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Applied Microbiology and Biotechnology (v.89, #1)

Acknowledgements and greetings from the Editor-in-Chief by Alexander Steinbüchel (pp. 1-1).

Acclimation of green algae to sulfur deficiency: underlying mechanisms and application for hydrogen production by Taras K. Antal; Tatyana E. Krendeleva; Andrew B. Rubin (pp. 3-15).

Hydrogen is definitely one of the most acceptable fuels in the future. Some photosynthetic microorganisms, such as green algae and cyanobacteria, can produce hydrogen gas from water by using solar energy. In green algae, hydrogen evolution is coupled to the photosynthetic electron transport in thylakoid membranes via reaction catalyzed by the specific enzyme, (FeFe)-hydrogenase. However, this enzyme is highly sensitive to oxygen and can be quickly inhibited when water splitting is active. A problem of incompatibility between the water splitting and hydrogenase reaction can be overcome by depletion of algal cells of sulfur which is essential element for life. In this review the mechanisms underlying sustained hydrogen photoproduction in sulfur deprived C. reinhardtii and the recent achievements in studying of this process are discussed. The attention is focused on the biophysical and physiological aspects of photosynthetic response to sulfur deficiency in green algae.

Keywords: Hydrogen photoproduction; Sulfur deprivation; Chlamydomonas reinhardtii

Biotechnological potential of the ethylmalonyl-CoA pathway by Birgit E. Alber (pp. 17-25).

The ethylmalonyl-CoA pathway is central to the carbon metabolism of many α-proteobacteria, like Rhodobacter sphaeroides and Methylobacterium extorquens as well as actinomycetes, like Streptomyces spp. Its function is to convert acetyl-CoA, a central carbon intermediate, to other precursor metabolites for cell carbon biosynthesis. In contrast to the glyoxylate cycle—another widely distributed acetyl-CoA assimilation strategy—the ethylmalonyl-CoA pathway contains many unique CoA-ester intermediates, such as (2R)- and (2S)-ethylmalonyl-CoA, (2S)-methylsuccinyl-CoA, mesaconyl-(C1)-CoA, and (2R, 3S)-methylmalyl-CoA. With this come novel catalysts that interconvert these compounds. Among these unique enzymes is a novel carboxylase that reductively carboxylates crotonyl-CoA, crotonyl-CoA carboxylase/reductase, and (3S)-malyl-CoA thioesterase. The latter represents the first example of a non-Claisen condensation enzyme of the malate synthase superfamily and defines a new class of thioesterases apart from the hotdog-fold and α/β-fold thioesterases. The biotechnological implications of the ethylmalonyl-CoA pathway are tremendous as one looks to tap into the potential of using these new intermediates and catalysts to produce value-added products.

Keywords: Ethylmalonyl-CoA pathway; Glyoxylate cycle; Claisen condensation; Thioesterase; Crotonyl-CoA carboxylase/reductase; 2-Methylfumaryl-CoA

Protein-based underwater adhesives and the prospects for their biotechnological production by Russell J. Stewart (pp. 27-33).

Biotechnological approaches to practical production of biological protein-based adhesives have had limited success over the last several decades. Broader efforts to produce recombinant adhesive proteins may have been limited by early disappointments. More recent synthetic polymer approaches have successfully replicated some aspects of natural underwater adhesives. For example, synthetic polymers, inspired by mussels, containing the catecholic functional group of 3,4-L-dihydroxyphenylalanine adhere strongly to wet metal oxide surfaces. Synthetic complex coacervates inspired by the Sandcastle worm are water-borne adhesives that can be delivered underwater without dispersing. Synthetic approaches offer several advantages, including versatile chemistries and scalable production. In the future, more sophisticated mimetic adhesives may combine synthetic copolymers with recombinant or agriculture-derived proteins to better replicate the structural and functional organization of natural adhesives.

