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Applied Microbiology and Biotechnology (v.87, #3)

Biology and biotechnology of Trichoderma by André Schuster; Monika Schmoll (pp. 787-799).

Fungi of the genus Trichoderma are soilborne, green-spored ascomycetes that can be found all over the world. They have been studied with respect to various characteristics and applications and are known as successful colonizers of their habitats, efficiently fighting their competitors. Once established, they launch their potent degradative machinery for decomposition of the often heterogeneous substrate at hand. Therefore, distribution and phylogeny, defense mechanisms, beneficial as well as deleterious interaction with hosts, enzyme production and secretion, sexual development, and response to environmental conditions such as nutrients and light have been studied in great detail with many species of this genus, thus rendering Trichoderma one of the best studied fungi with the genome of three species currently available. Efficient biocontrol strains of the genus are being developed as promising biological fungicides, and their weaponry for this function also includes secondary metabolites with potential applications as novel antibiotics. The cellulases produced by Trichoderma reesei, the biotechnological workhorse of the genus, are important industrial products, especially with respect to production of second generation biofuels from cellulosic waste. Genetic engineering not only led to significant improvements in industrial processes but also to intriguing insights into the biology of these fungi and is now complemented by the availability of a sexual cycle in T. reesei/Hypocrea jecorina, which significantly facilitates both industrial and basic research. This review aims to give a broad overview on the qualities and versatility of the best studied Trichoderma species and to highlight intriguing findings as well as promising applications.

Keywords: Trichoderma ; Hypocrea ; Cellulase; Biocontrol; Heterologous protein expression; Emerging human pathogen; Green mold disease; Biodiversity; Application; Biofuels

Oxalate decarboxylase: biotechnological update and prevalence of the enzyme in filamentous fungi by Miia R. Mäkelä; Kristiina Hildén; Taina K. Lundell (pp. 801-814).

Oxalate decarboxylase (ODC) is a manganese-containing, multimeric enzyme of the cupin protein superfamily. ODC is one of the three enzymes identified to decompose oxalic acid and oxalate, and within ODC catalysis, oxalate is split into formate and CO₂. This primarily intracellular enzyme is found in fungi and bacteria, and currently the best characterized enzyme is the Bacillus subtilis OxdC. Although the physiological role of ODC is yet unidentified, the feasibility of this enzyme in diverse biotechnological applications has been recognized for a long time. ODC could be exploited, e.g., in diagnostics, therapeutics, process industry, and agriculture. So far, the sources of ODC enzyme have been limited including only a few fungal and bacterial species. Thus, there is potential for identification and cloning of new ODC variants with diverse biochemical properties allowing e.g. more enzyme fitness to process applications. This review gives an insight to current knowledge on the biochemical characteristics of ODC, and the relevance of oxalate-converting enzymes in biotechnological applications. Particular emphasis is given to fungal enzymes and the inter-connection of ODC to fungal metabolism of oxalic acid.

Keywords: Oxalate decarboxylase; Wood-decaying fungi; Oxalic acid; Basidiomycetes; Cupin protein

Dipeptides in nutrition and therapy: cyanophycin-derived dipeptides as natural alternatives and their biotechnological production by Ahmed Sallam; Alexander Steinbüchel (pp. 815-828).

The numerous physiological functions of the nonessential amino acid L-aspartate, the semi-essential amino acid L-arginine, and the essential amino acid L-lysine, made them attractive for a wide range of nutritional and/or therapeutic applications. Furthermore, the administration of these amino acids as mixtures or as dipeptides for higher bioavailability is scientifically approved, and various commercial products of these forms are already available on the market. Although the industrial production of dipeptides is, with few exceptions, in an early stage, several strategies have been established and are compared in this review. Additionally, the recent developments in the technical production of aspartate–arginine and aspartate–lysine dipeptides from the biopolymer cyanophycin produced in microorganisms are discussed. Cyanophycin-derived dipeptides are produced exclusively by biotechnological procedures, probably possess higher bioavailability and may be used as better alternatives to the widely applied amino acid mixtures. Thus, the pivotal advantages and the potential applications of these dipeptides as well as of their constituting amino acids in nutrition and therapy are also discussed. Special emphasis is dedicated to arginine due to its numerous physiological roles in many cardiovascular, genitourinary, gastrointestinal, and immune disorders.

Keywords: Cyanophycin; Dipeptides; Arginine; Aspartic acid; Lysine

Mechanisms of ethanol tolerance in Saccharomyces cerevisiae by Menggen Ma; Z. Lewis Liu (pp. 829-845).

Saccharomyces cerevisiae is a superb ethanol producer, yet is also sensitive to higher ethanol concentrations especially under high gravity or very high gravity fermentation conditions. Ethanol tolerance is associated with interplay of complex networks at the genome level. Although significant efforts have been made to study ethanol stress response in past decades, mechanisms of ethanol tolerance are not well known. With developments of genome sequencing and genomic technologies, our understanding of yeast biology has been revolutionarily advanced. More evidence of mechanisms of ethanol tolerance have been discovered involving multiple loci, multi-stress, and complex interactions as well as signal transduction pathways and regulatory networks. Transcription dynamics and profiling studies of key gene sets including heat shock proteins provided insight into tolerance mechanisms. A transient gene expression response or a stress response to ethanol does not necessarily lead to ethanol tolerance in yeast. Reprogrammed pathways and interactions of cofactor regeneration and redox balance observed from studies of tolerant yeast demonstrated the significant importance of a time-course study for ethanol tolerance. In this review, we focus on current advances of our understanding for ethanol-tolerance mechanisms of S. cerevisiae including gene expression responses, pathway-based analysis, signal transduction and regulatory networks. A prototype of global system model for mechanisms of ethanol tolerance is presented.

Keywords: Gene expression; Genomic adaptation; Pathway analysis; Regulatory networks; Stress tolerance

Pretreatment of woody biomass for biofuel production: energy efficiency, technologies, and recalcitrance by J. Y. Zhu; Xuejun Pan; Ronald S. Zalesny Jr. (pp. 847-857).

This mini review discusses several key technical issues associated with cellulosic ethanol production from woody biomass: energy consumption for woody biomass pretreatment, pretreatment energy efficiency, woody biomass pretreatment technologies, and quantification of woody biomass recalcitrance. Both total sugar yield and pretreatment energy efficiency, defined as the total sugar recovery divided by total energy consumption for pretreatment, should be used to evaluate the performance of a pretreatment process. A post-chemical pretreatment wood size-reduction approach was proposed to significantly reduce energy consumption. The review also emphasizes using a low liquid-to-wood ratio (L/W) to reduce thermal energy consumption for any thermochemical/physical pretreatment in addition to reducing pretreatment temperature.

