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Applied Microbiology and Biotechnology (v.89, #5)

Special content of this issue by Alexander Steinbüchel (pp. 1265-1265).

Glucoamylases: structural and biotechnological aspects by Julia Marín-Navarro; Julio Polaina (pp. 1267-1273).

Glucoamylases, one of the main types of enzymes involved in starch hydrolysis, are exo-acting enzymes that release consecutive glucose units from the non-reducing ends of starch molecules. Glucoamylases are microbial enzymes, present in bacteria, archaea, and fungi but not in plants and animals. Structurally, they are classified in family 15 of glycoside hydrolases and characterised by the invariable presence of a catalytic domain with (α/α)₆-fold, often bound to a non-catalytic domain of diverse origin and function. Fungal glucoamylases are biotechnologically very important as they are used industrially in large amounts and have been extensively studied during the past 30 years. Prokaryotic glucoamylases are of biotechnological relevance for being generally thermophilic enzymes, active at elevated temperatures.

Keywords: Family 15 glycoside hydrolases; Industrial enzyme; Starch; Starch-binding domain

Light requirements in microalgal photobioreactors: an overview of biophotonic aspects by Ana P. Carvalho; Susana O. Silva; José M. Baptista; F. Xavier Malcata (pp. 1275-1288).

In order to enhance microalgal growth in photobioreactors (PBRs), light requirement is one of the most important parameters to be addressed; light should indeed be provided at the appropriate intensity, duration, and wavelength. Excessive intensity may lead to photo-oxidation and -inhibition, whereas low light levels will become growth-limiting. The constraint of light saturation may be overcome via either of two approaches: increasing photosynthetic efficiency by genetic engineering, aimed at changing the chlorophyll antenna size; or increasing flux tolerance, via tailoring the photonic spectrum, coupled with its intensity and temporal characteristics. These approaches will allow an increased control over the illumination features, leading to maximization of microalgal biomass and metabolite productivity. This minireview briefly introduces the nature of light, and describes its harvesting and transformation by microalgae, as well as its metabolic effects under excessively low or high supply. Optimization of the photosynthetic efficiency is discussed under the two approaches referred to above; the selection of light sources, coupled with recent improvements in light handling by PBRs, are chronologically reviewed and critically compared.

Keywords: Flashing light effect; Microalgae; Photosynthesis; Photobioreactor; Photo-inhibition; Photo-oxidation

Weedy lignocellulosic feedstock and microbial metabolic engineering: advancing the generation of ‘Biofuel’ by Anuj K. Chandel; Om V. Singh (pp. 1289-1303).

Lignocellulosic materials are the most abundant renewable organic resources (~200 billion tons annually) on earth that are readily available for conversion to ethanol and other value-added products, but they have not yet been tapped for the commercial production of fuel ethanol. The lignocellulosic substrates include woody substrates such as hardwood (birch and aspen, etc.) and softwood (spruce and pine, etc.), agro residues (wheat straw, sugarcane bagasse, corn stover, etc.), dedicated energy crops (switch grass, and Miscanthus etc.), weedy materials (Eicchornia crassipes, Lantana camara etc.), and municipal solid waste (food and kitchen waste, etc.). Despite the success achieved in the laboratory, there are limitations to success with lignocellulosic substrates on a commercial scale. The future of lignocellulosics is expected to lie in improvements of plant biomass, metabolic engineering of ethanol, and cellulolytic enzyme-producing microorganisms, fullest exploitation of weed materials, and process integration of the individual steps involved in bioethanol production. Issues related to the chemical composition of various weedy raw substrates for bioethanol formation, including chemical composition-based structural hydrolysis of the substrate, need special attention. This area could be opened up further by exploring genetically modified metabolic engineering routes in weedy materials and in biocatalysts that would make the production of bioethanol more efficient.

Keywords: Lignocellulose; Weed lignocelluloses; Bioethanol; Biorefinery; Fermentation

High hydrostatic pressure and biology: a brief history by Gérard Demazeau; Nolwennig Rivalain (pp. 1305-1314).

Pressure as a thermodynamical parameter was successively introduced in physics, hydrometallurgy, geochemistry, and biology. In all cases, the main objective was to recreate a natural phenomenon (gas or liquid compressibility, synthesis or crystal growth of minerals, survival of deep sea microorganisms…). The introduction of high hydrostatic pressure (HHP) in Biology was an important scientific feature over the last hundred years. This paper describes the different steps that have led to the spreading of pressure in biology and the opening of new frontiers either in basic and applied researches due to the specific characteristics of the pressure parameter. Because of the low energy conveyed by this parameter, leading to the preservation of most organoleptic properties of foods, and its ability to inactivate many pathogens, the use of HHP began to spread at the end of the twentieth century into the food industry, in particular for the development of pathogen inactivation processes. Today, even if this field is still the first application domain for HHP, more and more research works have shown that this parameter could be of great interest in health and medicine sciences.

Keywords: High hydrostatic pressure; Biology; History; Food processing; Decontamination processes; Health and medicine sciences

Strategies to extend the lifetime of bioelectrochemical enzyme electrodes for biosensing and biofuel cell applications by Stefanie Rubenwolf; Sven Kerzenmacher; Roland Zengerle; Felix von Stetten (pp. 1315-1322).

Enzymes are powerful catalysts for biosensor and biofuel cell electrodes due to their unique substrate specificity. This specificity is defined by the amino acid chain's complex three-dimensional structure based on non-covalent forces, being also responsible for the very limited enzyme lifetime of days to weeks. Many electrochemical applications, however, would benefit from lifetimes over months to years. This mini-review provides a critical overview of strategies and ideas dealing with the problem of short enzyme lifetime, which limits the overall lifetime of bioelectrochemical electrodes. The most common approaches aim to stabilize the enzyme itself. Various immobilization techniques have been used to reduce flexibility of the amino acid chain by introducing covalent or non-covalent binding forces to external molecules. The enzyme can also be stabilized using genetic engineering methods to increase the binding forces within the protein or by optimizing the environment in order to reduce destabilizing interactions. In contrast, renewing the inactivated catalyst decouples overall system lifetime from the limited enzyme lifetime and thereby promises theoretically unlimited electrode lifetimes. Active catalyst can be supplied by exchanging the electrolyte repeatedly. Alternatively, integrated microorganisms can display the enzymes on their surface or secrete them to the electrolyte, allowing unattended power supply for long-term applications.

Keywords: Enzyme inactivation; Biofuel cell; Biosensor; Amino acid replacement; Immobilization; Self-regeneration

Current findings, future trends, and unsolved problems in studies of medicinal mushrooms by Solomon P. Wasser (pp. 1323-1332).