Keywords: Bioadhesive; Sandcastle worm-inspired; Biomimetic; Complex coacervate

Hydrolysis of organophosphorus compounds by microbial enzymes by Casey M. Theriot; Amy M. Grunden (pp. 35-43).

There are classes of microbial enzymes that have the ability to degrade harmful organophosphorus (OP) compounds that are present in some pesticides and nerve agents. To date, the most studied and potentially important OP-degrading enzymes are organophosphorus hydrolase (OPH) and organophosphorus acid anhydrolase (OPAA), which have both been characterized from a number of organisms. Here we provide an update of what is experimentally known about OPH and OPAA to include their structures, substrate specificity, and catalytic properties. Current and future potential applications of these enzymes in the hydrolysis of OP compounds are also addressed.

Keywords: Organophosphorus compound; OP nerve agent; Pesticide; OPAA; OPH; Phosphotriesterase; Prolidase

Lectins: production and practical applications by Sze Kwan Lam; Tzi Bun Ng (pp. 45-55).

Lectins are proteins found in a diversity of organisms. They possess the ability to agglutinate erythrocytes with known carbohydrate specificity since they have at least one non-catalytic domain that binds reversibly to specific monosaccharides or oligosaccharides. This articles aims to review the production and practical applications of lectins. Lectins are isolated from their natural sources by chromatographic procedures or produced by recombinant DNA technology. The yields of animal lectins are usually low compared with the yields of plant lectins such as legume lectins. Lectins manifest a diversity of activities including antitumor, immunomodulatory, antifungal, HIV-1 reverse transcriptase inhibitory, and anti-insect activities, which may find practical applications. A small number of lectins demonstrate antibacterial and anti-nematode activities.

Keywords: Lectins; Hemagglutinins; Production; Practical applications

Construction of stress-induced metabolic pathway from glucose to 1,3-propanediol in Escherichia coli by Quanfeng Liang; Haojun Zhang; Shengnan Li; Qingsheng Qi (pp. 57-62).

1,3-Propanediol is an important chemical widely used in polymer production, but its availability is being restricted owing to its expensive synthesis. The aim of this study was to engineer an Escherichia coli strain that can produce 1,3-propanediol directly from glucose. We successfully constructed a stress-induced metabolic pathway from glucose to 1,3-propanediol in recombinant E. coli by the expression of gpd1 and gpp2 genes from Saccharomyces cerevisiae and dha operon from Klebsiella pneumoniae, respectively. Batch cultivation of the recombinant E. coli showed that 12.1 g/L 1,3-propanediol was accumulated in the culture without using any inducer.

Keywords: Escherichia coli ; 1,3-Propanediol; Glucose; Metabolic engineering

Cytosolic NADPH balancing in Penicillium chrysogenum cultivated on mixtures of glucose and ethanol by Zheng Zhao; Karel Kuijvenhoven; Walter M. van Gulik; Joseph J. Heijnen; Wouter A. van Winden; Peter J. T. Verheijen (pp. 63-72).

The in vivo flux through the oxidative branch of the pentose phosphate pathway (oxPPP) in Penicillium chrysogenum was determined during growth in glucose/ethanol carbon-limited chemostat cultures, at the same growth rate. Non-stationary ¹³C flux analysis was used to measure the oxPPP flux. A nearly constant oxPPP flux was found for all glucose/ethanol ratios studied. This indicates that the cytosolic NADPH supply is independent of the amount of assimilated ethanol. The cofactor assignment in the model of van Gulik et al. (Biotechnol Bioeng 68(6):602–618, 2000) was supported using the published genome annotation of P. chrysogenum. Metabolic flux analysis showed that NADPH requirements in the cytosol remain nearly the same in these experiments due to constant biomass growth. Based on the cytosolic NADPH balance, it is known that the cytosolic aldehyde dehydrogenase in P. chrysogenum is NAD⁺ dependent. Metabolic modeling shows that changing the NAD⁺-aldehyde dehydrogenase to NADP⁺-aldehyde dehydrogenase can increase the penicillin yield on substrate.