Keywords: Recalcitrance; Cellulosic ethanol; Woody/Forest biomass; SPORL; Enzymatic hydrolysis/saccharification; Pretreatment

Catabolism of citronellol and related acyclic terpenoids in pseudomonads by Karin Förster-Fromme; Dieter Jendrossek (pp. 859-869).

Terpenes are a huge group of natural compounds characterised by their predominantly pleasant smell. They are built up by isoprene units in cyclic or acyclic form and can be functionalised by carbonyl, hydroxyl or carboxyl groups and by presence of additional carbon–carbon double bonds (terpenoids). Currently, much more than 10,000 terpenoid compounds are known, and many thereof are present in different iso- and stereoforms. Terpenoids are secondary metabolites and can have important biological functions in living organisms. In many cases, the biological functions of terpenoids are not known at all. Nevertheless, terpenoids are used in large quantities as perfumes and aroma compounds for food additives. Terpenoids can be also precursors and building blocks for synthesis of complex chiral compounds in chemical and pharmaceutical industry. Unfortunately, only few terpenoids are available in large quantities at reasonable costs. Therefore, characterisation of suited biocatalysts specific for terpenoid compounds and development of biotransformation processes of abundant terpenoids to commercially interesting derivates becomes more and more important. This minireview summarises knowledge on catabolic pathways and biotransformations of acyclic monoterpenes that have received only little attention. Terpenoids with 20 or more carbon atoms are not a subject of this study.

Keywords: Acyclic terpenes; Monoterpenes; Citronellol; Citral; Pseudomonas aeruginosa ; Pseudomonas citronellolis ; Acyclic terpene utilisation (Atu) pathway; Leucine and isovalerate utilisation (Liu) pathway

New and classic families of secreted fungal heme peroxidases by Martin Hofrichter; René Ullrich; Marek J. Pecyna; Christiane Liers; Taina Lundell (pp. 871-897).

Heme-containing peroxidases secreted by fungi are a fascinating group of biocatalysts with various ecological and biotechnological implications. For example, they are involved in the biodegradation of lignocelluloses and lignins and participate in the bioconversion of other diverse recalcitrant compounds as well as in the natural turnover of humic substances and organohalogens. The current review focuses on the most recently discovered and novel types of heme-dependent peroxidases, aromatic peroxygenases (APOs), and dye-decolorizing peroxidases (DyPs), which catalyze remarkable reactions such as peroxide-driven oxygen transfer and cleavage of anthraquinone derivatives, respectively, and represent own separate peroxidase superfamilies. Furthermore, several aspects of the “classic” fungal heme-containing peroxidases, i.e., lignin, manganese, and versatile peroxidases (LiP, MnP, and VP), phenol-oxidizing peroxidases as well as chloroperoxidase (CPO), are discussed against the background of recent scientific developments.

Keywords: Peroxidases; Peroxygenases; P450; Oxygen transfer; Basidiomycota

Natural functions of mycotoxins and control of their biosynthesis in fungi by Massimo Reverberi; Alessandra Ricelli; Slaven Zjalic; Anna A. Fabbri; Corrado Fanelli (pp. 899-911).

Mycotoxins are harmful secondary metabolites produced by a range of widespread fungi belonging in the main to Fusarium, Aspergillus and Penicillium genera. But why should fungi produce toxins? And how is the biosynthesis of these toxins regulated? Several separate factors are now known to be capable of modulating mycotoxin synthesis; however, in this study, focussing just on mycotoxins whose regulatory mechanisms have already been established, we introduce a further factor based on a novel consideration. Various different mycotoxin biosynthetic pathways appear to share a common factor in that they are all susceptible to the influence of reactive oxygen species. In fact, when a fungus receives an external stimulus, it reacts by activating, through a well-defined signal cascade, a profound change in its lifestyle. This change usually leads to the activation of global gene regulators and, in particular, of transcription factors which modulate mycotoxin gene cluster expression. Some mycotoxins have a clear-cut role both in generating a pathogenetic process, i.e. fumonisins and some trichothecenes, and in competing with other organisms, i.e. patulin. In other cases, such as aflatoxins, more than one role can be hypothesised. In this review, we suggest an “oxidative stress theory of mycotoxin biosynthesis” to explain the role and the regulation of some of the above mentioned toxins.

Keywords: Mycotoxins; Oxidative stress; Oxylipins; Signal perception; Ecological role

The RNPP family of quorum-sensing proteins in Gram-positive bacteria by Jorge Rocha-Estrada; Angel E. Aceves-Diez; Gabriel Guarneros; Mayra de la Torre (pp. 913-923).

Quorum sensing is one of several mechanisms that bacterial cells use to interact with each other and coordinate certain physiological processes in response to cell density. This mechanism is mediated by extracellular signaling molecules; once a critical threshold concentration has been reached, a target sensor kinase or response regulator is activated (or repressed), facilitating the expression of quorum sensing-dependent genes. Gram-positive bacteria mostly use oligo-peptides as signaling molecules. These cells have a special kind of quorum-sensing systems in which the receptor protein interacts directly with its cognate signaling peptide. The receptors are either Rap phosphatases or transcriptional regulators and integrate the protein family RNPP, from Rap, Npr, PlcR, and PrgX. These quorum-sensing systems control several microbial processes, like sporulation, virulence, biofilm formation, conjugation, and production of extracellular enzymes. Insights of the mechanism of protein-signaling peptide binding as well as the molecular interaction among receptor protein, signaling peptide, and target DNA have changed some earlier perceptions. In spite of the increased knowledge and the potential biotechnological applications of these quorum-sensing systems, few examples on engineering for biotechnological applications have been published. Real applications will arise only when researchers working in applied microbiology and biotechnology are aware of the importance of quorum-sensing systems for health and bioprocess applications.

Keywords: Quorum sensing; Signaling peptides; RNPP protein family; Gram-positive bacteria

Effect of antibiotics in the environment on microbial populations by Chang Ding; Jianzhong He (pp. 925-941).