The target of the present review is to draw attention to many critically important unsolved problems in the future development of medicinal mushroom science in the twenty-first century. Special attention is paid to mushroom polysaccharides. Many, if not all, higher Basidiomycetes mushrooms contain biologically active polysaccharides in fruit bodies, cultured mycelium, and cultured broth. The data on mushroom polysaccharides are summarized for approximately 700 species of higher Hetero- and Homobasidiomycetes. The chemical structure of polysaccharides and its connection to antitumor activity, including possible ways of chemical modification, experimental testing and clinical use of antitumor or immunostimulating polysaccharides, and possible mechanisms of their biological action, are discussed. Numerous bioactive polysaccharides or polysaccharide–protein complexes from medicinal mushrooms are described that appear to enhance innate and cell-mediated immune responses and exhibit antitumor activities in animals and humans. Stimulation of host immune defense systems by bioactive polymers from medicinal mushrooms has significant effects on the maturation, differentiation, and proliferation of many kinds of immune cells in the host. Many of these mushroom polymers were reported previously to have immunotherapeutic properties by facilitating growth inhibition and destruction of tumor cells. While the mechanism of their antitumor actions is still not completely understood, stimulation and modulation of key host immune responses by these mushroom polymers appears central. Particularly and most importantly for modern medicine are polysaccharides with antitumor and immunostimulating properties. Several of the mushroom polysaccharide compounds have proceeded through phases I, II, and III clinical trials and are used extensively and successfully in Asia to treat various cancers and other diseases. A total of 126 medicinal functions are thought to be produced by medicinal mushrooms and fungi including antitumor, immunomodulating, antioxidant, radical scavenging, cardiovascular, antihypercholesterolemia, antiviral, antibacterial, antiparasitic, antifungal, detoxification, hepatoprotective, and antidiabetic effects.

Keywords: Medicinal mushrooms; Polysaccharides; beta-Glucans; Antitumor; Immunomodulating; Antioxidant activities

Chemical inhibitors of methanogenesis and putative applications by He Liu; Jin Wang; Aijie Wang; Jian Chen (pp. 1333-1340).

This mini-review summarizes the category, characteristics, and the application fields of the chemical methanogenic inhibitors. Usually, the chemical methanogenic inhibitors can be divided into “specific” and nonspecific inhibitors. The former group includes the structural analogs of coenzyme M and HMG-CoA inhibitors. The nonspecific group includes many chemicals which can inhibit the activity of both methanogens and non-methanogens. The chemical inhibitors of methanogenesis have been widely used in the fields of understanding methane production and consumption in pure culture or in complex natural environment, production of value-added substances, such as volatile fatty acids and hydrogen, and reduction of energy loss and improvement of the efficiency of ruminal energetic transformations. Finally, with an increasing understanding of the mechanistic effects of the chemical inhibitors of methanogenesis, it is possible that some could be used to develop into promising feed additives to reduce losses associated with enteric methane production or as useful tools to screen microbial consortia from various biotechnological applications to enhance hydrogen and acid production.

Keywords: Chemical inhibitor; Methanogenesis; Application

State of the art molecular markers for fecal pollution source tracking in water by Peter Roslev; Annette S. Bukh (pp. 1341-1355).

Most environmental waters are susceptible to fecal contamination from animal and/or human pollution sources. To attenuate or eliminate such contamination, it is often critical that the pollution sources are rapidly and correctly identified. Fecal pollution source tracking (FST) is a promising research area that aims to identify the origin(s) of fecal pollution in water. This mini-review focuses on the potentials and limitations of library independent molecular markers that are exclusively or strongly associated with fecal pollution from humans and different animals. Fecal-source-associated molecular markers include nucleic acid sequences from prokaryotes and viruses associated with specific biological hosts, but also sequences such as mitochondrial DNA retrieved directly from humans and animals. However, some fecal-source-associated markers may not be absolutely specific for a given source type, and apparent specificity and frequency established in early studies are sometimes compromised by new studies suggesting variation in specificity and abundance on a regional, global and/or temporal scale. It is therefore recommended that FST studies are based on carefully selected arrays of markers, and that identification of human and animal contributions are based on a multi-marker toolkit with several markers for each source category. Furthermore, future FST studies should benefit from increased knowledge regarding sampling strategies and temporal and spatial variability of marker ratios. It will also be important to obtain a better understanding of marker persistence and the quantitative relationship between marker abundance and the relative contribution from individual fecal pollution source types. A combination of enhanced pathogen screening methods, and validated quantitative source tracking techniques could then contribute significantly to future management of environmental water quality including improved microbial risk assessment.

Keywords: Fecal pollution; Microbial source tracking (MST); Host specific and host associated markers; mtDNA; Surface water quality; Microbial risk assessment

Bacteria-mediated PAH degradation in soil and sediment by Xiao-Ying Lu; Tong Zhang; Herbert Han-Ping Fang (pp. 1357-1371).

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous in the natural environment and easily accumulate in soil and sediment due to their low solubility and high hydrophobicity, rendering them less available for biological degradation. However, microbial degradation is a promising mechanism which is responsible for the ecological recovery of PAH-contaminated soil and sediment for removing these recalcitrant compounds compared with chemical degradation of PAHs. The goal of this review is to provide an outline of the current knowledge of biodegradation of PAHs in related aspects. Over 102 publications related to PAH biodegradation in soil and sediment are compiled, discussed, and analyzed. This review aims to discuss PAH degradation under various redox potential conditions, the factors affecting the biodegradation rates, degrading bacteria, the relevant genes in molecular monitoring methods, and some recent-year bioremediation field studies. The comprehensive understanding of the bioremediation kinetics and molecular means will be helpful for optimizing and monitoring the process, and overcoming its limitations in practical projects.

Keywords: Bioremediation; Biodegradation; PAHs; Microorganisms

Synthesis of polyhydroxyalkanoate from palm oil and some new applications by Kumar Sudesh; Kesaven Bhubalan; Jo-Ann Chuah; Yik-Kang Kek; Hanisah Kamilah; Nanthini Sridewi; Yan-Fen Lee (pp. 1373-1386).

Polyhydroxyalkanoate (PHA) is a potential substitute for some petrochemical-based plastics. This biodegradable plastic is derived from microbial fermentation using various carbon substrates. Since carbon source has been identified as one of the major cost-absorbing factors in PHA production, cheap and renewable substrates are currently being investigated as substitutes for existing sugar-based feedstock. Plant oils have been found to result in high-yield PHA production. Malaysia, being the world’s second largest producer of palm oil, is able to ensure continuous supply of palm oil products for sustainable PHA production. The biosynthesis and characterization of various types of PHA using palm oil products have been described in detail in this review. Besides, by-products and waste stream from palm oil industry have also demonstrated promising results as carbon sources for PHA biosynthesis. Some new applications in cosmetic and wastewater treatment show the diversity of PHA usage. With proper management practices and efficient milling processes, it may be possible to supply enough palm oil-based raw materials for human consumption and other biotechnological applications such as production of PHA in a sustainable manner.

