; Microbial conversion; Two-phase system; Immobilized cells; Sphingomonas sp. ZUTE03

Correlations between molecular and operational parameters in continuous lab-scale anaerobic reactors by Marta Carballa; Marianne Smits; Claudia Etchebehere; Nico Boon; Willy Verstraete (pp. 303-314).

In this study, the microbial community characteristics in continuous lab-scale anaerobic reactors were correlated to reactor functionality using the microbial resource management (MRM) approach. Two molecular techniques, denaturing gradient gel electrophoresis (DGGE) and terminal-restriction fragment length polymorphism (T-RFLP), were applied to analyze the bacterial and archaeal communities, and the results obtained have been compared. Clustering analyses showed a similar discrimination of samples with DGGE and T-RFLP data, with a clear separation between the meso- and thermophilic communities. Both techniques indicate that bacterial and mesophilic communities were richer and more even than archaeal and thermophilic communities, respectively. Remarkably, the community composition was highly dynamic for both Bacteria and Archaea, with a rate of change between 30% and 75% per 18 days, also in stable performing periods. A hypothesis to explain the latter in the context of the converging metabolism in anaerobic processes is proposed. Finally, a more even and diverse bacterial community was found to be statistically representative for a well-functioning reactor as evidenced by a low Ripley index and high biogas production.

Keywords: Anaerobic digestion; Community organization; Diversity; Dynamics; Microbial community structure; Richness

Cloning and characterization of a thermostable and halo-tolerant endoglucanase from Thermoanaerobacter tengcongensis MB4 by Chaoning Liang; Yanfen Xue; Marco Fioroni; Francisco Rodríguez-Ropero; Cheng Zhou; Ulrich Schwaneberg; Yanhe Ma (pp. 315-326).

A β-1,4-endoglucanase (Cel5A) was cloned from the genomic DNA of saccharolytic thermophilic eubacterium Thermoanaerobacter tengcongensis MB4 and functionally expressed in Escherichia coli. Substrate specificity analysis revealed that Cel5A cleaves specifically the β-1,4-glycosidic linkage in cellulose with high activity (294 U mg⁻¹; carboxymethyl cellulose sodium (CMC)). On CMC, kinetics of Cel5A was determined (K _m 1.39 ± 0.12 g l⁻¹; k _cat/K _m 1.41 ± 0.13 g⁻¹ s⁻¹). Cel5A displays an activity optimum between 75 and 80 °C. Residues Glu187 and Glu289 were identified as key catalytic amino acids by sequence alignment. Interestingly, derived from a non-halophilic bacterium, Cel5A exhibits high residual activities in molar concentration of NaCl (3 M, 49.3%) and KCl (4 M, 48.6%). In 1 M NaCl, 82% of Cel5A activity is retained after 24 h incubation. Molecular Dynamics studies performed at 0 and 3 M NaCl, correlate the Cel5A stability to the formation of R-COO⁻···Na⁺ ···⁻OOC-R salt bridges within the Cel5A tertiary structure, while activity possibly relates to the number of Na⁺ ions trapped into the negatively charged active site, involving a competition mechanism between substrate and Na⁺. Additionally, Cel5A is remarkably resistant in ionic liquids 1-butyl-3-methyllimidazolium chloride (1 M, 54.4%) and 1-allyl-3-methylimidazolium chloride (1 M, 65.1%) which are promising solvents for cellulose degradation and making Cel5A an attractive candidate for industrial applications.

Keywords: Endoglucanase; Characterization; Thermostability; Halotolerance; Molecular Dynamics

Identification of the membrane protein SucE and its role in succinate transport in Corynebacterium glutamicum by Stephanie Huhn; Elena Jolkver; Reinhard Krämer; Kay Marin (pp. 327-335).

