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Applied Microbiology and Biotechnology (v.84, #5)

Occurrence, properties, and applications of feruloyl esterases by Takuya Koseki; Shinya Fushinobu; Ardiansyah; Hitoshi Shirakawa; Michio Komai (pp. 803-810).

Feruloyl esterases hydrolyze the ester linkages of ferulic and diferulic acids present in plant cell walls. This interesting group of enzymes also has a potentially broad range of applications in the pharmaceutical and agri-food industries. An overview of the current knowledge of fungal feruloyl esterases focusing on the diverse of substrate specificity and potential applications is presented in this review. Furthermore, biological functions of ferulic acid are discussed.

Keywords: Feruloyl esterases; Ferulic acid; Fungi; Substrate specificity; Diversity; Applications

Efficient methods for large-area surface sampling of sites contaminated with pathogenic microorganisms and other hazardous agents: current state, needs, and perspectives by Jason M. Edmonds (pp. 811-816).

The recovery operations following the 2001 attacks with Bacillus anthracis spores were complicated due to the unprecedented need for large-area surface sampling and decontamination protocols. Since this event, multiple reports have been published describing recovery efficiencies of several surface sampling materials. These materials include fibrous swabs of various compositions, cloth wipes, vacuum socks, and adhesive tapes. These materials have reported recovery efficiencies ranging from approximately 20% to 90% due to the many variations in their respective studies including sampling material, composition of surface sampled, concentration of contaminant, and even the method of deposition and sample processing. Additionally, the term recovery efficiency is crudely defined and could be better constructed to incorporate variations in contaminated surface composition and end user needs. While significant efforts in devising protocols for large-area surface sampling have been undertaken in the years since the anthrax attacks, there is still a general lack of consensus in optimal sampling materials and the methodology in which they are evaluated. Fortunately, sampling efforts are continuing to be supported, and the knowledge gaps in our procedures, methodology, and general understanding of sampling mechanisms are being investigated which will leave us better prepared for the future.

Keywords: Surface; Sampling; Toxin; Spores; Anthrax; Large area

Uses and production of chiral 3-hydroxy-γ-butyrolactones and structurally related chemicals by Sang-Hyun Lee; Oh-Jin Park (pp. 817-828).

Enantiopure (S)-3-hydroxy-γ-butyrolactone (HGB) and its structurally related C3–C4 chemicals are an important target for chiral building blocks in synthetic organic chemistry. For the production of these compounds, more economical and practical synthetic routes are required. To date, chiral HGBs have been produced from petrochemicals and biomass, especially malic acids and carbohydrates. This report provides a short review on the production and application of enantiopure HGBs and their related compounds. Emphasis is focused mainly on synthetic routes using biocatalysis (microbial and chemoenzymatic) and application of these compounds. Biological methods have concentrated on devising different kinds of enzymes for the synthesis of the same compound as shown in the case of hydroxynitrile, a key intermediate of synthetic statins, and integrating unit processes for the optically active HGBs and 4-chloro-3-hydroxybutyrate with recombinant microorganisms expressing multiple enzymes. Chemical methods involve selective hydrogenation of carbohydrate-based starting materials. Both types of pathways will require further improvement to serve as a basis for a scalable route to HGBs and related compounds. Several of their synthetic applications are also introduced.

Keywords: (S)-3-hydroxy-γ-butyrolactone; Biocatalysis; Chiral; Enzymatic; Hydrogenation; Statin

Two-phase partitioning bioreactors in environmental biotechnology by Guillermo Quijano; María Hernandez; Frédéric Thalasso; Raúl Muñoz; Santiago Villaverde (pp. 829-846).

Two-phase partitioning bioreactors (TPPBs) in environmental biotechnology are based on the addition of a non-aqueous phase (NAP) into a biological process in order to overcome both mass-transfer limitations from the gas to aqueous phase and pollutant-mediated inhibitions. Despite constituting a robust and reliable technology in terms of pollutant biodegradation rates and process stability in wastewater, soil, and gas treatment applications, this superior performance only applies for a restricted number of pollutants or contamination events. Severe limitations such as high energy requirements, high costs of some NAPs, foaming, or pollutant sequestration challenge the full-scale application of this technology. The introduction of solid NAPs into this research field has opened a promising pathway for the future development of TPPBs. Finally, this work reviews fundamental aspects of NAP selection and mass transfer and identifies the niches for future research: low energy-demand bioreactor designs, experimental determination of partial mass transfers, and solid NAP tailoring.