Keywords: Palm oil; Polyhydroxyalkanoate; Spent cooking oil; Biodegradable; Bio-based

Production of serotonin by dual expression of tryptophan decarboxylase and tryptamine 5-hydroxylase in Escherichia coli by Sangkyu Park; Kiyoon Kang; Shin Woo Lee; Mi-Jeong Ahn; Jung-Myung Bae; Kyoungwhan Back (pp. 1387-1394).

A plant-specific biogenic amine, serotonin, was produced by heterologous expression of two key biosynthetic genes, tryptophan decarboxylase (TDC) and tryptamine 5-hydroxylase (T5H), in Escherichia coli. The native T5H, a cytochrome P450 enzyme, was unable to be functionally expressed in E. coli. Through a series of N-terminal deletions or additions of tagging proteins, we generated a functional T5H enzyme construct (GST∆37T5H) in which glutathione S transferase (GST) was translationally fused with the N-terminal 37 amino acid deleted T5H. Dual expression of GST∆37T5H and TDC using a pCOLADuet-1 E. coli vector produced serotonin at concentrations of approximately 24 mg l⁻¹ in the culture medium and 4 mg l⁻¹ in the cells. An optimum temperature of approximately 20°C was required to achieve peak serotonin production in E. coli because the low induction temperature gave rise to the highest soluble expression of GST∆37T5H.

Keywords: Tryptophan decarboxylase; Tryptamine 5-hydroxylase; Dual expression; Serotonin; Escherichia coli

Production of rhamnolipids in solid-state cultivation using a mixture of sugarcane bagasse and corn bran supplemented with glycerol and soybean oil by Doumit Camilios-Neto; Cryshelen Bugay; Arquimedes Paixão de Santana-Filho; Talita Joslin; Lauro Mera de Souza; Guilherme Lanzi Sassaki; David Alexander Mitchell; Nadia Krieger (pp. 1395-1403).

Rhamnolipid biosurfactants are attracting attention due to their low toxicity, high biodegradability, and good ecological acceptability. However, production in submerged culture is made difficult by severe foaming problems. Solid-state cultivation (SSC) is a promising alternative production method. In the current work, we report the optimization of rhamnolipid production by Pseudomonas aeruginosa UFPEDA 614 on a solid substrate containing sugarcane bagasse and corn bran. The best rhamnolipid production, 45 g/l of impregnating solution used, was obtained with a 50:50 (m/m) mixture of sugarcane bagasse and corn bran supplemented with an impregnating solution containing 6% (v/v) of each of glycerol and soybean oil. This level is comparable with those of previous studies undertaken in solid-state cultivation; the composition of the biosurfactant is similar, but our medium is cheaper. Our work therefore provides a suitable basis for future studies of the development of an SSC-based process for rhamnolipid production.

Keywords: Rhamnolipids; Biosurfactants; Solid-state cultivation; Solid-state fermentation; Pseudomonas aeruginosa

Metabolism of glucose and xylose as single and mixed feed in Debaryomyces nepalensis NCYC 3413: production of industrially important metabolites by Sawan Kumar; Sathyanarayana N. Gummadi (pp. 1405-1415).

Efficient conversion of hexose and pentose (glucose and xylose) by a single strain is a very important factor for the production of industrially important metabolites using lignocellulose as the substrate. The kinetics of growth and polyol production by Debaryomyces nepalensis NCYC 3413 was studied under single and mixed substrate conditions. In the presence of glucose, the strain produced ethanol (35.8 ± 2.3 g/l), glycerol (9.0 ± 0.2 g/l), and arabitol (6.3 ± 0.2 g/l). In the presence of xylose, the strain produced xylitol (38 ± 1.8 g/l) and glycerol (18 ± 1.0 g/l) as major metabolites. Diauxic growth was observed when the strain was grown with different combinations of glucose/xylose, and glucose was the preferred substrate. The presence of glucose enhanced the conversion of xylose to xylitol. By feeding a mixture of glucose at 100 g/l and xylose at 100 g/l, it was found that the strain produced a maximum of 72 ± 3 g/l of xylitol. A study of important enzymes involved in the synthesis of xylitol (xylose reductase (XR) and xylitol dehydrogenase (XDH)), glycerol (glycerol-3-phosphate dehydrogenase (G3PDH)) and ethanol (alcohol dehydrogenase (ADH)) in cells grown in the presence of glucose and xylose revealed high specific activity of G3PDH and ADH in cells grown in the presence of glucose, whereas high specific activity of XR, XDH, and G3PDH was observed in cells grown in the presence of xylose. To our knowledge, this is the first study to elaborate the glucose and xylose metabolic pathway in this yeast strain.

Keywords: Debaryomyces nepalensis ; Hemicellulose; Glucose; Xylose; Diauxic growth; Ethanol; Xylitol

Efficient and direct glutathione production from raw starch using engineered Saccharomyces cerevisiae by Hideyo Yoshida; Shogo Arai; Kiyotaka Y. Hara; Ryosuke Yamada; Chiaki Ogino; Hideki Fukuda; Akihiko Kondo (pp. 1417-1422).

Glutathione is a valuable tri-peptide that is widely used in the pharmaceutical, food, and cosmetic industries. Glutathione is produced industrially by fermentation using Saccharomyces cerevisiae. We demonstrated that expression of amylase genes in glutathione-producing S. cerevisiae enables direct use of starch as a carbon source, thus eliminating the Crabtree effect that is caused by excess glucose. Consequently, cell growth and glutathione productivity were significantly improved. This approach is potentially applicable to a variety of fermentative processes for production of value-added chemicals under aerobic conditions.

Keywords: Glutathione; Yeast; Amylase; Starch; Biorefinery

Sustained release of PI3K inhibitor from PHA nanoparticles and in vitro growth inhibition of cancer cell lines by Xiao-Yun Lu; Elisa Ciraolo; Rachele Stefenia; Guo-Qiang Chen; Yali Zhang; Emilio Hirsch (pp. 1423-1433).

The phosphoinositide-3-kinases (PI3Ks) are a conserved family of lipid kinases that phosphorylate the 3-hydroxyl group of phosphatidylinositols in response to extracellular stimuli. PI3K pathway is enrolled in different kinds of human cancer and plays a prominent role in cancer cell growth and survival. Several PI3K inhibitors have been recently identified but some PI3K inhibitors with high potency in vitro do not show satisfactory effects in animal cancer models because of the poor pharmaceutical properties in vivo such as poor solubility, instability, and fast plasma clearance rate. In this study, we developed a sustained release system of PI3K inhibitor (TGX221) based on polyhydroxyalkanoate nanoparticles (NP) and used it to block proliferation of cancer cell lines. TGX221 was gradually released from PHA-based NP and growth of cancer cell lines was significantly slower in NP-TGX221-treated cells than in either negative controls or in cells receiving free TGX221. Since poor bioavailability and limited in vivo half-life are common features of hydrophobic PI3K inhibitors, our results open the way to similar formulation of other PI3K blockers and to new strategies in cancer treatment.