Keywords: Isotopic non-stationary ¹³C flux analysis; Metabolic flux ratio analysis; Pentose phosphate pathway ; Metabolic engineering; NADPH metabolism

Improvement of l-lactic acid production by osmotic-tolerant mutant of Lactobacillus casei at high temperature by Xiang-Yang Ge; Jian Yuan; Hao Qin; Wei-Guo Zhang (pp. 73-78).

l-Lactic acid production by Lactobacillus casei was used as a model to study the mechanism of substrate inhibition and the strategy for enhancing l-lactic acid production. It was found that the concentration of cell growth and l-lactate decreased with the increase of glucose concentration and fermentation temperature. To enhance the osmotic stress resistance of the strain at high temperature, a mutant G-03 was screened and selected with 360 g/L glucose at 45°C as the selective criterion. To further increase the cell growth for lactic acid production, 3 g/L of biotin was supplemented to the medium. As a result, l-lactate concentration by the mutant G-03 reached 198.2 g/L (productivity of 5.5 g L⁻¹ h⁻¹) at 41°C in a 7-L fermentor with 210 g/L glucose as carbon source. l-Lactate concentration and productivity of mutant G-03 were 115.2% and 97.8% higher than those of the parent strain, respectively. The strategy for enhancing l-lactic acid production by increasing osmotic stress resistance at high temperature may provide an alternative approach to enhance organic acid production with other strains.

Keywords: l-Lactic acid; Lactobacillus casei ; Osmotic stress resistance; High temperature

Asymmetric reduction of activated alkenes using an enoate reductase from Gluconobacter oxydans by Nina Richter; Harald Gröger; Werner Hummel (pp. 79-89).

A recombinant enoate reductase from Gluconobacter oxydans was heterologously expressed, purified, characterised and applied in the asymmetric reduction of activated alkenes. In addition to the determination of the kinetic properties, the major focus of this work was to utilise the enzyme in the biotransformation of different interesting compounds such as 3,5,5-trimethyl-2-cyclohexen-1,4-dione (ketoisophorone) and (E/Z)-3,7-dimethyl-2,6-octadienal (citral). The reaction proceeded with excellent stereoselectivities (>99% ee) as well as absolute chemo- and regioselectivity, only the activated C═C bond of citral was reduced by the enoate reductase, while non-activated C═C bond and carbonyl moiety remained untouched. The described strategy can be used for the production of enantiomerically pure building blocks, which are difficult to prepare by chemical means. In general, the results show that the investigated enoate reductase is a promising catalyst for the use in asymmetric C═C bond reductions.

Keywords: Enoate reductase; Asymmetric reduction; Ketoisophorone; Enantioselectivity; Biocatalysis

A nitrilase from a metagenomic library acts regioselectively on aliphatic dinitriles by Sally Bayer; Claudia Birkemeyer; Meike Ballschmiter (pp. 91-98).

Several novel nitrilases were selected from metagenomic libraries using cinnamonitrile and a mixture of six different nitriles as substrates. The nitrilase gene nit1 was expressed in Escherichia coli and the resulting protein was further examined concerning its biochemical properties. Nit1 turned out to be an aliphatic nitrilase favoring dinitriles over mononitriles. Stereochemical analysis revealed that Nit1 converted the dinitrile 2-methylglutaronitrile regioselectively. Hydrolysis at the ω-nitrile group of a dinitrile, such as catalyzed by Nit1, leads to ω-cyanocarboxylic acids, which are important precursors for chemical and pharmaceutical products. Nit1 metabolized 2-methylglutaronitrile to the corresponding ω-cyanocarboxylic acid 4-cyanopentanoic acid can be used for the production of the fine chemical 1,5-dimethyl-2-piperidone.

Keywords: Aliphatic nitrilase; Dinitrile; Metagenome; Regioselectivity

Maximizing the native concentration and shelf life of protein: a multiobjective optimization to reduce aggregation by Saif Khan; Vinod Bhakuni; Vandana Praveen; R. Tewari; C. K. M. Tripathi; V. D. Gupta (pp. 99-108).