Succinic acid is excreted during anaerobiosis by many bacteria, and manifold applications are known making the biotechnological production of succinate attractive. Although the pathways for succinate formation are known, succinate export is not understood in most of the succinate producing bacteria. Here, we present a bioinformatic approach for identification of a putative succinate export system in Corynebacterium glutamicum. The subsequent screening revealed that a mutant in the gene cg2425 is impaired in succinate production or transport under anaerobic conditions. A function of the Cg2425 protein as import system was excluded. In contrast, a role of the Cg2425 protein as succinate export system was indicated by accumulation of increased amounts of internal succinate under anaerobic conditions in a Cg2425-dependent manner and a concomitant impairment of external succinate accumulation. In conclusion, we propose that Cg2425 participates in succinate export in C. glutamicum and suggest the name SucE for the protein.

Keywords: Corynebacterium glutamicum ; Substrate excretion; Transport; Succinate export; SucE

Identification of the membrane protein SucE and its role in succinate transport in Corynebacterium glutamicum by Stephanie Huhn; Elena Jolkver; Reinhard Krämer; Kay Marin (pp. 327-335).

Keywords: Corynebacterium glutamicum ; Substrate excretion; Transport; Succinate export; SucE

Covalent immobilization of β-1,4-glucosidase from Agaricus arvensis onto functionalized silicon oxide nanoparticles by Raushan Kumar Singh; Ye-Wang Zhang; Ngoc-Phuong-Thao Nguyen; Marimuthu Jeya; Jung-Kul Lee (pp. 337-344).

An efficient β-1,4-glucosidase (BGL) secreting strain, Agaricus arvensis, was isolated and identified. The relative molecular weight of the purified A. arvensis BGL was 98 kDa, as determined by sodium dodecylsulfate polyacrylamide gel electrophoresis, or 780 kDa by size exclusion chromatography, indicating that the enzyme is an octamer. Using a crude enzyme preparation, A. arvensis BGL was covalently immobilized onto functionalized silicon oxide nanoparticles with an immobilization efficiency of 158%. The apparent V _max (k _cat) values of free and immobilized BGL under standard assay conditions were 3,028 U mg protein⁻¹ (4,945 s⁻¹) and 3,347 U mg protein⁻¹ (5,466 s⁻¹), respectively. The immobilized BGL showed a higher optimum temperature and improved thermostability as compared to the free enzyme. The half-life at 65 °C showed a 288-fold improvement over the free BGL. After 25 cycles, the immobilized enzyme still retained 95% of the original activity, thus demonstrating its prospects for commercial applications. High specific activity, high immobilization efficiency, improved stability, and reusability of A. arvensis BGL make this enzyme of potential interest in a number of industrial applications.

Keywords: Covalent bonding; β-1,4-Glucosidase; Immobilization; Silicon oxide nanoparticles

An exopolysaccharide produced by the novel halophilic bacterium Halomonas stenophila strain B100 selectively induces apoptosis in human T leukaemia cells by Carmen Ruiz-Ruiz; Girish K. Srivastava; Diana Carranza; Juan A. Mata; Inmaculada Llamas; Manuel Santamaría; Emilia Quesada; Ignacio J. Molina (pp. 345-355).

Microbial exopolysaccharides (EPSs) are highly heterogeneous polymers produced by fungi and bacteria and have recently been attracting considerable attention from biotechnologists because of their potential applications in many fields, including biomedicine. We have screened the antitumoural activity of a panel of sulphated EPSs produced by a newly discovered species of halophilic bacteria. We found that the novel halophilic bacterium Halomonas stenophila strain B100 produced a heteropolysaccharide that, when oversulphated, exerted antitumoural activity on T cell lines deriving from acute lymphoblastic leukaemia (ALL). Only tumour cells were susceptible to apoptosis induced by the sulphated EPS (B100S), whilst primary T cells were resistant. Moreover, freshly isolated primary cells from the blood of patients with ALL were also susceptible to B100S-induced apoptosis. The newly discovered B100S is therefore the first bacterial EPS that has been demonstrated to exert a potent and selective pro-apoptotic effect on T leukaemia cells, and thus, we propose that the search for new antineoplastic drugs should include the screening of other bacterial EPSs, particularly those isolated from halophiles.