Keywords: Polyhydroxyalkanoate; PI3K inhibitor; TGX221; Nanoparticle; Drug delivery

Enhanced activity and stability of l-arabinose isomerase by immobilization on aminopropyl glass by Ye-Wang Zhang; Marimuthu Jeya; Jung-Kul Lee (pp. 1435-1442).

Immobilization of Bacillus licheniformis l-arabinose isomerase (BLAI) on aminopropyl glass modified with glutaraldehyde (4 mg protein g support⁻¹) was found to enhance the enzyme activity. The immobilization yield of BLAI was proportional to the quantity of amino groups on the surface of support. Reducing particle size increased the adsorption capacity (q _m) and affinity (k _a). The pH and temperature for immobilization were optimized to be pH 7.1 and 33°C using response surface methodology (RSM). The immobilized enzyme was characterized and compared to the free enzyme. There is no change in optimal pH and temperature before and after immobilization. However, the immobilized BLAI enzyme achieved 145% of the activity of the free enzyme. Correspondingly, the catalytic efficiency (k _cat/K _m) was improved 1.47-fold after immobilization compared to the free enzyme. The thermal stability was improved 138-fold (t _1/2 increased from 2 to 275 h) at 50°C following immobilization.

Keywords: Aminopropyl glass; l-Arabinose isomerase; Immobilization; Stability

The tyrosine O-prenyltransferase SirD catalyzes O-, N-, and C-prenylations by Hui-Xi Zou; Xiulan Xie; Xiao-Dong Zheng; Shu-Ming Li (pp. 1443-1451).

Recently, the prenyltransferase SirD was found to be responsible for the O-prenylation of tyrosine in the biosynthesis of sirodesmin PL in Leptosphaeria maculans. In this study, the behavior of SirD towards phenylalanine/tyrosine and tryptophan derivatives was investigated. Product formation has been observed with 12 of 19 phenylalanine/tyrosine derivatives. It was shown that the alanine structure attached to the benzene ring and an electron donor, e.g., OH or NH₂, at its para-position are essential for the enzyme activity. Modifications were possible both at the side chain and the benzene ring. Enzyme products from seven phenylalanine/tyrosine derivatives were isolated and characterized by MS and NMR analyses including HSQC and HMBC and proven to be O- or N-prenylated derivatives at position C4 of the benzene rings. K M values of six selected derivatives were found in the range of 0.10–0.68 mM. Catalytic efficiencies (K _cat/K M ) were determined in the range of 430–1,110 s⁻¹·M⁻¹ with l-tyrosine as the best substrate. In addition, 7 of 14 tested tryptophan analogs were also accepted by SirD and converted to C7-prenylated derivatives, which was confirmed by comparison with products obtained from enzyme assays using a 7-dimethylallyltryptophan synthase 7-DMATS from Aspergillus fumigatus.

Keywords: Leptosphaeria maculans ; Tyrosine O-prenyltransferase; N-prenylation; chemoenzymatic synthesis

A novel bifunctional endo-/exo-type cellulase from an anaerobic ruminal bacterium by Kyong-Cheol Ko; Yunjon Han; Jong Hyun Choi; Geun-Joong Kim; Seung-Goo Lee; Jae Jun Song (pp. 1453-1462).

An anaerobic microorganism termed AN-C16-KBRB was isolated from the bovine rumen and demonstrated cellulolytic activity on a NB agar plate containing azo-carboxymethyl cellulose. The 16S rRNA gene of the strain was 98% similar to that of Clostridiaceae bacterium SK082 (AB298754) as the highest homology. A novel celEdx16 gene encoding a bifunctional endo-/exocellulase (CelEdx16) was cloned by the shotgun method from AN-C16-KBRB, and the enzyme was characterized. The celEdx16 gene had an open reading frame of 1,104-base pairs, which encoded 367 amino acids to yield a protein of molecular mass 40.4 kDa. The amino acid sequence was 53% identical to that of an endoglucanase from Clostridium thermocellum. CelEdx16 was overexpressed in Escherichia coli and purified using Ni-NTA affinity chromatography. The specific endocellulase and exocellulase activities of CelEdx16 were 15.9 and 3.6 × 10⁻² U mg⁻¹, respectively. The Michaelis–Menten constant (K _m values) and the maximal reaction velocities (V _max values) of CelEdx16 were 47.1 μM and 9.6 × 10⁻³ μmole min⁻¹ when endocellulase activity was measured and 106.3 μM and 2.1 × 10⁻⁵ μmole min⁻¹ when exocellulase activity was assessed. CelEdx16 was optimally active at pH 5.0 and 40°C.

Keywords: Screening; Korean bovine ruminal bacterium; Bifunctional endo-/exo-type cellulase; Cloning; Expression

Fervidobacterium changbaicum Lip1: identification, cloning, and characterization of the thermophilic lipase as a new member of bacterial lipase family V by Jingang Cai; Yuan Xie; Bo Song; Yanping Wang; Zuoming Zhang; Yan Feng (pp. 1463-1473).

A novel lipase gene encoded 315 amino acid residues was obtained using lipase-prospecting primers and genome walking from hyperthermophilic bacterium Fervidobacterium changbaicum CBS-1. Sequence alignment and phylogenetic analysis revealed this novel lipase is a new member of bacterial lipase family V. The recombinant enzyme F. changbaicum lipase 1 (FCLip1) showed maximum activity at 78°C and pH 7.8. It displayed extreme thermostability at 70°C and was also stable across a wide pH range from 6.0 to 12.0. Kinetic study demonstrated FCLip1 preferentially hydrolyzed middle-length acyl chains, especially p-nitrophenyl caprate and tricaprylin. With p-nitrophenyl caprate as a substrate, the enzyme exhibited a K _m and k _cat of 4.67 μM and 22.7/s, respectively. In addition, FCLip1 was resistant to various detergents and organic solvents. This enzyme is the first reported thermophilic lipase from bacterial family Thermotogaceae. Its extreme stability with respect to temperature and pH, along with its triglyceride hydrolysis activity, indicate that FCLip1 has high potential for future application.

Keywords: Thermophilic lipases; Bacterial lipase family V; Fervidobacterium changbaicum ; Lipase-prospecting primers; Genome walking

Characterization of cytochrome P450 monooxygenase CYP154H1 from the thermophilic soil bacterium Thermobifida fusca by Anett Schallmey; Gijs den Besten; Ite G. P. Teune; Roga F. Kembaren; Dick B. Janssen (pp. 1475-1485).

Cytochrome P450 monooxygenases are valuable biocatalysts due to their ability to hydroxylate unactivated carbon atoms using molecular oxygen. We have cloned the gene for a new cytochrome P450 monooxygenase, named CYP154H1, from the moderately thermophilic soil bacterium Thermobifida fusca. The enzyme was overexpressed in Escherichia coli at up to 14% of total soluble protein and purified to homogeneity in three steps. CYP154H1 activity was reconstituted using putidaredoxin reductase and putidaredoxin from Pseudomonas putida DSM 50198 as surrogate electron transfer partners. In biocatalytic reactions with different aliphatic and aromatic substrates of varying size, the enzyme converted small aromatic and arylaliphatic compounds like ethylbenzene, styrene, and indole. Furthermore, CYP154H1 also accepted different arylaliphatic sulfides as substrates chemoselectively forming the corresponding sulfoxides and sulfones. The enzyme is moderately thermostable with an apparent melting temperature of 67°C and exhibited still 90% of initial activity after incubation at 50°C.