Antibiotics act as an ecological factor in the environment that could potentially affect microbial communities. The effects include phylogenetic structure alteration, resistance expansion, and ecological function disturbance in the micro-ecosystem. Numerous studies have detected changes of microbial community structure upon addition of antibiotics in soil and water environment. However, the causal relationship between antibiotic input and resistance expansion is still under debate, with evidence either supporting or declining the contribution of antibiotics on alteration of antibiotic resistance. Effects of antibiotics on ecological functions have also been discovered, including nitrogen transformation, methanogenesis, and sulfate reduction. In the latter part, this review discusses in detail on factors that influence antibiotic effects on microbial communities in soil and aquatic environment, including concentration of antibiotics, exposure time, added substrates, as well as combined effects of multiple antibiotics. In all, recent research progress offer an outline of effects of antibiotics in the natural environment. However, questions raised in this review need further investigation in order to provide a comprehensive risk assessment on the consequence of anthropogenic antibiotic input.

Keywords: Antibiotics; Resistance gene; Microbial community; Soil; Aquatic environment

Enhanced production of 2,3-butanediol from glycerol by forced pH fluctuations by Kaloyan Petrov; Penka Petrova (pp. 943-949).

The glycerol fermentation by Klebsiella pneumoniae occurs by receiving more than five liquid products—organic acids, diols, and ethanol. Aiming to direct the glycerol conversion towards predominant production of 2,3-butanediol (2,3-BD), the main influencing parameters (the aeration and the pH) were investigated during fed-batch processes. The regime of intensive aeration (2.2 vvm air supply) was evaluated as most favorable for 2,3-BD synthesis and ensured the decrease of all other metabolites. Thus, without pH control, 52.5 g/l 2,3-BD were produced, as the carbon conversion of glycerol into 2,3-BD reached 60.6%. Additional enhancement in 2,3-BD production (by significant increase of glycerol utilization) was achieved by the development of a new method of “forced pH fluctuations”. It was realized by consecutive raisings of pH using definite ΔpH value, at exact time intervals, allowing multiple variations. Thus, the optimal conditions for maximal glycerol consumption were defined, and 70 g/l 2,3-BD were produced, which is the highest amount obtained from glycerol as a sole carbon source until now. The forced pH fluctuations emphasized pH as a governing factor in microbial conversion processes.

Keywords: 2,3-Butanediol; Glycerol; Klebsiella pneumoniae ; pH fluctuation

Bacillus methanolicus pyruvate carboxylase and homoserine dehydrogenase I and II and their roles for l-lysine production from methanol at 50°C by Trygve Brautaset; Øyvind M. Jakobsen; Kristin F. Degnes; Roman Netzer; Ingemar Nærdal; Anne Krog; Rick Dillingham; Michael C. Flickinger; Trond E. Ellingsen (pp. 951-964).

We here present the pyc gene encoding pyruvate carboxylase (PC), and the hom-1 and hom-2 genes encoding two active homoserine dehydrogenase (HD) proteins, in methylotrophic Bacillus methanolicus MGA3. In general, both PC and HD are regarded as key targets for improving bacterial l-lysine production; PC plays a role in precursor oxaloacetate (OAA) supply while HD controls an important branch point in the l-lysine biosynthetic pathway. The hom-1 and hom-2 genes were strongly repressed by l-threonine and l-methionine, respectively. Wild-type MGA3 cells secreted 0.4 g/l l-lysine and 59 g/l l-glutamate under optimised fed batch methanol fermentation. The hom-1 mutant M168-20 constructed herein secreted 11 g/l l-lysine and 69 g/l of l-glutamate, while a sixfold higher l-lysine overproduction (65 g/l) of the previously constructed classical B. methanolicus mutant NOA2#13A52-8A66 was accompanied with reduced l-glutamate production (28 g/l) and threefold elevated pyc transcription level. Overproduction of PC and its mutant enzyme P455S in M168-20 had no positive effect on the volumetric l-lysine yield and the l-lysine yield on methanol, and caused significantly reduced volumetric l-glutamate yield and l-glutamate yield on methanol. Our results demonstrated that hom-1 represents one key target for achieving l-lysine overproduction, PC activity plays an important role in controlling l-glutamate production from methanol, and that OAA precursor supply is not a major bottleneck for l-lysine overproduction by B. methanolicus.

Keywords: Thermotolerant; Methylotrophy; Quantitative PCR; Anaplerotic; Fed batch fermentation; Metabolic engineering

Production of 2,3-butanediol from corncob molasses, a waste by-product in xylitol production by Ailong Wang; Yu Wang; Tianyi Jiang; Lixiang Li; Cuiqing Ma; Ping Xu (pp. 965-970).

Corncob molasses, a waste by-product in xylitol production, contains high concentrations of mixed sugars. In the present study, corncob molasses was used to produce 2,3-butanediol (BD) using Klebsiella pneumoniae SDM. This was the first report on the use of corncob molasses to produce bulk chemicals. Our results indicated that K. pneumoniae SDM can utilize various sugars contained in the corncob molasses in a preferential manner: glucose > arabinose > xylose. It was shown that high sugars concentration had an inhibitory effect on the cells growth and BD production. The maximum concentration of BD was 78.9 g/l after 61 h of fed-batch fermentation, giving a BD productivity of 1.3 g/l h and a yield of 81.4%. The present study suggests that the low-cost corncob molasses could be used as an alternative substrate for the production of BD by K. pneumoniae SDM, as well as a potential carbon source for production of other high-value chemicals.

Keywords: 2,3-Butanediol; Klebsiella pneumoniae ; Corncob molasses; Fermentation

Citric acid production from glycerol-containing waste of biodiesel industry by Yarrowia lipolytica in batch, repeated batch, and cell recycle regimes by Waldemar Rymowicz; Alina R. Fatykhova; Svetlana V. Kamzolova; Anita Rywińska; Igor G. Morgunov (pp. 971-979).

Yarrowia lipolytica A-101-1.22 produces high citric acid (112 g l⁻¹) with a yield of 0.6 g g⁻¹ and a productivity of 0.71 g l⁻¹ h⁻¹ during batch cultivation in the medium with glycerol-containing waste of biodiesel industry. However, it was observed that the specific citric acid production rate, which was maximal at the beginning of the biosynthesis, gradually decreases in the late production phase and it makes continuation of the process over 100 h pointless. The cell recycle and the repeated batch regimes were performed as ways for prolongation of citric acid synthesis by yeast. Using cell recycle, the active citric acid biosynthesis (96–107 g l⁻¹) with a yield of 0.64 g g⁻¹ and a productivity of 1.42 g l⁻¹ h⁻¹ was prolongated up to 300 h. Repeated batch culture remained stable for over 1000 h; the RB variant of 30% feed every 3 days showed the best results: 124.2 g l^-1 citric acid with a yield of 0.77 g g^-1 and a productivity of 0.85 g l^-1 h^-1.