Keywords: Apoptosis; Exopolysaccharides; T cell leukaemia; Halomonas stenophila B100; Antitumoural activity

Effects of a defective ERAD pathway on growth and heterologous protein production in Aspergillus niger by Neuza D. S. P. Carvalho; Mark Arentshorst; Rolf Kooistra; Hein Stam; Cees M. Sagt; Cees A. M. J. J. van den Hondel; Arthur F. J. Ram (pp. 357-373).

Endoplasmic reticulum associated degradation (ERAD) is a conserved mechanism to remove misfolded proteins from the ER by targeting them to the proteasome for degradation. To assess the role of ERAD in filamentous fungi, we have examined the consequences of disrupting putative ERAD components in the filamentous fungus Aspergillus niger. Deletion of derA, doaA, hrdC, mifA, or mnsA in A. niger yields viable strains, and with the exception of doaA, no significant growth phenotype is observed when compared to the parental strain. The gene deletion mutants were also made in A. niger strains containing single- or multicopies of a glucoamylase–glucuronidase (GlaGus) gene fusion. The induction of the unfolded protein response (UPR) target genes (bipA and pdiA) was dependent on the copy number of the heterologous gene and the ERAD gene deleted. The highest induction of UPR target genes was observed in ERAD mutants containing multiple copies of the GlaGus gene. Western blot analysis revealed that deletion of the derA gene in the multicopy GlaGus overexpressing strain resulted in a 6-fold increase in the intracellular amount of GlaGus protein detected. Our results suggest that impairing some components of the ERAD pathway in combination with high expression levels of the heterologous protein results in higher intracellular protein levels, indicating a delay in protein degradation.

Keywords: Aspergillus niger ; ERAD; UPR; Heterologous protein

Heterologous expression of a thermophilic esterase in Kluyveromyces yeasts by Saul Nitsche Rocha; José Abrahão-Neto; María Esperanza Cerdán; Andreas Karoly Gombert; María Isabel González-Siso (pp. 375-385).

In the present work, a thermophilic esterase from Thermus thermophilus HB27 was cloned into Kluyveromyces marxianus and into Kluyveromyces lactis using two different expression systems, yielding four recombinant strains. K. lactis showed the highest esterase expression levels (294 units per gram dry cell weight, with 65% of cell-bound enzyme) using an episomal system with the PGK promoter and terminator from Saccharomyces cerevisiae combined with the K. lactis k1 secretion signal. K. marxianus showed higher secretion efficiency of the heterologous esterase (56.9 units per gram dry cell weight, with 34% of cell-bound enzyme) than K. lactis. Hydrolytic activities for the heterologous esterases were maximum at pH values between 8.0 and 9.0 for both yeast species and at temperatures of 50 °C and 45 °C for K. marxianus and K. lactis, respectively. When compared to previously published data on this same esterase produced in the original host or in S. cerevisiae, our results indicate that Kluyveromyces yeasts can be considered good hosts for the heterologous secretion of thermophilic esterases, which have a potential application in biodiesel production or in resolving racemates.

Keywords: Thermophilic esterase; Kluyveromyces marxianus ; Kluyveromyces lactis ; Heterologous expression

Comparative studies on the metabolism of linoleic acid by rumen bacteria, protozoa, and their mixture in vitro by Mamun M. Or-Rashid; Ousama AlZahal; Brian W. McBride (pp. 387-395).