Keywords: Cytochrome P450 monooxygenase; Thermobifida fusca ; Enzyme catalysis; Thermostable enzyme; Hydroxylation

Biochemical characterization of a new type of intracellular PHB depolymerase from Rhodospirillum rubrum with high hydrolytic activity on native PHB granules by Anna Sznajder; Dieter Jendrossek (pp. 1487-1495).

A Rhodospirillum rubrum gene that is predicted to code for an extracellular poly(3-hydroxybutyrate) (PHB) depolymerase by the recently published polyhydroxyalkanoates (PHA) depolymerase engineering database was cloned. The gene product (PhaZ3 Rru) was expressed in recombinant E. coli, purified and biochemically characterized. PhaZ3 Rru turned out, however, to share characteristics of intracellular PHB depolymerases and revealed a combination of properties that have not yet been described for other PHB depolymerases. A fusion of PhaZ3 Rruwith the enhanced cyan fluorescent protein was able to bind to PHB granules in vivo and supported the function as an intracellular PHB depolymerase. Purified PhaZ3 Rru was specific for short-chain-length polyhydroxyalkanoates (PHA_SCL) and hydrolysed both untreated native PHB granules as well as trypsin-activated native PHB granules to a mixture of mono- and dimeric 3-hydroxybutyrate. Crystalline (denatured) PHB granules were not hydrolysed by PhayZ3 Rru. Low concentrations of calcium or magnesium ions (1–5 mM) reversibly (EDTA) inhibited the enzyme. Our data suggest that PhaZ3 Rru is the representative of a new type of the growing number of intracellular PHB depolymerases.

Keywords: PHB; PHA; Intracellular PHB depolymerase; Extracellular PHB depolymerase native; Denatured and artificial PHB; PHB depolymerase products

Biosynthesis of polyhydroxyalkanoate homopolymers by Pseudomonas putida by Hong-hui Wang; Xin-rong Zhou; Qian Liu; Guo-Qiang Chen (pp. 1497-1507).

Pseudomonas putida KT2442 has been a well-studied producer of medium-chain-length (mcl) polyhydroxyalkanoate (PHA) copolymers containing C6 ~ C14 monomer units. A mutant was constructed from P. putida KT2442 by deleting its phaG gene encoding R-3-hydroxyacyl-ACP-CoA transacylase and several other β-oxidation related genes including fadB, fadA, fadB2x, and fadAx. This mutant termed P. putida KTHH03 synthesized mcl homopolymers including poly(3-hydroxyhexanoate) (PHHx) and poly(3-hydroxyheptanoate) (PHHp), together with a near homopolymer poly(3-hydroxyoctanoate-co-2 mol% 3-hydroxyhexanoate) (PHO*) in presence of hexanoate, heptanoate, and octanoate, respectively. When deleted with its mcl PHA synthase genes phaC1 and phaC2, the recombinant mutant termed P. putida KTHH08 harboring pZWJ4-31 containing PHA synthesis operon phaPCJ from Aeromonas hydrophila 4AK4 accumulated homopolymer poly(3-hydroxyvalerate) (PHV) when valerate was used as carbon source. The phaC deleted recombinant mutant termed P. putida KTHH06 harboring pBHH01 holding PHA synthase PhbC from Ralstonia eutropha produced homopolymers poly(3-hydroxybutyrate) (PHB) and poly(4-hydroxybutyrate) using γ-butyrolactone was added as precursor. All the homopolymers were physically characterized. Their weight average molecular weights ranged from 1.8 × 10⁵ to 1.6 × 10⁶, their thermal stability changed with side chain lengths. The derivatives of P. putida KT2442 have been developed into a platform for production of various PHA homopolymers.

Keywords: PHB; Polyhydroxyalkanoates; Homopolymer; Pseudomonas putida ; Beta-oxidation

Propeptide of Bacillus subtilis amylase enhances extracellular production of human interferon-α in Bacillus subtilis by Hiroshi Kakeshita; Yasushi Kageyama; Katsutoshi Ara; Katsuya Ozaki; Kouji Nakamura (pp. 1509-1517).

The Gram-positive bacterium, Bacillus subtilis and related species are widely used industrially as hosts for producing enzymes. These species possess a high potential to produce secreted proteins into the culture medium. Nevertheless, the secretion of heterologous proteins by these species is frequently inefficient. In this study, the human interferon-α2b (hIFN-α2b) was used as a heterologous model protein, to investigate the effect of B. subtilis AmyE propeptide in enhancing the secretion of heterologous proteins in B. subtilis. We found that the secretion production and activity of hIFN-α2b with AmyE propeptide increased by more than threefold, compared to that without AmyE propeptide. The maximum amount of secreted hIFN-α2b with propeptide was 14.8 ± 0.6 μg ml⁻¹. In addition, the pro-hIFN-α2b bioactivity reached 5.4 ± 0.5 × 10⁷ U mg⁻¹, which is roughly the same level as that of the non-propeptide hIFN-α2b. These results indicated that AmyE propeptide enhanced the secretion of the hIFN-α2b protein from B. subtilis. This study provides a useful method to enhance the extracellular production of heterologous proteins in B. subtilis.

Keywords: Bacillus subtilis ; Protein secretion; Propeptide; Signal peptide; Heterologous proteins; Human interferon α

Overexpression of alpha-ketoglutarate dehydrogenase in Yarrowia lipolytica and its effect on production of organic acids by Martina Holz; Christina Otto; Anne Kretzschmar; Venelina Yovkova; Andreas Aurich; Markus Pötter; Achim Marx; Gerold Barth (pp. 1519-1526).

The yeast Yarrowia lipolytica is one of the most intensively studied “non-conventional” yeast species. Its ability to secrete various organic acids, like pyruvic (PA), citric, isocitric, and alpha-ketoglutaric (KGA) acid, in large amounts is of interest for biotechnological applications. We have studied the effect of the alpha-ketoglutarate dehydrogenase (KGDH) complex on the production process of KGA. Being well studied in Saccharomyces cerevisiae this enzyme complex consists of three subunits: alpha-ketoglutarate dehydrogenase, dihydrolipoyl transsuccinylase, and lipoamide dehydrogenase. Here we report the effect of overexpression of these subunits encoding genes and resulting increase of specific KGDH activity on organic acid production under several conditions of growth limitation and an excess of carbon source in Y. lipolytica. The constructed strain containing multiple copies of all three KGDH genes showed a reduced production of KGA and an elevated production of PA under conditions of KGA production. However, an increased activity of the KGDH complex had no influence on organic acid production under citric acid production conditions.