A multiobjective optimization was performed to maximize native protein concentration and shelf life of ASD, using artificial neural network (ANN) and genetic algorithm (GA). Optimum pH, storage temperature, concentration of protein, and protein stabilizers (Glycerol, NaCl) were determined satisfying the twin objective: maximum relative area of the dimer peak (native state) after 48 h of storage, and maximum shelf life. The relative area of the dimer peak, obtained from size exclusion chromatography performed as per the central composite design (CCD), and shelf life (obtained as turbidity change) served as training targets for the ANN. The ANN was used to establish mathematical relationship between the inputs and targets (from CCD). GA was then used to optimize the above determinants of aggregation, maximizing the twin objectives of the network. An almost fourfold increase in shelf life (~196 h) was observed at the GA-predicted optimum (protein concentration: 6.49 mg/ml, storage temperature: 20.8 °C, Glycerol: 10.02%, NaCl: 51.65 mM and pH: 8.2). Since no aggregation was observed at the optimum till 48 h, all the protein was found at the dimer position with maximum relative area (64.49). Predictions of the finally adapted network also reveal that storage temperature and solvent glycerol concentration plays key role in deciding the degree of ASD aggregation. This multiobjective optimization strategy was also successfully applied in minimizing the batch culture period and determining optimum combination of medium components required for most economical production of actinomycin D.

Keywords: Multiobjective optimization; Neural network; Genetic algorithm; Shelf life; Aggregation

A molecular approach to optimize hIFN α2b expression and secretion in Yarrowia lipolytica by Najla Gasmi; Franck Fudalej; Héla Kallel; Jean-Marc Nicaud (pp. 109-119).

In this work, we investigated the effect of codon bias and consensus sequence (CACA) at the translation initiation site on the expression level of heterologous proteins in Yarrowia lipolytica; human interferon alpha 2b (hIFN-α2b) was studied as an example. A codon optimized hIFN-α2b gene was synthesized according to the frequency of codon usage in Y. lipolytica. Both wild-type (IFN-wt) and optimized hIFN-α2b (IFN-op) genes were expressed under the control of a strong inducible promoter acyl-co-enzyme A oxidase (POX2). Protein secretion was directed by the targeting sequence of the extracellular lipase (LIP2): pre–proLIP2. Codon optimization increased protein production by 11-fold, whereas the insertion of CACA sequence upstream of the initiation codon of IFN-op construct resulted in 16.5-fold increase of the expression level; this indicates that translational efficiency plays an important part in the increase of hIFN-α2b production level. The replacement of the pre–proLIP2 signal secretion with the LIP2 pre-region sequence followed by the X-Ala/X-Pro stretch but without the pro-region also increased the secretion of the target protein by twofold, suggesting therefore that the LIP2 pro-region is not necessary for extracellular secretion of small heterologous proteins in Yarrowia lipolytica.

Keywords: Yarrowia lipolytica ; hIFNα2b ; Targeting sequence; Codon optimization; Heterologous expression

Structural and functional analysis of hybrid enzymes generated by domain shuffling between Saccharomyces cerevisiae (var. diastaticus) Sta1 glucoamylase and Saccharomycopsis fibuligera Bgl1 β-glucosidase by Julia Marín-Navarro; Leontina Gurgu; Santiago Alamar; Julio Polaina (pp. 121-130).

Saccharomyces cerevisiae Sta1 glucoamylase and Saccharomycopsis fibuligera Bgl1 β-glucosidase, two relevant enzymes from a biotechnological point of view, are proteins with multidomain structure. Starting with homology-based structural models of Sta1 and Bgl1, we have constructed a series of hybrid enzymes by interchanging domains of the two proteins. The first purpose of these constructs was to check available hypotheses about the uncertain biological functions of two domains: the serine/threonine-rich domain (STRD) of Sta1 and a β-sandwich domain present in Bgl1 that we have designated fibronectin-like domain (FLD). While, according to the initial hypothesis, proteins carrying the FLD tend to adhere to the cell wall, our results argued against the idea of an involvement of the STRD in protein secretion that stemmed from the presence of similar domains in different proteins secreted by yeast. The second objective of this work was to increase the enzymatic repertoire by generating enzymes with new structural and functional properties.