Keywords: Mass transfer; Soil treatment; Toxic pollutants; Two-phase partitioning bioreactors; Volatile organic contaminants

Environmental and industrial applications of Yarrowia lipolytica by Ashok V. Bankar; Ameeta R. Kumar; Smita S. Zinjarde (pp. 847-865).

Yarrowia lipolytica is a fungus that degrades hydrophobic substrates very efficiently. The fungus displays several important characteristics that have encouraged researchers to study various basic biological and biotechnological applications in detail. Although the organism has been used as model system for studying dimorphism, salt tolerance, heterologous protein expression, and lipid accumulation, there are no recent reviews on the environmental and industrial applications of this organism. Included here are applications in bioremediation of environments contaminated with aliphatic and aromatic compounds, organic pollutants, 2,4,6-trinitrotoluene, and metals. A variety of industrially important recent processes for the synthesis of β-hydroxy butyrate, l-dopa, and emulsifiers have also been reviewed. Production of unique inherent enzymes (inulinases, α-mannosidases), novel applications of esterases and lipases, and the use of the fungus for heterologous expression of biotechnologically relevant products have also been highlighted. The review while entailing a general overview focuses critically on some of the recent advances on the applications of this yeast. The examples cited here demonstrate the use of wild-type, mutant as well as genetically manipulated strains of Y. lipolytica for the development of different products, processes, and technologies. This also throws light on how a single organism can be versatile with respect to its metabolic abilities and how it can be exploited for a variety of purposes. This review will thus form a base for future developments in this field.

Keywords: Yarrowia lipolytica ; Hydrocarbons bioremediation; Wastewaters; Emulsifier; Industrial enzymes; Enantiomer resolution

Bioproduction of lauryl lactone and 4-vinyl guaiacol as value-added chemicals in two-phase biotransformation systems by Jianzhong Yang; Shaozhao Wang; Marie-Josée Lorrain; Denis Rho; Kofi Abokitse; Peter C. K. Lau (pp. 867-876).

Recombinant Escherichia coli whole-cell biocatalysts harboring either a Baeyer–Villiger monooxygenase or ferulic acid decarboxylase were employed in organic-aqueous two-phase bioreactor systems. The feasibility of the bioproduction of water-insoluble products, viz., lauryl lactone from cyclododecanone and 4-vinyl guaiacol from ferulic acid were examined. Using hexadecane as the organic phase, 10∼16 g of lauryl lactone were produced in a 3-l bioreactor that operated in a semicontinuous mode compared to 2.4 g of product in a batch mode. For the decarboxylation of ferulic acid, a new recombinant biocatalyst, ferulic acid decarboxylase derived from Bacillus pumilus, was constructed. Selected solvents as well as other parameters for in situ recovery of vinyl guaiacol were investigated. Up to 13.8 g vinyl guaiacol (purity of 98.4%) were obtained from 25 g of ferulic acid in a 2-l working volume bioreactor by using octane as organic phase. These selected examples highlight the superiority of the two-phase biotransformations systems over the conventional batch mode.

Keywords: Ferulic acid; Baeyer–Villiger monooxygenase; Phenolic acid decarboxylase; Two-phase biotransformation; Green chemistry

High level expression and purification of bioactive human α-defensin 5 mature peptide in Pichia pastoris by Aiping Wang; Song Wang; Mingqiang Shen; Fang Chen; Zhongmin Zou; Xinze Ran; Tianmin Cheng; Yongping Su; Junping Wang (pp. 877-884).

Human α-defensin 5 (HD₅), a small cysteine-rich peptide expressed predominantly in small intestine and female reproductive tissues, plays an important role in innate and adaptive immunity. It is a worthy yet challenging work to produce bioactive recombinant HD₅ through the use of bioengineering techniques. Here, we present the expression and purification of recombinant HD₅ mature peptide (rmHD₅) in Pichia pastoris. To avoid generating unfavorable extra N-terminal amino acids, Red/ET homologous recombination was applied to construct the expression vector pPIC9K-mHD₅ by insertion of a polymerase chain reaction-amplified DNA fragment coding for mHD₅ into the plasmid pPIC9K, at a position right after the cleavage sequence of Kex2. The pPIC9K-mHD₅ vector was transformed into P. pastoris GS115 cells, and positive colonies harboring genomic integration of the multicopy mHD₅ nucleotide sequence were screened out and used for fermentation. After high-cell density fermentation of P. pastoris GS115-HD₅, a two-step purification strategy of macroporous resin adsorption chromatography followed by cation exchange chromatography was performed to obtain purified rmHD₅. The results showed that about 165.0 mg/l of rmHD₅ with its intact N-terminal amino acid sequence as revealed by mass spectrometry analysis and amino acid sequencing was produced under optimal bioreactor-culture conditions and that approximately 50% of the initial rmHD₅ was recovered after purification. The in vitro experiments revealed that rmHD₅ exhibited a prominent antibacterial activity and potency to block human papillomavirus infection. This is the first report on the production and purification of bioactive rmHD₅ in P. pastoris. This study also provides considerations for production of other antimicrobial peptides using the P. pastoris expression system.