Keywords: Alpha-ketoglutarate dehydrogenase; Alpha-ketoglutaric acid; Pyruvic acid; Isocitric acid; Citric acid; Yarrowia lipolytica

Evaluation of promoters for gene expression in polyhydroxyalkanoate-producing Cupriavidus necator H16 by Toshiaki Fukui; Kei Ohsawa; Jun Mifune; Izumi Orita; Satoshi Nakamura (pp. 1527-1536).

Five kinds of promoters were evaluated as tools for regulated gene expression in the PHA-producing bacterium Cupriavidus necator. Several broad-host-range expression vectors were constructed by which expression of a reporter gene gfp was controlled by P lac, P tac, or P BAD derived from Escherichia coli, or promoter regions of phaC1 (P phaC) or phaP1 (P phaP) derived from C. necator. Then, the gfp-expression profiles were determined in C. necator strains harboring the constructed vectors when the cells were grown on fructose or soybean oil. P lac, P tac, P phaC, and P phaP mediated constitutive gene expression, among which P tac was the strongest promoter. lacI-P tac was not thoroughly functional even after addition of isopropyl-β-d-thiogalactopyranoside (IPTG), probably due to inability of C. necator to uptake IPTG. Gene expression by araC-P BAD could be regulated by varying l-arabinose concentration in the medium, although P(3HB) production rate was slightly decreased in the recombinant. phaR-P phaP exhibited an expression profile tightly coupled with P(3HB) accumulation, suggesting application of the vector harboring phaR-P phaP for gene expression specific at the PHA-biosynthesis phase. The properties of these promoters were expected to be useful for effective engineering of PHA biosynthesis in C. necator.

Keywords: Polyhydroxyalkanoate; Poly(3-hydroxybutyrate); Promoter; Cupriavidus necator

Effects of nitrogen catabolite repression and di-ammonium phosphate addition during wine fermentation by a commercial strain of S. cerevisiae by Nathan K. Deed; Hennie J. J. van Vuuren; Richard C. Gardner (pp. 1537-1549).

Two deletion mutants expected to be defective in nitrogen catabolite repression (NCR) were constructed in a commercial wine yeast background M2: a ure2 mutant and a dal80 gzf3 double mutant. Wild-type and both mutant strains were fermented in Sauvignon Blanc grape juice with and without addition of di-ammonium phosphate (DAP). The dal80 gzf3 double mutant exhibited a long fermentative lag phase, which was offset by DAP addition (corresponding to 300 mg/L of N). Neither the NCR mutations nor DAP addition affected the content of volatile thiols in the final wine. Microarray analyses of transcripts in the wild-type and dal80 gzf3 double-mutant strains were performed after 2% and 70% sugars were fermented. Of 80 genes previously identified as NCR-regulated, only 13 were upregulated during fermentation of the dal80 gzf3 double-mutant strain in grape juice. Following DAP addition, 34 of the known NCR genes were downregulated, including 17 that were downregulated even in the NCR mutant strain. The results demonstrate an unexpected complexity of the NCR response that may reflect differences between strains of yeast or differences in gene regulation during alcoholic fermentation compared with standard aerobic growth.

Keywords: Saccharomyces cerevisiae ; Wine; Fermentation; Nitrogen catabolite repression; Microarrays; Varietal thiols

Towards an understanding of the adaptation of wine yeasts to must: relevance of the osmotic stress response by Elena Jiménez-Martí; Mercè Gomar-Alba; Antonio Palacios; Anne Ortiz-Julien; Marcel-li del Olmo (pp. 1551-1561).

During the transformation of grape must sugars in ethanol, yeasts belonging to Saccharomyces cerevisiae strains are particularly involved. One of the stress conditions that yeast cells have to cope with during vinification, especially at the time of inoculation into must, is osmotic stress caused by high sugar concentrations. In this work, we compare several laboratory and wine yeast strains in terms of their ability to start growth in must. By means of transcriptomic approaches and the determination of glycerol intracellular content, we propose several clues for yeast strains to adapt to the wine production conditions: the high expression of genes involved in both biosynthetic processes and glycerol biosynthesis, and the appropriate levels of intracellular glycerol. Besides, we demonstrate that the pre-adaptation of the wine yeast strains showing growth problems at the beginning of vinification in a rehydration medium containing 2% or 5% glucose (depending on the yeast strain considered) may increase their vitality when inoculated into high sugar media.

Keywords: Wine yeasts; Saccharomyces cerevisiae ; Osmotic stress; Vinification; Glycerol; Gene expression; Vitality

Characterization of extracellular polymeric substances from biofilm in the process of starting-up a partial nitrification process under salt stress by Zhao-Ji Zhang; Shao-Hua Chen; Shu-Mei Wang; Hong-Yuan Luo (pp. 1563-1571).

In this study, the characteristics of extracellular polymeric substance (EPS) fractions of biofilm during the process of establishing a partial nitrification under salt stress were analyzed in terms of concentrations, molecular weight distribution, and three-dimensional excitation–emission matrix (EEM) fluorescence spectroscopy. A partial nitrification was formed successfully with a salinity of 1%. Results indicated that the amount of total EPS increased from 54.2 mg g⁻¹ VSS⁻¹ on day 1 to 99.6 mg g⁻¹ VSS⁻¹ on day 55 due to the NaCl concentration changed from 0 to 10.0 g L⁻¹ in a biofilm reactor. The changes of loosely bound EPS (LB-EPS) compounds under different salt concentrations appeared to be more significant than those of the tightly bound EPS. A clear release of polysaccharides in the LB-EPS fraction was detected during the enhancement of salinity. This was considered as a protective response of bacteria to the salinity. Three fluorescence peaks were identified in the EEM fluorescence spectra of the EPS fraction samples. Two peaks were assigned to the protein-like fluorophores, and the third peak was located at the excitation/emission wavelengths of 275 nm/425–435 nm of the spectra of EPS fractions till the salinity maintained constant at 1%. This information is valuable for understanding the characteristics of EPS isolated from biomass in a saline nitrogen removal system.

Keywords: Extracellular polymeric substances; Salinity; Partial nitrification; Excitation–emission matrix

Long-term influence of the presence of a non-aqueous phase on the cell surface hydrophobicity of Pseudomonas in two-phase partitioning bioreactors by María Hernández; Raúl Muñoz Torre (pp. 1573-1581).