Keywords: Cellobiose; Fibronectin-like domain; Glycoside hydrolases; Protein engineering; Starch

Use of the usp45 lactococcal secretion signal sequence to drive the secretion and functional expression of enterococcal bacteriocins in Lactococcus lactis by Juan Borrero; Juan J. Jiménez; Loreto Gútiez; Carmen Herranz; Luis M. Cintas; Pablo E. Hernández (pp. 131-143).

Replacement of the signal peptide (SP) of the bacteriocins enterocin P (EntP) and hiracin JM79 (HirJM79), produced by Enterococcus faecium P13 and Enterococcus hirae DCH5, respectively, by the signal peptide of Usp45 (SP usp45), the major Sec-dependent protein secreted by Lactococcus lactis, permits the production, secretion, and functional expression of EntP and HirJM79 by L. lactis. Chimeric genes encoding the SP usp45 fused to either mature EntP (entP), with or without the immunity gene (entiP) or to mature HirJM79 (hirJM79), with or without the immunity gene (hiriJM79), were cloned into the expression vector pMG36c, carrying the P₃₂ constitutive promoter, and into pNZ8048 under control of the inducible PnisA promoter. The production of EntP and HirJM79 by most of the L. lactis recombinant strains was 1.5- to 3.7-fold higher and up to 3.6-fold higher than by the E. faecium P13 and E. hirae DCH5 control strains, respectively. However, the specific antimicrobial activity of the recombinant EntP was 1.1- to 6.2-fold higher than that produced by E. faecium P13, while that of the HirJM79 was a 40% to an 89% of that produced by E. hirae DCH5. Chimeras of SP usp45 fused to mature EntP or HirJM79 drive the production and secretion of these bacteriocins in L. lactis in the absence of specific immunity and secretion proteins. The supernatants of the recombinant L. lactis NZ9000 strains, producers of EntP, showed a much higher antimicrobial activity against Listeria spp. than that of the recombinant L. lactis NZ9000 derivatives, producers of HirJM79.

Keywords: Bacteriocins; Enterocin P; Hiracin JM79; Heterologous production; Sec-dependent secretion; Lactic acid bacteria (BAL); Lactococcus lactis

Proteomic analysis in non-denaturing condition of the secretome reveals the presence of multienzyme complexes in Penicillium purpurogenum by Alvaro Gonzalez-Vogel; Jaime Eyzaguirre; Gabriela Oleas; Eduardo Callegari; Mario Navarrete (pp. 145-155).

Proteins secreted by filamentous fungi play key roles in different aspects of their biology. The fungus Penicillium purpurogenum, used as a model organism, is able to degrade hemicelluloses and pectins by secreting a variety of enzymes to the culture medium. This work shows that these enzymes interact with each other to form high molecular weight, catalytically active complexes. By using a proteomics approach, we were able to identify several protein complexes in the secretome of this fungus. The expression and assembly of these complexes depend on the carbon source used and display molecular masses ranging from 300 to 700 kDa. These complexes are composed of a variety of enzymes, including arabinofuranosidases, acetyl xylan esterases, feruloyl esterases, β-glucosidases and xylanases. The protein–protein interactions in these multienzyme complexes were confirmed by coimmunoprecipitation assays. One of the complexes was purified from sugar beet pulp cultures and the subunits identified by tandem mass spectrometry. A better understanding of the biological significance of these kinds of interactions will help in the comprehension of the degradation mechanisms used by fungi and may be of special interest to the biotechnology industry.