Keywords: Human α defensin 5; Pichia pastoris ; Expression; Purification; Bioactivity

Molecular characterization of aromatic peroxygenase from Agrocybe aegerita by Marek J. Pecyna; René Ullrich; Britta Bittner; André Clemens; Katrin Scheibner; Roland Schubert; Martin Hofrichter (pp. 885-897).

Recently, a novel group of fungal peroxidases, known as the aromatic peroxygenases (APO), has been discovered. Members of these extracellular biocatalysts produced by agaric basidiomycetes such as Agrocybe aegerita or Coprinellus radians catalyze reactions—for example, the peroxygenation of naphthalene, toluene, dibenzothiophene, or pyridine—which are actually attributed to cytochrome P450 monooxygenases. Here, for the first time, genetic information is presented on this new group of peroxide-consuming enzymes. The gene of A. aegerita peroxygenase (apo1) was identified on the level of messenger RNA and genomic DNA. The gene sequence was affirmed by peptide sequences obtained through an Edman degradation and de novo peptide sequencing of the purified enzyme. Quantitative real-time reverse transcriptase polymerase chain reaction demonstrated that the course of enzyme activity correlated well with that of mRNA signals for apo1 in A. aegerita. The full-length sequences of A. aegerita peroxygenase as well as a partial sequence of C. radians peroxygenase confirmed the enzymes’ affiliation to the heme-thiolate proteins. The sequences revealed no homology to classic peroxidases, cytochrome P450 enzymes, and only little homology (<30%) to fungal chloroperoxidase produced by the ascomycete Caldariomyces fumago (and this only in the N-terminal part of the protein comprising the heme-binding region and part of the distal heme pocket). This fact reinforces the novelty of APO proteins. On the other hand, homology retrievals in genetic databases resulted in the identification of various APO homologous genes and transcripts, particularly among the agaric fungi, indicating APO’s widespread occurrence in the fungal kingdom.

Keywords: Peroxygenase; Chloroperoxidase; Cytochrome P450; Heme-thiolate; Oxygenation; Coprinellus

Expression of four β-galactosidases from Bifidobacterium bifidum NCIMB41171 and their contribution on the hydrolysis and synthesis of galactooligosaccharides by Theodoros Goulas; Athanasios Goulas; George Tzortzis; Glenn R. Gibson (pp. 899-907).

This paper deals with two aspects tightly related to the enzymatic characteristics and expression of four β-galactosidases (BbgI, BbgII, BbgIII and BbgIV) from Bifidobacterium bifidum NCIMB41171. The growth patterns of this strain indicated a preference towards complex (i.e. lactose, galactooligosaccharides (GOSs)) rather than simple carbohydrates (i.e. glucose and galactose) and a collaborative action and synergistic relation of more than one β-galactosidase isoenzyme for either lactose or GOS hydrolysis and subsequent assimilation. Native polyacrylamide gel electrophoresis analysis of protein extracts from cells growing on different carbohydrates (i.e. glucose, lactose or GOS) indicated that two lactose hydrolysing enzymes (BbgI and BbgIII) and one GOS hydrolysing enzyme (BbgII) were constitutively expressed, whereas a fourth lactose hydrolysing enzyme (BbgIV) was induced in the presence of lactose or different GOS fractions. Furthermore, the β-galactosidase expression profiles of B. bifidum cells and the transgalactosylating properties of each individual isoenzyme, with lactose as substrate, clearly indicated that mainly three isoenzymes (BbgI, BbgIII and BbgIV) are implicated in GOS synthesis when whole B. bifidum cells are utilised. Two of the isoenzymes (BbgI and BbgIV) proved to have better transgalactosylating properties giving yields ranging from 42% to 47% whereas the rest (BbgI and BbgIII) showed lower yields (15% and 29%, respectively).