Linoleic acid was differentially catabolized by the various rumen microbial fractions, such as rumen bacteria (B), protozoa (P), and their mixture (BP). The predominant isomer of conjugated linoleic acids (CLA) synthesized by B, P, and BP from linoleic acid was 9c11t-CLA. The formation of 9c11t-CLA was higher (P < 0.05) in P suspension (53.6 μg/mg microbial nitrogen) compared with B (38.3 μg/mg microbial nitrogen) and BP (28.8 μg/mg microbial nitrogen) suspensions by 12 h of incubation. The second most abundant CLA isomer was 10t12c. The accumulation of 10t12c-CLA in BP suspension was 2.3 times lower (P < 0.05) than that in B suspension (84.8 μg/mg microbial nitrogen) by 12 h of incubation. The accumulation of 10t-18:1 in BP suspension during 6- and 12-h incubation periods were not different (P > 0.05) than that in B suspension (6.8 and 14.0 μg/mg microbial nitrogen, respectively). However, the accumulation of 11t-18:1 in BP suspension at 6- and 12-h incubations were 2.7 and 3.3 times higher (P < 0.05), respectively, than that in B suspension. There were no significant accumulations of 11t-18:1, 10t-18:1, and 18:0 in P suspension throughout the incubation period. It was concluded that B, P, and BP metabolized linoleic acid to different isomers of CLA, whereas B, including BP, was only capable of biohydrogenating the CLA isomers to 18:0 by the reduction of 18:1 isomers. P was incapable of biohydrogenating LA, but its association with B in the BP suspension altered the biohydrogenation of LA significantly compared with B alone.

Keywords: CLA; Rumen bacteria and protozoa; Linoleic acid; Vaccenic acid

Comparative studies on the metabolism of linoleic acid by rumen bacteria, protozoa, and their mixture in vitro by Mamun M. Or-Rashid; Ousama AlZahal; Brian W. McBride (pp. 387-395).

Keywords: CLA; Rumen bacteria and protozoa; Linoleic acid; Vaccenic acid

Uniform stable-isotope labeling in mammalian cells: formulation of a cost-effective culture medium by Tatiana A. Egorova-Zachernyuk; Giel J. C. G. M. Bosman; Willem J. DeGrip (pp. 397-406).

Uniform stable-isotope labeling of mammalian cells is achieved via a novel formulation of a serum-free cell culture medium that is based on stable-isotope-labeled autolysates and lipid extracts of various microbiological origin. Yeast autolysates allow complete replacement of individual amino acids and organic acids in a chemically defined medium (DMEM/F12), enabling a cost-effective formulation of a stable-isotope-labeled culture medium for mammalian cells. In addition, biomass-derived hydrolysates, autolysates, and lipid extracts of various classes of algae were explored as cell culture components, both separately and in combination with yeast autolysates. Optimal autolysate concentrations were established. Such novel medium formulations were tested on mammalian cell lines, often used for recombinant protein production, i.e., Chinese hamster ovary (CHO) and human embryonic kidney (HEK 293). Special attention was paid to the adaptation of these mammalian cell lines to serum-free media. Formulation of the novel proprietary cell culture medium PLIm, based on yeastolates instead of individual amino acids and organic acids, allows a four- to eightfold cost reduction for ¹⁵N and ¹³C,¹⁵N stable-isotope-labeling, respectively, in CHO cells and a three- to sixfold cost reduction in HEK 293 cells. A high level of stable-isotope enrichment of mammalian cells (>90%) was achieved within four passages by complete replacement of carbon and nitrogen sources in the medium with their stable-isotope-labeled analogs. These conditions can be used to more cost-effectively produce labeled recombinant proteins in mammalian cells.

Keywords: Stable-isotope labeling; Mammalian cells; Metabolomics; Culture medium formulation; Extracts; Autolysates; Biodiversity

Uniform stable-isotope labeling in mammalian cells: formulation of a cost-effective culture medium by Tatiana A. Egorova-Zachernyuk; Giel J. C. G. M. Bosman; Willem J. DeGrip (pp. 397-406).

Keywords: Stable-isotope labeling; Mammalian cells; Metabolomics; Culture medium formulation; Extracts; Autolysates; Biodiversity

Mechanisms of degradation of DNA standards for calibration function during storage by Peter Rossmanith; Barbara Röder; Karin Frühwirth; Claus Vogl; Martin Wagner (pp. 407-417).