Keywords: Xylanosome; Penicillium purpurogenum ; Sugar beet pulp; Hemicellulose degradation; Multienzyme complexes; Secretome

Production of antioxidant and antitumor metabolites by submerged cultures of Inonotus obliquus cocultured with Phellinus punctatus by Weifa Zheng; Yanxia Zhao; Xin Zheng; Yubing Liu; Shenyuan Pan; Yucheng Dai; Fuming Liu (pp. 157-167).

While Inonotus obliquus produces a diverse range of bioactive metabolites in its natural habitats, it accumulates less in its submerged cultures. We show here that coculture of I. obliquus with Phellinus punctatus resulted in less production of mycelial biomass but an increased accumulation of phenolic compounds, melanins, and lanostane-type triterpenoids. Metabolites increased in production by coculture include phelligridin C, phelligridin H, methyl inoscavin A, inoscavin C, inoscavin B, davallialactone, methyl davallialactone, foscoparianol D, 21,24-cyclopentalanosta-3β,21,25-triol-8-en, lanosta-7,9(11),23-triene-3β,22,25-triol, and inotodisaccharide and melanins. Metabolites from coculture also showed an increased potential for scavenging free radicals and inhibiting the proliferation of HeLa 229 cells. Davallialactone, methyl davallialactone, and minor phenolic components are the major contributors for scavenging DPPH and hydroxyl radical in monoculture, and phelligridin C, phelligridin H, methyl inoscavin A, inoscavin C, methyl davallialactone, foscoparianol D, and inotodisaccharide are those for scavenging the tested radicals in coculture. Lanostane-type triterpenoids indicated limited roles in scavenging free radicals. Nearly all the detected metabolites correlate positively with inhibiting proliferation of HeLa 229 cells. Thus, coculture of I. obliquus with other fungi seems to be a cost-effective strategy for upregulating biosynthesis of bioactive metabolites.

Keywords: Inonotus obliquus ; Phellinus punctatus ; Incompatible interactions; Coculture; NMR-based metabolomic analysis; Bioactive metabolites

Enhanced production of 2-hydroxyphenazine in Pseudomonas chlororaphis GP72 by Ling Huang; Ming-Min Chen; Wei Wang; Hong-Bo Hu; Hua-Song Peng; Yu-Quan Xu; Xue-Hong Zhang (pp. 169-177).

Pseudomonas chlororaphis GP72 is a root-colonizing biocontrol strain isolated from the green pepper rhizosphere that synthesizes two phenazine derivatives: phenazine-1-carboxylic acid (PCA) and 2-hydroxyphenazine (2-OH-PHZ). The 2-OH-PHZ derivative shows somewhat stronger broad-spectrum antifungal activity than PCA, but its conversion mechanism has not yet been clearly revealed. The aim of this study was to clone and analyze the phenazine biosynthesis gene cluster in this newly found strain and to improve the production of 2-OH-PHZ by gene disruption and precursor addition. The conserved phenazine biosynthesis core operon in GP72 was cloned by PCR, and the unknown sequences located upstream and downstream of the core operon were detected by random PCR gene walking. This led to a complete isolation of the phenazine biosynthesis gene cluster phzIRABCDEFG and phzO in GP72. Gene rpeA and phzO were insertionally mutated to construct GP72AN and GP72ON, respectively, and GP72ANON collectively. The inactivation of rpeA resulted in a fivefold increase in the production of PCA, as well as 2-OH-PHZ. The addition of exogenous precursor PCA to the broth culture, to determine the conversion efficiency of PCA to 2-OH-PHZ under current culture conditions, revealed that PCA had a positive feedback effect on its own accumulation, leading to enhanced synthesis of both PCA and 2-OH-PHZ. The production of 2-OH-PHZ by GP72AN increased to about 170 μg ml⁻¹, compared with just 5 μg ml⁻¹ for the wild type. The hypothesis of biosynthetic pathway for 2-OH-PHZ from PCA was confirmed by identification of 2-hydroxyphenazine-1-carboxylic acid as an intermediate in the culture medium of the high-phenazine producing GP72AN mutant.