The long-term influence of silicone oil 200 cSt (SO200) and 2, 2, 4, 4, 6, 8, 8-heptamethylnonane (HMN) on the cell surface hydrophobicity (CSH) of a hexane-degrading Pseudomonas aeruginosa strain and a toluene-degrading Pseudomonas putida strain was assessed in two-phase partitioning bioreactors under batch and continuous operation. CSH was evaluated using a modified BATH method based on optical density (CSH_OD) and colony-forming unit (CSH_CFU) measurements. In the presence of HMN, P. aeruginosa turned hydrophobic over the time course as shown by the gradual increase in CSH_OD (61 ± 1%) and CSH_CFU (53 ± 3%) under batch degradation and in CSH_OD (49 ± 0%) under continuous operation. However, P. putida turned hydrophobic only under continuous operation ( $$ {hbox{CS}}{{hbox{H}}_{ m{OD}}} = 28 pm 2% $$ ). On the other hand, no significant CSH enhancement was observed in both Pseudomonas strains in the presence of SO200. These results suggested that CSH is species, non-aqueous phase, and cultivation mode dependant, and an inducible property of bacteria. Maximum hexane elimination capacities increased by 2 and 3 in the presence of SO200 and HMN, respectively. Based on the absence of CSH in P. aeruginosa in the presence of SO200, the higher elimination capacities recorded were likely due to an improved hexane mass transfer (physical effect). However, in the presence of HMN, a direct hexane uptake from the non-aqueous phase (biological effect) might have also contributed to this enhancement.

Keywords: Cell surface hydrophobicity; 2, 2, 4, 4, 6, 8, 8-heptamethylnonane; Pseudomonas ; Silicone oil; Two-phase partitioning bioreactors

Disruption of β-oxidation pathway in Pseudomonas putida KT2442 to produce new functionalized PHAs with thioester groups by Isabel F. Escapa; Valle Morales; Verónica P. Martino; Eric Pollet; Luc Avérous; José L. García; María A. Prieto (pp. 1583-1598).

This work describes the generation of novel PHAs (named PHACOS) with a new monomer composition containing thioester groups in the side chain, which confers new properties and made them suitable for chemical modifications after their biosynthesis. We have analyzed the PHACOS production abilities of the wild-type strain Pseudomonas putida KT2442 vs. its derived strain P. putida KT42FadB, mutated in the fadB gene from the central metabolic β-oxidation pathway involved in the synthesis of medium-chain-length PHA (mcl-PHA). Different fermentation strategies based on one- or two-stage cultures have been tested resulting in PHACOS with different monomer composition. Using decanoic acid as inducer of the growth and polymer synthesis and 6-acetylthiohexanoic acid as PHA precursor in a two-stage strategy, the maximum yield was obtained by culturing the strain KT42FadB. Nuclear magnetic resonance and gas chromatography coupled to mass spectrometry showed that polymers obtained from the wild-type and KT42FadB strains, included 6-acetylthio-3-hydroxyhexanoic acid (OH-6ATH) and the shorter derivative 4-acetylthio-3-hydroxybutanoic acid (OH-4ATB) in their composition, although in different ratios. While the polymer obtained from KT42FadB strain contained mainly OH-6ATH monomer units, mcl-PHA produced by the wild-type strain contained OH-6ATH and OH-4ATB. Furthermore, polyesters showed differences in the OH-alkyl derivates moiety. The strain KT42FadB overproduced PHACOS when compared to the production rate of the control strain in one- and two-stage cultures. Thermal properties obtained by differential scanning calorimetry indicated that both polymers have different glass transition temperatures related to their composition.

Keywords: Pseudomonas putida KT2442; Polyhydroxyalkanoates; Functionalized PHA; Thioester side chain

Formation of new polyhydroxyalkanoate containing 3-hydroxy-4-methylvalerate monomer in Burkholderia sp. by Nyok-Sean Lau; Takeharu Tsuge; Kumar Sudesh (pp. 1599-1609).

Burkholderia sp. synthase has been shown to polymerize 3-hydroxybutyrate (3HB), 3-hydroxyvalerate, and 3-hydroxy-4-pentenoic acid monomers. This study was carried out to evaluate the ability of Burkholderia sp. USM (JCM 15050) and its transformant harboring the polyhydroxyalkanoate (PHA) synthase gene of Aeromonas caviae to incorporate the newly reported 3-hydroxy-4-methylvalerate (3H4MV) monomer. Various culture parameters such as concentrations of nutrient rich medium, fructose and 4-methylvaleric acid as well as harvesting time were manipulated to produce P(3HB-co-3H4MV) with different 3H4MV compositions. The structural properties of PHA containing 3H4MV monomer were investigated by using nuclear magnetic resonance and Fourier transform infrared spectroscopy (FTIR). The relative intensities of the bands at 1,183 and 1,228 cm⁻¹ in the FTIR spectra enabled the rapid detection and differentiation of P(3HB-co-3H4MV) from other types of PHA. In addition, the presence of 3H4MV units in the copolymer was found to considerably lower the melting temperature and enthalpy of fusion values compared with poly(3-hydroxybutyrate) (P(3HB)). The copolymer exhibited higher thermo-degradation temperature but similar molecular weight and polydispersity compared with P(3HB).

Keywords: Polyhydroxyalkanoate; 3-Hydroxy-4-methylvalerate; 4-Methylvaleric acid; Burkholderia sp.; Polymer characterization

Growth and polyhydroxybutyrate production by Ralstonia eutropha in emulsified plant oil medium by Charles F. Budde; Sebastian L. Riedel; Florian Hübner; Stefan Risch; Milan K. Popović; ChoKyun Rha; Anthony J. Sinskey (pp. 1611-1619).

Polyhydroxyalkanoates (PHAs) are natural polyesters synthesized by bacteria for carbon and energy storage that also have commercial potential as bioplastics. One promising class of carbon feedstocks for industrial PHA production is plant oils, due to the high carbon content of these compounds. The bacterium Ralstonia eutropha accumulates high levels of PHA and can effectively utilize plant oil. Growth experiments that include plant oil, however, are difficult to conduct in a quantitative and reproducible manner due to the heterogeneity of the two-phase medium. In order to overcome this obstacle, a new culture method was developed in which palm oil was emulsified in growth medium using the glycoprotein gum arabic as the emulsifying agent. Gum arabic did not influence R. eutropha growth and could not be used as a nutrient source by the bacteria. R. eutropha was grown in the emulsified oil medium and PHA production was measured over time. Additionally, an extraction method was developed to monitor oil consumption. The new method described in this study allows quantitative, reproducible R. eutropha experiments to be performed with plant oils. The method may also prove useful for studying growth of different bacteria on plant oils and other hydrophobic carbon sources.

Keywords: Ralstonia eutropha ; Polyhydroxyalkanoate; Polyhydroxybutyrate; Plant oil; Palm oil

Microvinification—how small can we go? by Tommaso Liccioli; Tina M. T. Tran; Daniel Cozzolino; Vladimir Jiranek; Paul J. Chambers; Simon A. Schmidt (pp. 1621-1628).