Keywords: Yarrowia lipolytica ; Citric acid production; Glycerol-containing waste of biodiesel industry; Crude glycerol; Repeated batch; Cell recycle

A chromosomally based luminescent bioassay for mercury detection in red soil of China by He Wei; Han Cheng; Mao Ting; Zhong Wen-Hui; Lin Xian-Gui (pp. 981-989).

A luminescent reporter gene system was constructed by fusing the mercury-inducible promoter, P merT, and its regulatory gene, merR, with a promoterless reporter gene EGFP. A stable and nonantibiotic whole-cell reporter (BMB-ME) was created by introducing the system cassette into the chromosome of Pseudomonas putida strain and then applied it for mercury detection in the red soil of China. Spiked with 10 and 100 μg g⁻¹ Hg²⁺ and after 15 and 30 days incubation, soil samples were extracted and evaluated water soluble, bioavailable, organic matter bound, and residual fractions of mercury by both BMB-ME and chemical way. The expression of EGFP was confirmed in soil extraction, and fluorescence intensity was quantified by luminescence spectrometer. The sensor strain BMB-ME appeared to have a detection range similar to that of reversed-phase high-performance liquid chromatography method. The optimal temperature for EGFP expression was 35°C and the lowest detectable concentration of Hg²⁺ 200 nM. Cu²⁺, Fe²⁺, Mn²⁺, Sn²⁺, Zn²⁺, Co²⁺, Ag⁺, Ba²⁺, Mg²⁺, and Pb²⁺ ions at nanomolar level did not interfere with the measurement. These results showed that the BMB-ME constitute an adaptable system for easy sensing of small amounts of mercury in the red soil of China.

Keywords: Mercury; Biosensor; EGFP; Red soil

Heterologous expression, characterization and site-directed mutagenesis of cutinase CUTAB1 from Alternaria brassicicola by Katja Koschorreck; Danni Liu; Christian Kazenwadel; Rolf D. Schmid; Bernhard Hauer (pp. 991-997).

The cutinase CUTAB1 was cloned from a cutin induced culture of Alternaria brassicicola and heterologously expressed in Pichia pastoris under the control of the methanol-inducible AOX1 promoter. From a 400-ml culture, 36 mg of purified recombinant enzyme were obtained. Biochemical characterization revealed highest catalytic activity of the enzyme at 40°C and pH 7-9 using p-nitrophenyl palmitate (p-NPP) as substrate. Among several fatty acid methyl and ethyl esters, glycerol esters and p-nitrophenyl esters tested, CUTAB1 showed highest activity towards tributyrin (3,302 ± 160 U mg⁻¹) and the activity decreased with increase in chain length of the investigated esters. Lowest activity was found for p-NPP. Replacing Leu80, Leu181 and Ile183, respectively, by the smaller alanine in the hydrophobic binding loop of CUTAB1, drastically reduced the overall activity of the enzyme. On the other hand, mutation A84F located in the small helical flap of CUTAB1 significantly increased the activity of the enzyme towards longer chain substrates like p-NPP.

Keywords: Cutinase; Alternaria brassicicola ; Expression; Substrate specificity; Site-directed mutagenesis

Enhancement of keratinolytic activity of a thermophilic subtilase by improving its autolysis resistance and thermostability under reducing conditions by Xiaoliang Liang; Yan Bian; Xiao-Feng Tang; Gengfu Xiao; Bing Tang (pp. 999-1006).

WF146 protease, a thermophilic subtilase from thermophile Bacillus sp. WF146, suffers excessive autolysis in the presence of reducing agents. In this report, two autolytic sites of WF146 protease were modified by site-directed mutagenesis. The introduction of prolines into the autolytic sites increased the autolysis resistance of the enzyme under reducing conditions. The double mutant N63P/A66P displayed a 2.8-fold longer half-life at 80°C and higher hydrolytic activities than wild-type enzyme toward soluble (casein) and insoluble (keratin azure) substrates at high temperatures. In the presence of reducing agents, N63P/A66P was able to degrade feather at 80°C (∼3 h), with hydrolysis efficiency comparable to that of proteinase K at 50°C (∼24 h). Meanwhile, the mutant N63P/A66P had the ability to hydrolyze PrP^Sc-like prion protein at high temperatures. In virtue of these properties, N63P/A66P is of great interest to be used in recycling of keratinous wastes, such as feather, and disinfection of medical apparatus. In addition, our study may provide useful information needed to explore keratinolytic potential of thermophilic subtilases, even if they are produced by non-keratinolytic microorganisms.

Keywords: Keratinase; Autolysis; Thermophilic subtilase; Feather; Prion

Characterization of the family GH54 α-l-arabinofuranosidases in Penicillium funiculosum, including a novel protein bearing a cellulose-binding domain by Olivier Guais; Olivier Tourrasse; Marion Dourdoigne; Jean Luc Parrou; Jean Marie Francois (pp. 1007-1021).

The soil deuteromycete Penicillium funiculosum is characterized by its remarkable capacity to produce a wide variety of cellulolytic and hemicellulolytic enzymes. In the course of the genome sequencing of this industrial fungus, four different genes encoding glycosyl hydrolase family 54 (GH54)22 α-l-arabinofuranosidases were identified. Three of them termed PfabfB1, PfabfB3, and PfabfB4 were highly similar, encoding proteins of 507, 508, and 505 amino acids, respectively. They exhibited structural features typical of GH54 enzymes, including an N-terminal catalytic domain connected to a C-terminal arabinose-binding domain (ABD). The fourth gene termed PfafbB2 codes for an unusual 400 amino acid length GH54 α-l-arabinofuranosidase, in which the ABD was replaced by a fungal cellulose-binding domain (fCBD). This domain was shown to be functional since it allowed this protein to be retained onto microcrystalline cellulose, and the fusion of this CBD to the C-terminal end of PfAbfB1 allowed this protein to bind to cellulose. Expression analysis of the four PfabfB genes during an industrial-like process fermentation on complex carbohydrates revealed that PfafB2 was expressed more than 20,000-fold, while PfabfB3 and PfabfB4 were increased moderately at the end of the fermentation. In contrast, the transcript levels of PfabfB1 remained unchanged throughout the process. This new type of GH54 α-arabinofuranosidase encoded by PfabfB2 showed enzymatic properties slightly different to those of other GH54 enzymes characterized so far, including a higher thermostability, an optimum pH, and temperature of 2.6 and 50 °C, instead of 3.5 and 60 °C as found for PfAbfB1. Nonetheless, like other GH54 α-arabinofuranosidases, PfAbfB2 was able to release arabinose from various sources of branched arabinoxylan and arabinan.