Keywords: Phenazine-1-carboxylic acid (PCA); 2-Hydroxyphenazine (2-OH-PHZ); Pseudomonas chlororaphis ; Gene disruption; Gene repression; Phenazine biosynthesis gene cluster

Appraising bacterial strains for rapid BOD sensing—an empirical test to identify bacterial strains capable of reliably predicting real effluent BODs by Judith B. Webber; Mike Noonan; Neil F. Pasco; Joanne M. Hay (pp. 179-188).

The measured response of rapid biochemical oxygen demand (BOD) biosensors is often not identical to those measured using the conventional 5-day BOD assay. This paper highlights the efficacy of using both glucose–glutamic acid (GGA) and Organisation for Economic Cooperation and Development (OECD) BOD standards as a rapid screen for microorganisms most likely to reliably predict real effluent BODs when used in rapid BOD devices. Using these two synthetic BOD standards, a microorganism was identified that produced comparable BOD response profiles for two assays, the MICREDOX® assay and the conventional 5-day BOD₅ test. A factorial experimental design systematically evaluated the impact of four factors (microbial strain, growth media composition, media strength, and microbial growth phase) on the BOD response profiles using GGA and OECD synthetic standard substrates. An outlier was identified that showed an improved correlation between the MICREDOX® BOD (BOD_sens) and BOD₅ assays for both the synthetic standards and for real wastewater samples. Microbial strain was the dominant factor influencing BOD_sens values, with Arthrobacter globiformis single cultures clearly demonstrating superior rapid BOD_sens response profiles for both synthetic and real waste samples. It was the only microorganism to approach the BOD₅ response for the OECD substrate (171 mg O₂ L⁻¹), and also reported BOD values for real waste samples that were comparable to those produced by the BOD₅ test, including discriminating between filtered and unfiltered samples.

Keywords: Biochemical oxygen demand; Rapid BOD screen; Microorganisms; Arthrobacter globiformis

Field application of nitrogen and phenylacetylene to mitigate greenhouse gas emissions from landfill cover soils: effects on microbial community structure by Jeongdae Im; Sung-Woo Lee; Levente Bodrossy; Michael J. Barcelona; Jeremy D. Semrau (pp. 189-200).

Landfills are large sources of CH₄, but a considerable amount of CH₄ can be removed in situ by methanotrophs if their activity can be stimulated through the addition of nitrogen. Nitrogen can, however, lead to increased N₂O production. To examine the effects of nitrogen and a selective inhibitor on CH₄ oxidation and N₂O production in situ, 0.5 M of NH₄Cl and 0.25 M of KNO₃, with and without 0.01% (w/v) phenylacetylene, were applied to test plots at a landfill in Kalamazoo, MI from 2007 November to 2009 July. Nitrogen amendments stimulated N₂O production but had no effect on CH₄ oxidation. The addition of phenylacetylene stimulated CH₄ oxidation while reducing N₂O production. Methanotrophs possessing particulate methane monooxygenase and archaeal ammonia-oxidizers (AOAs) were abundant. The addition of nitrogen reduced methanotrophic diversity, particularly for type I methanotrophs. The simultaneous addition of phenylacetylene increased methanotrophic diversity and the presence of type I methanotrophs. Clone libraries of the archaeal amoA gene showed that the addition of nitrogen increased AOAs affiliated with Crenarchaeal group 1.1b, while they decreased with the simultaneous addition of phenylacetylene. These results suggest that the addition of phenylacetylene with nitrogen reduces N₂O production by selectively inhibiting AOAs and/or type II methanotrophs.

Keywords: Methane; Nitrous oxide; Methanotrophs; Ammonia-oxidizing archaea

Enhanced reductive degradation of methyl orange in a microbial fuel cell through cathode modification with redox mediators by Rong-Hua Liu; Guo-Ping Sheng; Min Sun; Guo-Long Zang; Wen-Wei Li; Zhong-Hua Tong; Fang Dong; Michael Hon-Wah Lam; Han-Qing Yu (pp. 201-208).