Establishment of molecular diagnostics offering quantitative technology is directly associated with real-time polymerase chain reaction (PCR). This rapid, accurate and sensitive method requires careful execution, including reliable calibration standards. The storage of such standards is crucial to prevent nucleic acid decay and to ensure stable results using real-time PCR. In this study, a broad investigation of possible causes of DNA degradation during storage was performed, including GC-content of the fragments, long-term storage, rapid freeze-and-thaw experiments, genomic DNA and short DNA fragments of different species, the influence of shear stress and the effect of nuclease remaining after DNA isolation. Several known chemical DNA degradation mechanisms have been matched with the experimental data through a process of elimination. Protocols for practical application, as well as a theoretical model describing the underlying mechanisms of deviation of real-time PCR results due to decay of standard DNA, have been developed. Primary amines in the buffer composition, which enhance depurination of the DNA helix, and shear stress due to ice crystal formation, could be identified as major sources of interaction. This results in degradation of the standard DNA, as well as in the probability of occurrence of mismatches affecting real-time PCR performance.

Keywords: DNA degradation; DNA storage; Depurination; Shearing of DNA; Calibration function; Real-time PCR

Mechanisms of degradation of DNA standards for calibration function during storage by Peter Rossmanith; Barbara Röder; Karin Frühwirth; Claus Vogl; Martin Wagner (pp. 407-417).

Keywords: DNA degradation; DNA storage; Depurination; Shearing of DNA; Calibration function; Real-time PCR

Nocardioides sp. strain WSN05-2, isolated from a wheat field, degrades deoxynivalenol, producing the novel intermediate 3-epi-deoxynivalenol by Yoko Ikunaga; Ikuo Sato; Stephanie Grond; Nobutaka Numaziri; Shigenobu Yoshida; Hiroko Yamaya; Syuntaro Hiradate; Morifumi Hasegawa; Hiroaki Toshima; Motoo Koitabashi; Michihiro Ito; Petr Karlovsky; Seiya Tsushima (pp. 419-427).

The mycotoxin deoxynivalenol (DON) causes serious problems worldwide in the production of crops such as wheat and barley because of its toxicity toward humans and livestock. A bacterial culture capable of degrading DON was obtained from soil samples collected in wheat fields using an enrichment culture procedure. The isolated bacterium, designated strain WSN05-2, completely removed 1,000 μg/mL of DON from the culture medium after incubation for 10 days. On the basis of phylogenetic studies, WSN05-2 was classified as a bacterium belonging to the genus Nocardioides. WSN05-2 showed significant growth in culture medium with DON as the sole carbon source. High-performance liquid chromatography analysis indicated the presence of a major initial metabolite of DON in the culture supernatant. The metabolite was identified as 3-epi-deoxynivalenol (3-epi-DON) by mass spectrometry and ¹H and ¹³C nuclear magnetic resonance analysis. The amount of DON on wheat grain was reduced by about 90% at 7 days after inoculation with WSN05-2. This is the first report of a Nocardioides sp. strain able to degrade DON and of the yet unknown 3-epi-DON as an intermediate in the degradation of DON by a microorganism.

Keywords: Fusarium graminearum ; Trichothecenes; Deoxynivalenol; Mycotoxin degradation; Nocardioides

Enhancement of the antimicrobial performance of biocidal formulations used for the preservation of white mineral dispersions by Nicola Di Maiuta; Patrick Schwarzentruber; Crawford S. Dow (pp. 429-439).

Biocides play an important role in the preservation of white mineral dispersions (WMD). Due to the occurrence of biocide-resistant bacteria and technical limitations in the use of biocides, new preservation strategies are required—like the enhancement of biocides by non-biocidal compounds. The aim of this study was to evaluate the biocide enhancement performance of lithium against various biocide-resistant bacteria in WMD. Subsequently, the minimal enhancing concentration (MEC) of lithium and the bioavailability of lithium in respect to the mode of introduction into WMD were investigated. The antimicrobial performance of biocidal formulations comprising isothiazolinones and formaldehyde releasers or isothiazolinones and glutaraldehyde has been evaluated against the related resistant bacterial spectrum in the presence of lithium. The MEC of lithium ranged from 1,350 to 1,500 ppm (based on the liquid phase weight of a WMD with 75% solids) for formaldehyde releasers and glutaraldehyde-based biocidal formulations, respectively. The biocide enhancing property of lithium was independent of whether lithium was introduced into WMD via a lithium-neutralised dispersant, added during the calcium carbonate grinding step, or dosed into the final product. Lithium is a non-biocidal compound which has been discovered to be a potent and universal biocide enhancer. Lithium boosts the biocidal activity of various biocides and provides a novel technique to overcome biocide resistance in WMD. Such a biocide enhancer represents a breakthrough that offers a potential tool to revolutionise the consumption of biocidal agents in the WMD producing industry.