Keywords: Arabinofuranosidase; Hemicelluloses; Carbohydrate module; Protein secretion; Filamentous fungi

Molecular cloning and characterization of a novel metagenome-derived multicopper oxidase with alkaline laccase activity and highly soluble expression by Mao Ye; Gang Li; Wei Qu Liang; Yu Huan Liu (pp. 1023-1031).

Lac591, a gene encoding a novel multicopper oxidase with laccase activity, was identified through activity-based functional screening of a metagenomic library from mangrove soil. Sequence analysis revealed that lac591 encodes a protein of 500 amino acids with a predicted molecular mass of 57.4 kDa. Lac591 was overexpressed heterologously as soluble active enzyme in Escherichia coli and purified, giving rise to 380 mg of purified enzyme from 1 l induced culture, which is the highest expression report for bacterial laccase genes so far. Furthermore, the recombinant enzyme demonstrated activity toward classical laccase substrates syringaldazine (SGZ), guaiacol, and 2, 6-dimethoxyphenol (2, 6-DMP). The purified Lac591 exhibited maximal activity at 55°C and pH 7.5 with guaiacol as substrate and was found to be stable in the pH range of 7.0–10.0. The substrate specificity on different substrates was studied with the purified enzyme, and the optimal substrates were in the order of 2, 6-DMP > catechol > α-naphthol > guaiacol > SGZ > 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid). The alkaline activity and highly soluble expression of Lac591 make it a good candidate of laccases in industrial applications for which classical laccases are unsuitable, such as biobleaching of paper pulp and dyestuffs processing.

Keywords: Multicopper oxidase; Alkaline laccase; Gene cloning; Enzyme characterization; Metagenome

Catalase overexpression reduces the germination time and increases the pathogenicity of the fungus Metarhizium anisopliae by Claudia Erika Morales Hernandez; Israel Enrique Padilla Guerrero; Gloria Angelica Gonzalez Hernandez; Eduardo Salazar Solis; Juan Carlos Torres Guzman (pp. 1033-1044).

Catalases and peroxidases are the most important enzymes that degrade hydrogen peroxide into water and oxygen. These enzymes and superoxide dismutase are the first lines of cell defense against reactive oxygen species. Metarhizium anisopliae displays an increase in catalase–peroxidase activity during germination and growth. To determine the importance of catalase during the invasion process of M. anisopliae, we isolated the cat1 gene. cat1 cDNA expression in Escherichia coli and the subsequent purification of the protein confirmed that the cat1 gene codes for a monofunctional catalase. Expression analysis of this gene by RT-PCR from RNA isolated from fungus grown in liquid cultures showed a decrease in the expression level of the cat1 gene during germination and an increase during mycelium growth. The expression of this gene in the fungus during the infection process of the larvae of Plutella xylostella also showed a significant increase during invasive growth. Transgenic strains overexpressing the cat1 gene had twice the catalase activity of the wild-type strain. This increase in catalase activity was accompanied by a higher level of resistance to exogenous hydrogen peroxide and a reduction in the germination time. This improvement was also observed during the infection of P. xylostella larvae. M. anisopliae transgenic strains overexpressing the cat1 gene grew and spread faster in the soft tissue of the insect, reducing the time to death of the insect by 25% and the dose required to kill 50% of the population 14-fold.

Keywords: Catalase; Metarhizium anisopliae ; Entomopathogenic fungi; Biocontrol

Engineering Corynebacterium glutamicum for isobutanol production by Kevin Michael Smith; Kwang-Myung Cho; James C. Liao (pp. 1045-1055).

The production of isobutanol in microorganisms has recently been achieved by harnessing the highly active 2-keto acid pathways. Since these 2-keto acids are precursors of amino acids, we aimed to construct an isobutanol production platform in Corynebacterium glutamicum, a well-known amino-acid-producing microorganism. Analysis of this host’s sensitivity to isobutanol toxicity revealed that C. glutamicum shows an increased tolerance to isobutanol relative to Escherichia coli. Overexpression of alsS of Bacillus subtilis, ilvC and ilvD of C. glutamicum, kivd of Lactococcus lactis, and a native alcohol dehydrogenase, adhA, led to the production of 2.6 g/L isobutanol and 0.4 g/L 3-methyl-1-butanol in 48 h. In addition, other higher chain alcohols such as 1-propanol, 2-methyl-1-butanol, 1-butanol, and 2-phenylethanol were also detected as byproducts. Using longer-term batch cultures, isobutanol titers reached 4.0 g/L after 96 h with wild-type C. glutamicum as a host. Upon the inactivation of several genes to direct more carbon through the isobutanol pathway, we increased production by ∼25% to 4.9 g/L isobutanol in a ∆pyc∆ldh background. These results show promise in engineering C. glutamicum for higher chain alcohol production using the 2-keto acid pathways.

Keywords: Biofuel; Isobutanol; C. glutamicum

Overexpression of aveBIV leading to the improvement of 4′-epidaunorubicin production in Streptomyces coeruleorubidus strain SIPI-A0707 by Lei Shao; Jia Huang; Lan Jing; Ji-Ye Chen; Shi-Dong Kan; Min Wang; Ji-An Li; Dai-Jie Chen (pp. 1057-1064).

The 4′-epidaunorubicin is the semisynthesis precursor of epirubicin which is a clinically useful antitumor drug thought to have slightly less cardiotoxicity than doxorubin. The 4′-epidaunorubicin was formed by overexpression of heterologous Streptomyces avermitilis aveBIV in Streptomyces coeruleorubidus SIPI-A0707 dnmV mutant blocked in the biosynthesis of daunosamine, the deoxysugar component of daunorubicin. But there was a low-yield production of 4′-epidaunorubicin. In our study, product yields were enhanced considerably by increasing aveBIV gene copy number or changing means of aveBIV integration. The 4′-epidaunorubicin titer was improved by around threefold in the recombinant strain DYG1006 with the aveBIV increased threefold copy numbers. The transcript levels of aveBIV gene meet the expectation of fermentation levels of 4′-epidaunorubicin. The method described here provides the means to produce 4′-epidaunorubicin efficiently on an industrial scale.

Keywords: Epidaunorubicin; Daunorubicin; Biosynthesis; Gene replacement; Overexpression

Development of a highly efficient gene targeting system allowing rapid genetic manipulations in Penicillium decumbens by Zhong-Hai Li; Chun-Mei Du; Yao-Hua Zhong; Tian-Hong Wang (pp. 1065-1076).