High-throughput methodologies to screen large numbers of microorganisms necessitate the use of small-scale culture vessels. In this context, an increasing number of researchers are turning to microtiter plate (MTP) formats to conduct experiments. MTPs are now widely used as a culturing vessel for phenotypic screening of aerobic laboratory cultures, and their suitability has been assessed for a range of applications. The work presented here extends these previous studies by assessing the metabolic footprint of MTP fermentation. A comparison of Chardonnay grape juice fermentation in MTPs with fermentations performed in air-locked (self-induced anaerobic) and cotton-plugged (aerobic) flasks was made. Maximum growth rates and biomass accumulation of yeast cultures grown in MTPs were indistinguishable from self-induced anaerobic flask cultures. Metabolic profiles measured differed depending on the metabolite. While glycerol and acetate accumulation mirrored that of self-induced anaerobic cultures, ethanol accumulation in MTP ferments was limited by the increased propensity of this volatile metabolite for evaporation in microlitre-scale culture format. The data illustrates that microplate cultures can be used as a replacement for self-induced anaerobic flasks in some instances and provide a useful and economical platform for the screening of industrial strains and culture media.

Keywords: Yeast; Microplates; Grape juice; Fermentation; High-throughput

Microorganism-immobilized carbon nanoparticle anode for microbial fuel cells based on direct electron transfer by Yong Yuan; Shungui Zhou; Nan Xu; Li Zhuang (pp. 1629-1635).

A fast and convenient bacterial immobilization method was proposed as an attempt to improve the anode efficiency of a microbial fuel cell, in which bacteria were entrapped into carbon nanoparticle matrix. The direct electron transfer from the entrapped bacterial cells to the anode was verified using cyclic voltammogram (CV). Using the immobilized bioanode, the start-up time of the MFC was greatly reduced. Meanwhile, the maximum power density of 1,947 mW m⁻² with the modified anode was much higher than that with the biofilm-based carbon cloth anode (1,479 mW m⁻²). Impedance measurements suggested that performance improvement resulted from the decrease in charge transfer and diffusion resistances. The results demonstrated that bacteria immobilization using carbon nanoparticle matrix was a simple and efficient approach for improving the anodes performances in MFCs.

Keywords: Microbial fuel cell; Immobilization; Carbon nanoparticle; Mixed culture; Biofilm

Isolation and characterization of endophytic bacterium LRE07 from cadmium hyperaccumulator Solanum nigrum L. and its potential for remediation by Shenglian Luo; Yong Wan; Xiao Xiao; Hanjun Guo; Liang Chen; Qiang Xi; Guangming Zeng; Chengbin Liu; Jueliang Chen (pp. 1637-1644).

Valuable endophytic strains facilitating plants growth and detoxification of heavy metals are required because the application of plant–endophyte symbiotic system is a promising potential technique to improve efficiency of phytoremediation. In this study, endophytic bacterium LRE07 was isolated from cadmium hyperaccumulator Solanum nigrum L. It was identified as Serratia sp. by 16S rRNA sequence analysis. The endophytic bacterium LRE07 was resistant to the toxic effects of heavy metals, solubilized mineral phosphate, and produced indoleacetic acid and siderophore. The heavy metal detoxification was studied in growing LRE07 cells. The strain bound over 65% of cadmium and 35% of zinc in its growing cells from single metal solutions 72 h after inoculation. Besides the high removal efficiencies in single-ion system, an analogous removal phenomenon was also observed in multi-ions system, indicating that the endophyte possesses specific and remarkable heavy metal remediation abilities.

Keywords: Endophyte; Hyperaccumulator; Heavy metal; Detoxification; Phytoremediation

Comparison of biological removal via nitrite with real-time control using aerobic granular sludge and flocculent activated sludge by Dawen Gao; Xiangjuan Yuan; Hong Liang; Wei-Min Wu (pp. 1645-1652).

The process of nitrification–denitrification via nitrite for nitrogen removal under real-time control mode was tested in two laboratory-scale sequencing batch reactors (SBRs) with flocculent activated sludge (R1) and aerobic granular sludge (R2) to compare operational performance and real-time control strategies. The results showed that the average ammonia nitrogen, total inorganic nitrogen (TIN), and chemical oxygen demand (COD) removal during aeration phase were 97.6%, 57.0%, and 90.1% in R2 compared with 98.6%, 48.7%, and 88.1% in R1. The TIN removed in both SBRs was partially due to the presence of simultaneous nitrification–denitrification via nitrite, especially in R2. The specific nitrification and denitrification rates in R2 were 0.0416 mgNH ₄ ⁺ –N/gSS-min and 0.1889 mgNO _X ⁻ –N/gSS-min, which were 1.48 times and 1.35 times that of R1. The higher rates for COD removal, nitrification, and denitrification were achieved in R2 than R1 with similar influent quality. Dissolved oxygen (DO), pH, and oxidization reduction potential, corresponding to nutrient variations, were used as diagnostic parameters to control the organic carbon degradation and nitrification–denitrification via nitrite processes in both SBRs. The online control strategy of granular SBR was similar to that of the SBR with flocculent activated sludge. However, a unique U-type pattern on the DO curve in granular SBR was different from SBR with flocculent activated sludge in aerobic phase.

Keywords: Nitrification–denitrification via nitrite; Aerobic granular sludge; Flocculent activated sludge; Real-time control; SBR

Trichoderma harzianum SQR-T037 rapidly degrades allelochemicals in rhizospheres of continuously cropped cucumbers by Lihua Chen; Xingming Yang; Waseem Raza; Junhua Li; Yanxia Liu; Meihua Qiu; Fengge Zhang; Qirong Shen (pp. 1653-1663).

To alleviate the stress of continuous cropping for cucumber continuous cropping (CCC) system, a beneficial fungus Trichoderma harzianum SQR-T037 (SQR-T037) was isolated and applied to soil to degrade allelochemicals exuded from cucumber plants in a Rhizobox experiment. The following phenolic acids (PAs), classified as allelochemicals, were isolated and identified from cucumber rhizospheres: 4-hydroxybenzoic acid, vanillic acid, ferulic acid, benzoic acid, 3-phenylpropionic acid, and cinnamic acid. Mixed PAs added in potato dextrose broth, each with 0.2 gram per liter, were completely degraded by SQR-T037 after 170 h of incubation. In Rhizobox experiments, inoculation of SQR-T037 in the CCC soil also degraded the PAs exuded from cucumber plant roots. This degradation was 88.8% for 4-hydroxybenzoic acid, 90% for vanillic acid, 95% for benzoic acid, and 100% for ferulic acid, 3-phenylpropionic acid, and cinnamic acid at 45 days after plantation. Simultaneously, a significant (p ≥ 0.05) decrease in the disease index of Fusarium wilt and an increase in dry weights of cucumber plants were obtained in pot experiments by application of SQR-T037. This was mostly attributed to degradation of PAs exuded from cucumber roots in CCC soil by SQR-T037 and alleviation of the allelopathic stress. Application of beneficial microorganisms, such as SQR-T037 that biodegrades allelochemicals, is a highly efficient way to resolve the problems associated with continuous cropping system.

Keywords: Continuous cropping; Phenolic acid; Fusarium wilt; Root exudate; Rhizobox

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Applied Microbiology and Biotechnology (v.89, #5)

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Applied Microbiology and Biotechnology (v.89, #5)