Keywords: Biocide; Enhancement; Resistance; Bacteria; Lithium; Calcium carbonate slurry; White mineral dispersion

Relationship between bioleaching performance, bacterial community structure and mineralogy in the bioleaching of a copper concentrate in stirred-tank reactors by Pauline Spolaore; Catherine Joulian; Jérôme Gouin; Dominique Morin; Patrick d’Hugues (pp. 441-448).

During the Bioshale European project, a techno-economic study of the bioleaching of a copper concentrate originating from a black shale ore was carried out. This concentrate is a multi-mineral resource in which the copper sulphides are mainly chalcocite, covellite, bornite and chalcopyrite. The experiments undertaken to produce the techno-economic data were also an opportunity to carry out more fundamental research. The objective of this work was to combine the results of the bioleaching experiments, in terms of copper recovery, with the results of bacterial community monitoring and mineralogy residue analysis. Batch and continuous bioleaching tests were carried out with 10% solids, at 42 °C and with a pH between 1.2 and 1.6. Final copper recovery was higher in batch cultures than in continuous mode (>95% vs. 91%). Mineralogical analysis showed that the limiting factor for copper recovery was incomplete chalcopyrite dissolution in both cases. However, chalcopyrite was even less dissolved in continuous conditions. This was also related to a variation in bacterial community structure. The population in all tests was composed of Acidithiobacillus caldus, Leptospirillum ferriphilum and one or two species of Sulfobacillus (Sulfobacillus thermosulfidooxidans and sometimes Sulfobacillus benefaciens), but Sulfobacillus and more generally sulphur oxidizers were more represented in batch mode. It was proposed that due to their capacity to reduce inorganic compounds, sulphur oxidizers may be efficient in limiting chalcopyrite surface hindering. It may help to better dissolve this mineral and reach a better copper recovery.

Keywords: Bioleaching; Black shale; Copper; Stirred-tank reactor; Sulfobacillus

Bioelectrochemical system accelerates microbial growth and degradation of filter paper by Kengo Sasaki; Shin-ichi Hirano; Masahiko Morita; Daisuke Sasaki; Norio Matsumoto; Naoya Ohmura; Yasuo Igarashi (pp. 449-455).

Bioelectrochemical reactors (BERs) with a cathodic working potential of −0.6 or −0.8 V more efficiently degraded cellulosic material, i.e., filter paper (57.4–74.1% in 3 days and 95.9–96.3% in 7 days) than did control reactors without giving exogenous potential (15.4% in 3 days and 64.2% in 7 days). At the same time, resultant conversions to methane and carbon dioxide in cathodic working chamber of BERs by application of electrochemical reduction in 3 days of operation were larger than control reactors. However, cumulative methane production in cathodic BERs was similar to those in control reactors after 7 days of operation. Microscopic observation and 16S rRNA gene analysis showed that microbial growth in the entire consortium was higher after 2 days of operation of cathodic BERs as compared with the control reactors. In addition, the number of methanogenic 16S rRNA gene copies in cathodic BERs was higher than in control reactors. Moreover, archaeal community structures constructed in cathodic BERs consisted of hydrogenotrophic methanogen-related organisms and differed from those in control reactors after 2 days of operation. Specifically, the amount of Methanothermobacter species in cathodic BERs was higher within archaeal communities than in those control reactors after 2 days of operation. Electrochemical reduction may be effective for accelerating microbial growth in the start-up period and thereby increasing microbial treatment of cellulosic waste and methane production.

Keywords: Bioelectrochemical reactor; Anaerobic degradation; Microbial community; Cathodic reaction

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Applied Microbiology and Biotechnology (v.89, #2)

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Applied Microbiology and Biotechnology (v.89, #2)