Penicillium decumbens is an important industrial filamentous fungus and has been widely used in biorefinery due to its high production of cellulase and hemicellulase. However, molecular engineering has still rarely been applied for strain improvement in P. decumbens. It has been proven that gene targeting manipulation in many filamentous fungi is hampered by nonhomologous end-joining (NHEJ) pathway. To improve gene targeting efficiency in P. decumbens, the putative pku70 encoding the Ku70 homologue involved in the NHEJ pathway was identified and deleted. The Δpku70 strain showed no apparent defect in vegetative growth, conidiation, and cellulase production, and displayed similar sensitivity to chemical agents of hygromycin B, ethyl methane sulfonate, and H₂O₂ at different concentrations compared with the wild-type strain. The effect of the absence of pku70 on gene targeting was tested by disruption of creA encoding a putative carbon catabolite repressor and xlnR encoding a putative transcriptional activator. Efficiency of gene targeting for both genes was 100% in the Δpku70 strain, compared with the low efficiency in the wild-type recipient. Furthermore, the integration types for three single targeting cassettes and the cotransformation of two independent targeting cassettes were primarily investigated in P. decumbens. The highly efficient gene targeting system established in this study will open the way to large-scale functional genomic analysis in P. decumbens and contribute to the study of the mechanism of lignocellulose degradation by P. decumbens.

Keywords: Penicillium decumbens ; Pku70; Gene targeting; Homologous recombination; Nonhomologous end-joining

Cloning and functional analysis of the second geranylgeranyl diphosphate synthase gene influencing helvolic acid biosynthesis in Metarhizium anisopliae by Suthitar Singkaravanit; Hiroshi Kinoshita; Fumio Ihara; Takuya Nihira (pp. 1077-1088).

A gene (ggs2) having high similarity to the geranylgeranyl diphosphate synthase (GGPP synthase) gene was cloned from Metarhizium anisopliae NAFF635007. The ggs2 gene (1,239-bp open reading frame with no intron) encoded a protein of 412 amino acids, and the transcription occurred only after late log-phase during the growth. Gene disruption of ggs2, performed to clarify the function in M. anisopliae, resulted in decreased GGPP synthase activity together with a slight delay of sporulation. An high performance liquid chromatography (HPLC) comparison of compound profiles between the wild-type strain and the disruptant revealed that a compound was abolished by the ggs2 disruption. Purification and structural elucidation by ¹H-NMR and mass spectrometry analyses revealed that the lost compound is helvolic acid. Furthermore, the pathogenicity assay against two species of insect larvae revealed that the ggs2-disruptant possessed much weaker toxicity than the wild-type strain. Based on these results, it was concluded that ggs2 encodes the GGPP synthase influencing the biosynthesis of secondary metabolites in various species, including helvolic acid in M. anisopliae. To the best of our knowledge, this is the first report to identify a GGPP synthase gene related to secondary metabolism in entomopathogenic fungi.

Keywords: Geranylgeranyl diphosphate synthase; Metarhizium anisopliae ; Helvolic acid; Secondary metabolism

New insights into the effect of medium-chain-length lactones on yeast membranes. Importance of the culture medium by Thi Minh Ngoc Ta; Lan Cao-Hoang; Hanh Phan-Thi; Hai Dang Tran; Nadhuirata Souffou; Joseph Gresti; Pierre-André Marechal; Jean-François Cavin; Yves Waché (pp. 1089-1099).

In hydrophobic compounds biotechnology, medium-chain-length metabolites often perturb cell activity. Their effect is usually studied in model conditions of growth in glucose media. Here, we study whether culture on lipids has an impact on the resistance of Yarrowia lipolytica to such compounds: Cells were cultured on glucose or oleate and submitted to γ-dodecalactone. After a 60-min exposure to 3 g L⁻¹, about 80% of the glucose-grown cells (yeast extract peptone dextrose (YPD) cells) had lost their cultivability, 38% their membrane integrity, and 31% their reducing capacity as shown with propidium iodide and methylene blue, respectively. For oleate-grown cells, treatment at 6 g L⁻¹ did not alter cultivability despite some transient loss of membrane integrity from 3 g L⁻¹. It was shown with diphenylhexatriene and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene that oleate-grown cells had membranes more fluid and less sensitive to the lactone-induced fluidization. Analyses revealed also higher contents of ergosterol but, for YPD- and minimum-oleate-grown cells (YNBO cells), the addition of lactone provoked a decrease in the concentration of ergosterol in a way similar to the depletion by methyl-β-cyclodextrin and an important membrane fluidization. Ergosterol depletion or incorporation increased or decreased, respectively, cell sensitivity to lactone. This study shows that the embedment of oleate moieties into membranes as well as higher concentrations of sterol play a role in the higher resistance to lactone of oleate-grown cells (YPO cells). Similar oleate-induced increase in resistance was also observed for Rhodotorula and Candida strains able to grow on oleate as the sole carbon source whereas Saccharomyces and Sporidiobolus cells were more sensitive after induction.

Keywords: Membrane; Fluidity; Ergosterol; Yarrowia lipolytica ; Stress; Lipid metabolism; Lactone

Anti-yeast activity of a food-grade dilution-stable microemulsion by Hui Zhang; Yaoqi Xu; Lijiang Wu; Xiaodong Zheng; Songming Zhu; Fengqin Feng; Lirong Shen (pp. 1101-1108).

The anti-yeast activities of a food-grade dilution-stable microemulsion against Candida albicans and Saccharomyces cerevisiae have been studied. The weight ratio of the formulated microemulsion is glycerol monolaurate (GML)/propionic acid/Tween 80/sodium benzoate (SB)/water = 3:9:14:14:24. Results of anti-yeast activity on solid medium by agar diffusion method showed that the anti-yeast activity of the microemulsion at 4.8 mg/ml was comparable to that of natamycin at 0.1 mg/ml as positive control. Results of anti-yeast activity in liquid medium by broth dilution method showed that the growth of both C. albicans and S. cerevisiae was completely inhibited when the liquid medium containing 10⁶ cfu/ml was treated with 1.2 mg/ml microemulsion, which was determined as minimum fungicidal concentration. The kinetics of killing results showed that the microemulsion killed over 90% yeast cells rapidly within 15 min and caused a complete loss of viability in 120 min. Among the components, SB and GML had a similar anti-yeast activity, followed by propionic acid, while Tween 80 exhibited no activity and could not enhance the anti-yeast activities of these components, and it was revealed that the anti-yeast activity of the microemulsion was attributed to a combination of propionic acid, GML, and SB. The anti-yeast activity of the microemulsion was in good agreement with the leakage of 260-nm absorbing materials and the observation of transmission electron microscopy, indicating that the microemulsion induced the disruption and dysfunction of the cell membrane.