Keywords: Bifidobacterium bifidum ; β-Galactosidase; Prebiotic

Microbial production of 4-hydroxybutyrate, poly-4-hydroxybutyrate, and poly(3-hydroxybutyrate-co-4-hydroxybutyrate) by recombinant microorganisms by Lei Zhang; Zhen-Yu Shi; Qiong Wu; Guo-Qiang Chen (pp. 909-916).

4-Hydroxybutyrate (4HB) was produced by Aeromonas hydrophila 4AK4, Escherichia coli S17-1, or Pseudomonas putida KT2442 harboring 1,3-propanediol dehydrogenase gene dhaT and aldehyde dehydrogenase gene aldD from P. putida KT2442 which are capable of transforming 1,4-butanediol (1,4-BD) to 4HB. 4HB containing fermentation broth was used for production of homopolymer poly-4-hydroxybutyrate [P(4HB)] and copolymers poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-4HB)]. Recombinant A. hydrophila 4AK4 harboring plasmid pZL-dhaT-aldD containing dhaT and aldD was the most effective 4HB producer, achieving approximately 4 g/l 4HB from 10 g/l 1,4-BD after 48 h of incubation. The strain produced over 10 g/l 4HB from 20 g/l 1,4-BD after 52 h of cultivation in a 6-L fermenter. Recombinant E. coli S17-1 grown on 4HB containing fermentation broth was found to accumulate 83 wt.% of intracellular P(4HB) in shake flask study. Recombinant Ralstonia eutropha H16 grew to over 6 g/l cell dry weight containing 49 wt.% P(3HB-13%4HB) after 72 h.

Keywords: 1,3-Propanediol dehydrogenase; 4-Hydroxybutyrate; PHB; P(4HB); Poly-4-hydroxybutyrate; Poly(3-hydroxybutyrate-co-4-hydroxybutyrate)

Molecular characterization of StcI esterase from Aspergillus nidulans by Carolina Peña-Montes; Stefan Lange; Idalia Flores; Denise Castro-Ochoa; Rolf Schmid; Felipe Cruz-García; Amelia Farrés (pp. 917-926).

Aspergillus nidulans produces StcI esterase, which is involved in the biosynthesis of sterigmatocystin, a precursor of aflatoxins. Previous reports of this esterase in A. nidulans suggest that it is composed of 286 amino acid residues with a theoretical molecular mass of 31 kDa. Various conditions were evaluated to determine the optimal expression conditions for StcI; the highest level was observed when A. nidulans was cultured in solid oat media. Various esterases were expressed differentially according to the culture media used. However, specific antibodies designed to detect StcI reacted with a protein with an unexpected molecular mass of 35 kDa in cell extracts from all expression conditions. Analysis of the gene sequence and already reported expressed sequence tags indicated the presence of an additional 29-amino-acid N-terminal region of StcI, which is not a signal peptide and which has not been previously reported. We also detected the presence of this additional N-terminal region using reverse-transcriptase polymerase chain reaction. The complete protein (NStcI) was cloned and successfully expressed in Pichia pastoris.

Keywords: Aspergillus nidulans ; Esterase; Cloning; Sterigmatocystin; N terminus

Heterologous expression of a Tpo1 homolog from Arabidopsis thaliana confers resistance to the herbicide 2,4-D and other chemical stresses in yeast by Tânia R. Cabrito; Miguel C. Teixeira; Alexandra A. Duarte; Paula Duque; Isabel Sá-Correia (pp. 927-936).

The understanding of the molecular mechanisms underlying acquired herbicide resistance is crucial in dealing with the emergence of resistant weeds. Saccharomyces cerevisiae has been used as a model system to gain insights into the mechanisms underlying resistance to the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). The TPO1 gene, encoding a multidrug resistance (MDR) plasma membrane transporter of the major facilitator superfamily (MFS), was previously found to confer resistance to 2,4-D in yeast and to be transcriptionally activated in response to the herbicide. In this work, we demonstrate that Tpo1p is required to reduce the intracellular concentration of 2,4-D. ScTpo1p homologs encoding putative plasma membrane MFS transporters from the plant model Arabidopsis thaliana were analyzed for a possible role in 2,4-D resistance. At5g13750 was chosen for further analysis, as its transcript levels were found to increase in 2,4-D stressed plants. The functional heterologous expression of this plant open reading frame in yeast was found to confer increased resistance to the herbicide in Δtpo1 and wild-type cells, through the reduction of the intracellular concentration of 2,4-D. Heterologous expression of At5g13750 in yeast also leads to increased resistance to indole-3-acetic acid (IAA), Al³⁺ and Tl³⁺. At5g13750 is the first plant putative MFS transporter to be suggested as possibly involved in MDR.