A model azo dye, methyl orange (MO), was reduced through in situ utilization of the electrons derived from the anaerobic conversion of organics in a microbial fuel cell (MFC). The MO reduction process could be described by a pseudo first-order kinetic model with a rate constant of 1.29 day⁻¹. Electrochemical impedance spectroscopic analysis shows that the cathode had a high polarization resistance, which could decrease the reaction rate and limit the electron transfer. To improve the MO reduction efficiency, the cathode was modified with redox mediators to enhance the electron transfer. After modification with thionine, the polarization resistance significantly decreased by over 50%. As a consequence, the MO decolorization rate increased by over 20%, and the power density was enhanced by over three times. Compared with thionine, anthraquinone-2, 6-disulfonate modified cathode has less positive effect on the MFC performance. These results indicate that the electrode modification with thionine is a useful approach to accelerate the electrochemical reactions. This work provides useful information about the key factors limiting the azo dye reduction in the MFC and how to improve such a process.

Keywords: Cathode; Methyl orange (MO); Microbial fuel cell (MFC); Redox mediator; Reduction; Thionine

Degradation of cresols by phenol-acclimated aerobic granules by Duu-Jong Lee; Kuo-Ling Ho; Yu-You Chen (pp. 209-215).

High-strength cresol isomers were treated with phenol-acclimated granules in batch experiments. The aerobic granules effectively metabolized cresol isomers at concentrations up to 1,500 mg l⁻¹. The modified Haldane kinetic model, used to assess the kinetic behavior during cresol degradation by granule cells, yielded a high maximum specific growth rate (1.13–1.45 h⁻¹) and inhibition constant (617–952 mg l⁻¹). The microbial community structure, which was stable under cresol stress, was principally composed of genera Bacillus, Acinetobacter, Corynebacterium, and Nocardioides. Enzyme assay results suggest simultaneous expression of ortho- and meta-cleavage pathways during cresol degradation. Under high cresol concentrations, however, cresol isomers were largely degraded via the meta-cleavage pathway, likely attributable to the activity of Bacillus. The aerobic granular sludge system is a promising biotechnology for degrading wastewater containing high-strength cresols.

Keywords: Granules; Haldane model; Kinetics; Cresol

Fluorescence spectral characteristics of the supernatants from an anaerobic hydrogen-producing bioreactor by Wei-Hua Li; Guo-Ping Sheng; Rui Lu; Han-Qing Yu; Yu-You Li; Hideki Harada (pp. 217-224).

Microbial products formed in biological wastewater treatment systems are closely related to system performance and status, and many of them have fluorescence spectral characteristics. In this work, the fluorescence spectral characteristics of the supernatants from an anaerobic hydrogen-producing bioreactor were studied using three-dimensional excitation–emission matrix (EEM) fluorescence spectroscopy. Since the components of the microbial products are complex, the parallel factor analysis (PARAFAC) method was used to extract the real spectra from the overlapped spectra. Two principal components were identified from the EEM spectra. The peaks at excitation–emission maxima of 280/350 and 350/440 nm were, respectively, attributed to the fluorescence of proteins and NADH. Their real concentrations were quantified using the PARAFAC coupled with the second-order calibration method. Results show that the formation rate of proteins was correlated to the production rate of hydrogen and volatile fatty acids, as well as the substrate degradation rate. A close correlation between the hydrogen partial pressure and the two fluorophores was found out. This study provides a reliable and convenient approach, which could be potentially used for monitoring the wastewater treatment reactor performance through measuring the fluorescence spectra of the supernatant.

Keywords: Anaerobic; Excitation–emission matrix (EEM) fluorescence spectroscopy; Hydrogen production; PARAFAC; Proteins; Second-order calibration method

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Applied Microbiology and Biotechnology (v.89, #1)