Keywords: Microemulsion; Anti-yeast activity; Leakage of 260-nm absorbing materials; Transmission electron microscopy

Effects of biocides on gene expression in the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough by Meng-Hsin Phoebe Lee; Sean M. Caffrey; Johanna K. Voordouw; Gerrit Voordouw (pp. 1109-1118).

Although sulfate-reducing bacteria (SRB), such as Desulfovibrio vulgaris Hildenborough (DvH) are often eradicated in oil and gas operations with biocides, such as glutaraldehyde (Glut), tetrakis (hydroxymethyl) phosphonium sulfate (THPS), and benzalkonium chloride (BAC), their response to these agents is not well known. Whole genome microarrays of D. vulgaris treated with biocides well below the minimum inhibitory concentration showed that 256, 96, and 198 genes were responsive to Glut, THPS, and BAC, respectively, and that these three commonly used biocides affect the physiology of the cell quite differently. Glut induces expression of genes required to degrade or refold proteins inactivated by either chemical modification or heat shock, whereas BAC appears to target ribosomal structure. THPS appears to primarily affect energy metabolism of SRB. Mutants constructed for genes strongly up-regulated by Glut, were killed by Glut to a similar degree as the wild type. Hence, it is difficult to achieve increased sensitivity to this biocide by single gene mutations, because Glut affects so many targets. Our results increase understanding of the biocide's mode of action, allowing a more intelligent combination of mechanistically different agents. This can reduce stress on budgets for chemicals and on the environment.

Keywords: Biocide; Souring; Sulfide; Sulfate; SRB; Gene expression; Microarray

Identification of polyhydroxyalkanoates in Halococcus and other haloarchaeal species by Andrea Legat; Claudia Gruber; Klaus Zangger; Gerhard Wanner; Helga Stan-Lotter (pp. 1119-1127).

Polyhydroxyalkanoates (PHAs) are accumulated in many prokaryotes. Several members of the Halobacteriaceae produce poly-3-hydroxybutyrate (PHB), but it is not known if this is a general property of the family. We evaluated identification methods for PHAs with 20 haloarchaeal species, three of them isolates from Permian salt. Staining with Sudan Black B, Nile Blue A, or Nile Red was applied to screen for the presence of PHAs. Transmission electron microscopy and ¹H-nuclear magnetic resonance spectroscopy were used for visualization of PHB granules and chemical confirmation of PHAs in cell extracts, respectively. We report for the first time the production of PHAs by Halococcus sp. (Halococcus morrhuae DSM 1307^T, Halococcus saccharolyticus DSM 5350^T, Halococcus salifodinae DSM 8989^T, Halococcus dombrowskii DSM 14522^T, Halococcus hamelinensis JCM 12892^T, Halococcus qingdaonensis JCM 13587^T), Halorubrum sp. (Hrr. coriense DSM 10284^T, Halorubrum chaoviator DSM 19316^T, Hrr. chaoviator strains NaxosII and AUS-1), haloalkaliphiles (Natronobacterium gregoryi NCMB 2189^T, Natronococcus occultus DSM 3396^T) and Halobacterium noricense DSM 9758^T. No PHB was detected in Halobacterium salinarum NRC-1 ATCC 700922, Hbt. salinarum R1 and Haloferax volcanii DSM 3757^T. Most species synthesized PHAs when growing in synthetic as well as in complex medium. The polyesters were generally composed of PHB and poly-ß-hydroxybutyrate-co-3-hydroxyvalerate (PHBV). Available genomic data suggest the absence of PHA synthesis in some haloarchaea and in all other Euryarchaeota and Crenarchaeota. Homologies between haloarchaeal and bacterial PHA synthesizing enzymes had indicated to some authors probable horizontal gene transfer, which, considering the data obtained in this study, may have occurred already before Permian times.

Keywords: Polyhydroxybutyrate; Haloarchaea; Halococcus ; Halobacterium ; Haloalkaliphile

The role of acyl-coenzyme A carboxylase complex in lipstatin biosynthesis of Streptomyces toxytricini by Atanas V. Demirev; Anamika Khanal; Bhishma R. Sedai; Si Kyu Lim; Min Kyun Na; Doo Hyun Nam (pp. 1129-1139).

Streptomyces toxytricini produces lipstatin, a specific inhibitor of pancreatic lipase, which is derived from two fatty acid moieties with eight and 14 carbon atoms. The pccB gene locus in 10.6 kb fragment of S. toxytricini chromosomal DNA contains three genes for acyl-coenzyme A carboxylase (ACCase) complex accA3, pccB, and pccE that are presumed to be involved in secondary metabolism. The pccB gene encoding a β subunit of ACCase [carboxyltransferase (CT)] was identified upstream of pccE gene for a small protein of ε subunit. The accA3 encoding the α subunit of ACCase [biotin carboxylase (BC)] was also identified downstream of pccB gene. When the pccB and pccE genes were inactivated by homologous recombination, the lipstatin production was reduced as much as 80%. In contrast, the accumulation of another compound, tetradeca-5.8-dienoic acid (the major lipstatin precursor), was 4.5-fold increased in disruptant compared with wild-type. It implies that PccB of S. toxytricini is involved in the activation of octanoic acid to hexylmalonic acid for lipstatin biosynthesis.

Keywords: Acyl-CoA carboxylase; pccB ; Lipstatin; Octanoic acid; Tetradeca-5.8-dienoic acid; Streptomyces toxytricini

Direct and efficient cloning of full-length genes from environmental DNA by RT-qPCR and modified TAIL-PCR by Huoqing Huang; Guozeng Wang; Yanyu Zhao; Pengjun Shi; Huiying Luo; Bin Yao (pp. 1141-1149).

Environmental DNA (eDNA) is defined as the total DNA that can be isolated from environmental samples. In total, therefore, eDNA includes a vast functional genes, and various approaches have been developed to retrieve full-length functional genes from eDNA. The efficiency of PCR amplification of eDNA is limited, however, because in truth, the net content of actual target functional genes is rather low in eDNA. To address this technical challenge, we developed a fast and effective approach to cloning full-length functional genes from eDNA. Two important modifications were made to existing PCR-based methods: first, a real-time quantitative PCR step was added to assess the difficulty of obtaining full-length genes; second, we improved the thermal asymmetric interlaced PCR program to make it more effective for cloning the flanking regions of known fragments that are present at low abundance in eDNA. Using this approach, five novel full-length functional genes with very low identity to known genes were cloned from environmental samples. This approach has great potential for allowing discovery of functional genes from environmental sources and may be broadly applicable to molecular biology research.