Keywords: Arabidopsis thaliana ; Saccharomyces cerevisiae ; Herbicide resistance; Plant MFS transporter; At5g13750 ; 2,4-D; IAA and metal resistance

Comparing the transcriptomes of wine yeast strains: toward understanding the interaction between environment and transcriptome during fermentation by Debra Rossouw; Florian F. Bauer (pp. 937-954).

System-wide “omics” approaches have been widely applied to study a limited number of laboratory strains of Saccharomyces cerevisiae. More recently, industrial S. cerevisiae strains have become the target of such analyses, mainly to improve our understanding of biotechnologically relevant phenotypes that cannot be adequately studied in laboratory strains. Most of these studies have investigated single strains in a single medium. This experimental layout cannot differentiate between generally relevant molecular responses and strain- or media-specific features. Here we analyzed the transcriptomes of two phenotypically diverging wine yeast strains in two different fermentation media at three stages of wine fermentation. The data show that the intersection of transcriptome datasets from fermentations using either synthetic MS300 (simulated wine must) or real grape must (Colombard) can help to delineate relevant from “noisy” changes in gene expression in response to experimental factors such as fermentation stage and strain identity. The differences in the expression profiles of strains in the different environments also provide relevant insights into the transcriptional responses toward specific compositional features of the media. The data also suggest that MS300 is a representative environment for conducting research on wine fermentation and industrially relevant properties of wine yeast strains.

Keywords: Wine; Yeast; Transcriptomics; Fermentation; Colombard; MS300

Behavioral response of dissimilatory perchlorate-reducing bacteria to different electron acceptors by Yvonne Sun; Ruth L. Gustavson; Nadia Ali; Karrie A. Weber; Lacey L. Westphal; John D. Coates (pp. 955-963).

The response behavior of three dissimilatory perchlorate-reducing bacteria to different electron acceptors (nitrate, chlorate, and perchlorate) was investigated with two different assays. The observed response was species-specific, dependent on the prior growth conditions, and was inhibited by oxygen. We observed attraction toward nitrate when Dechloromonas aromatica strain RCB and Azospira suillum strain PS were grown with nitrate. When D. aromatica and Dechloromonas agitata strain CKB were grown with perchlorate, both responded to nitrate, chlorate, and perchlorate. When A. suillum was grown with perchlorate, the organism responded to chlorate and perchlorate but not nitrate. A gene replacement mutant in the perchlorate reductase subunit (pcrA) of D. aromatica resulted in a loss of the attraction response toward perchlorate but had no impact on the nitrate response. Washed-cell suspension studies revealed that the perchlorate grown cells of D. aromatica reduced both perchlorate and nitrate, while A. suillum cells reduced perchlorate only. Based on these observations, energy taxis was proposed as the underlying mechanism for the responses to (per)chlorate by D. aromatica. To the best of our knowledge, this study represents the first investigation of the response behavior of perchlorate-reducing bacteria to environmental stimuli. It clearly demonstrates attraction toward chlorine oxyanions and the unique ability of these organisms to distinguish structurally analogous compounds, nitrate, chlorate, and perchlorate and respond accordingly.

Keywords: Dechloromonas ; Dechlorosoma ; Azospira ; Chemotaxis; Perchlorate

Formation of coumarines during the degradation of alkyl substituted aromatic oil components by the yeast Trichosporon asahii by Susanne Awe; Annett Mikolasch; Frieder Schauer (pp. 965-976).

In this study, we investigated the ability of the yeast Trichosporon asahii SBUG-Y 833 to assimilate phenylalkanes with alkyl chain lengths from 7 to 12 carbon atoms, and we describe for the first time the formation of coumarines via a novel degradation pathway other than the normal terminal and ß-oxidation pathway of the alkyl residues. Besides benzoic acid and its further oxidation products, six new metabolites were identified. These were the three coumarines—4-hydroxycoumarin, 4,6-dihydroxycoumarin, 4,8-dihydroxycoumarin—and the three alkyl substituted aromatic acids—7-phenylheptanoic acid, 2-hydroxyphenylheptanoic acid, and 2-hydroxyphenylpropanoic acid. 4-Hydroxycoumarin was the main extracellular metabolite during the degradation of both odd- and even-chain phenylalkanes and was also produced during further biotransformation of 2-hydroxyphenylpropanoic acid and trans-2-hydroxycinnamic acid. Due to the ability of T. asahii to form hydroxylated coumarines, the transformation of 7-hydroxycoumarin and 2,4-dihydroxyphenylpropanoic acid was investigated. Yeast cells supplemented with 7-hydroxycoumarin formed 6,7-dihydroxycoumarin and 4,7-dihydroxycoumarin. The transformation of 2,4-dihydroxyphenylpropanoic acid yielded to 4,7-dihydroxycoumarin as the main product. All hydroxylated coumarines were continuously accumulated and are very resistant to further oxidation. The high potential of the yeast T. asahii SBUG-Y 833 to form different hydroxylated coumarines from alkylaromatics suggests possible applications in the biotechnological production of coumarine structures with medical potential as anticoagulative and antitumor pharmaceutical.