Keywords: Environmental DNA; Xylanase; Phytase; TAIL-PCR; RT-qPCR

Sulfonylurea resistance as a new selectable marker for the entomopathogenic fungus Beauveria bassiana by Shizhu Zhang; Yanhua Fan; Yu Xian Xia; Nemat O. Keyhani (pp. 1151-1156).

Beauveria bassiana is a filamentous ascomycete that is pathogenic towards a broad host range of insect targets and is increasingly serving as a model for examining fungal development and host-pathogen interactions. B. bassiana displays a prohibitive level of resistance against many current fungal and/or yeast selection markers including hygromycin, neomycin, and zeocin. A genetic transformation system for B. bassiana based upon the use of a sulfonylurea resistance cassette derived from the Magnaporthe grisea, acetolactate synthase gene (sur) was developed. The transformation frequency ranged from 100–150 transformants per microgram DNA/10⁸ cells and Southern blot analysis indicated that the plasmid vector was randomly integrated into the genome of B. bassiana. In addition, a construct bearing the sur gene and the enhanced green fluorescent protein gene egfp as a visual marker was used to successfully transform B. bassiana. Over 95% of the transformants retained the sulfonylurea resistance phenotype under non-selective conditions. The described transformation method increases opportunities for the genetic manipulation of B. bassiana.

Keywords: Beauveria bassiana ; Sulfonylurea; Fungal transformation; Egfp; Pathogen

Factors influencing antibiotic resistance burden in municipal wastewater treatment plants by Ana Novo; Célia M. Manaia (pp. 1157-1166).

Municipal wastewater treatment plants are recognized reservoirs of antibiotic-resistant bacteria. Three municipal wastewater treatment plants differing on the dimensions and bio-treatment processes were compared for the loads of amoxicillin-, tetracycline-, and ciprofloxacin-resistant heterotrophic bacteria, enterobacteria, and enterococci in the raw inflow and in the treated effluents. The sewage received by each plant, in average, corresponded to 85,000 inhabitant equivalents (IE), including pretreated industrial effluents (≤30%) in plant activated sludge, 105,000 IE, including pretreated hospital effluents (≤15%) in plant trickling filter, and 2,000 IE, exclusively of domestic sewage, in plant submerged aerated filter. The presence of pretreated industrial effluents or of pretreated hospital sewage in the raw inflow did not imply significantly higher densities (per milliliter or per IE) of antibiotic-resistant bacteria in the raw wastewater. Longer hydraulic residence periods (24 h) corresponded to higher bacterial removal rates than shorter periods (12 and 9 h), although such efficiency did not imply significant average decreases in the antibiotic resistance prevalence of the treated effluent. The bacterial loads in the treated effluent could be ranked according to the treatment efficiency, suggesting that the characteristics of the raw inflow may have less relevance on the quality of the treated wastewater than other aspects, such as the inflow volume, the type of biological treatment, or the hydraulic residence time.

Keywords: Wastewater treatment; Antibiotic resistance; Amoxicillin; Tetracycline; Ciprofloxacin

Characterization and quantification of ammonia-oxidizing archaea (AOA) and bacteria (AOB) in a nitrogen-removing reactor using T-RFLP and qPCR by Tao Jin; Tong Zhang; Qingmei Yan (pp. 1167-1176).

Using ammonia monooxygenase α-subunit (amoA) gene and 16S rRNA gene, the community structure and abundance of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) in a nitrogen-removing reactor, which was operated for five phases, were characterized and quantified by cloning, terminal restriction fragment length polymorphism (T-RFLP), and quantitative polymerase chain reaction (qPCR). The results suggested that the dominant AOB in the reactor fell to the genus Nitrosomonas, while the dominant AOA belonged to Crenarchaeotal Group I.1a in phylum Crenarchaeota. Real-time PCR results demonstrated that the levels of AOB amoA varied from 2.9 × 10³ to 2.3 × 10⁵ copies per nanogram DNA, greatly (about 60 times) higher than those of AOA, which ranged from 1.7 × 10² to 3.8 × 10³ copies per nanogram DNA. This indicated the possible leading role of AOB in the nitrification process in this study. T-RFLP results showed that the AOB community structure significantly shifted in different phases while AOA only showed one major peak for all the phases. The analyses also suggested that the AOB community was more sensitive than that of AOA to operational conditions, such as ammonia loading and dissolved oxygen.

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

Efficient conversion of lactic acid to butanol with pH-stat continuous lactic acid and glucose feeding method by Clostridium saccharoperbutylacetonicum by Mugihito Oshiro; Katsuhiro Hanada; Yukihiro Tashiro; Kenji Sonomoto (pp. 1177-1185).

In order to achieve high butanol production by Clostridium saccharoperbutylacetonicum N1-4, the effect of lactic acid on acetone–butanol–ethanol fermentation and several fed-batch cultures in which lactic acid is fed have been investigated. When a medium containing 20 g/l glucose was supplemented with 5 g/l of closely racemic lactic acid, both the concentration and yield of butanol increased; however, supplementation with more than 10 g/l lactic acid did not increase the butanol concentration. It was found that when fed a mixture of lactic acid and glucose, the final concentration of butanol produced by a fed-batch culture was greater than that produced by a batch culture. In addition, a pH-controlled fed-batch culture resulted in not only acceleration of lactic acid consumption but also a further increase in butanol production. Finally, we obtained 15.5 g/l butanol at a production rate of 1.76 g/l/h using a fed-batch culture with a pH-stat continuous lactic acid and glucose feeding method. To confirm whether lactic acid was converted to butanol by the N1-4 strain, we performed gas chromatography–mass spectroscopy (GC-MS) analysis of butanol produced by a batch culture during fermentation in a medium containing [1,2,3-¹³C₃] lactic acid as the initial substrate. The results of the GC-MS analysis confirmed the bioconversion of lactic acid to butanol.

Keywords: Acetone–butanol–ethanol fermentation; Lactic acid; Butanol; Clostridium saccharoperbutylacetonicum N1-4; pH-stat fed-batch culture; GC-MS analysis

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Applied Microbiology and Biotechnology (v.87, #3)

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Applied Microbiology and Biotechnology (v.87, #3)