Keywords: Degradation; Alkylbenzenes; Coumarines; 4-Hydroxycoumarin; Yeasts

Nitrogen removal by co-occurring methane oxidation, denitrification, aerobic ammonium oxidation, and anammox by Miyoko Waki; Tomoko Yasuda; Hiroshi Yokoyama; Dai Hanajima; Akifumi Ogino; Kazuyoshi Suzuki; Takao Yamagishi; Yuichi Suwa; Yasuo Tanaka (pp. 977-985).

The pathway for removing NO ₃ ⁻ and NH ₄ ⁺ from wastewater in the presence of both CH₄ and O₂ was clarified by studying microbial activity and community. Batch incubation tests were performed to characterize the microbial activity of the sludge, which was acclimatized in a bioreactor in which O₂ and CH₄ were supplied to treat wastewater containing NO ₃ ⁻ and NH ₄ ⁺ . The tests showed that the sludge removed significant amounts of NO ₃ ⁻ and NH ₄ ⁺ in the presence of CH₄ and O₂, and the presence of the activity of methane oxidation, denitrification, nitrification, and anammox in the sludge. It was estimated that the total inorganic nitrogen removal was attributed to denitrification associated with methane oxidation as 53.4%, microbial assimilation as 37.9%, and anammox as 8.7%. Nitrification also contributed to NH ₄ ⁺ decrease as 34.5% and anammox as 6.4%. Anammox activity was unambiguously demonstrated by ²⁹N₂ production in anaerobic batch incubation with ¹⁵N-labeled inorganic nitrogen compounds. The presence of methane-oxidizing bacteria and candidate denitrifiers in the sludge was shown by denaturing gradient gel electrophoresis of 16S rRNA gene fragments. Clone library analysis of the PCR-amplified 16S rRNA gene fragment using specific primers for aerobic ammonium oxidizer and anammox revealed the presence of these bacteria. The results reveal that complex nitrogen-removal processes occur in the presence of CH₄ and O₂ by methanotroph, denitrifier, aerobic ammonium oxidizer, and anammox.

Keywords: Methanotroph; Denitrifier; Nitrifier; Anammox; Simultaneous removal of nitrate and ammonium

Investigation of factors influencing biogas production in a large-scale thermophilic municipal biogas plant by Agnes Weiss; Valérie Jérôme; Diana Burghardt; Likke Likke; Stefan Peiffer; Eugen M. Hofstetter; Ralf Gabler; Ruth Freitag (pp. 987-1001).

A continuously operated, thermophilic, municipal biogas plant was observed over 26 months (sampling twice per month) in regard to a number of physicochemical parameters and the biogas production. Biogas yields were put in correlation to parameters such as the volatile fatty acid concentration, the pH and the ammonium concentration. When the residing microbiota was classified via analysis of the 16S rRNA genes, most bacterial sequences matched with unidentified or uncultured bacteria from similar habitats. Of the archaeal sequences, 78.4% were identified as belonging to the genus Methanoculleus, which has not previously been reported for biogas plants, but is known to efficiently use H₂ and CO₂ produced by the degradation of fatty acids by syntrophic microorganisms. In order to further investigate the influence of varied amounts of ammonia (2–8 g/L) and volatile fatty acids on biogas production and composition (methane/CO₂), laboratory scale satellite experiments were performed in parallel to the technical plant. Finally, ammonia stripping of the process water of the technical plant was accomplished, a measure through which the ammonia entering the biogas reactor via the mash could be nearly halved, which increased the energy output of the biogas plant by almost 20%.

Keywords: Ammonia stripping; Biogas; Energy; Inhibition; Acetoclastic methanogenesis; Volatile fatty acids

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Applied Microbiology and Biotechnology (v.84, #5)