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Applied Microbiology and Biotechnology (v.91, #6)

Violacein and related tryptophan metabolites produced by Chromobacterium violaceum: biosynthetic mechanism and pathway for construction of violacein core by Tsutomu Hoshino (pp. 1463-1475).

Violacein is a natural violet pigment produced by several Gram-negative bacteria, including Chromobacterium violaceum, Janthinobacterium lividum, and Pseudoalteromonas tunicata D2, among others. This pigment has potential medical applications as antibacterial, anti-trypanocidal, anti-ulcerogenic, and anticancer drugs. The structure of violacein consists of three units: a 5-hydroxyindole, an oxindole, and a 2-pyrrolidone. The biosynthetic origins of hydrogen, nitrogen, and carbon in the pyrrolidone nucleus were established by feeding experiments using various stable isotopically labeled tryptophans (Trps). Pro-S hydrogen of CH₂ at the 3-position of Trp is retained during biosynthesis. The nitrogen atom is exclusively from the α-amino group, and the skeletal carbon atoms originate from the side chains of the two Trp molecules. All three oxygen atoms in the violacein core are derived from molecular oxygen. The most interesting biosynthetic mechanism is the 1,2-shift of the indole nucleus on the left side of the violacein scaffold. The alternative Trp molecule is directly incorporated into the right side of the violacein core. This indole shift has been observed only in violacein biosynthesis, despite the large number of natural products having been isolated. There were remarkable advances in biosynthetic studies in 2006–2008. During the 3 years, most of the intermediates and the complete pathway were established. Two independent processes are involved: the enzymatic process catalyzed by the five proteins VioABCDE or the alternative nonenzymatic oxidative decarboxylation reactions. The X-ray crystallographic structure of VioE that mediates the indole rearrangement reaction was recently identified, and the mechanism of the indole shift is discussed here.

Keywords: Violacein; Bisindole; Chromobacterium violaceum ; VioABCDE; Indole shift

Fungal enzyme sets for plant polysaccharide degradation by Joost van den Brink; Ronald P. de Vries (pp. 1477-1492).

Enzymatic degradation of plant polysaccharides has many industrial applications, such as within the paper, food, and feed industry and for sustainable production of fuels and chemicals. Cellulose, hemicelluloses, and pectins are the main components of plant cell wall polysaccharides. These polysaccharides are often tightly packed, contain many different sugar residues, and are branched with a diversity of structures. To enable efficient degradation of these polysaccharides, fungi produce an extensive set of carbohydrate-active enzymes. The variety of the enzyme set differs between fungi and often corresponds to the requirements of its habitat. Carbohydrate-active enzymes can be organized in different families based on the amino acid sequence of the structurally related catalytic modules. Fungal enzymes involved in plant polysaccharide degradation are assigned to at least 35 glycoside hydrolase families, three carbohydrate esterase families and six polysaccharide lyase families. This mini-review will discuss the enzymes needed for complete degradation of plant polysaccharides and will give an overview of the latest developments concerning fungal carbohydrate-active enzymes and their corresponding families.

Keywords: Polysaccharides; Plant biomass; Fungal enzymes; Aspergillus ; Trichoderma

In situ sensor techniques in modern bioprocess monitoring by Sascha Beutel; Steffen Henkel (pp. 1493-1505).

New reactor concepts as multi-parallel screening systems or disposable bioreactor systems for decentralized and reproducible production increase the need for new and easy applicable sensor technologies to access data for process control. These sophisticated reactor systems require sensors to work with the lowest sampling volumes or, even better, to measure directly in situ, but in situ sensors are directly incorporated into a reactor or fermenter within the sterility barrier and have therefore to stand the sterilization procedures. Consequently, these in situ sensor technologies should enable the measurement of multi-analytes simultaneously online and in real-time at a low price for the robust sensing element. Current research therefore focuses on the implementation of noninvasive spectroscopic and optical technologies, and tries to employ them through fiber optics attached to disposable sensing connectors. Spectroscopic methods reach from ultraviolet to infrared and further comprising fluorescence and Raman spectroscopy. Also, optic techniques like microscopy are adapted for the direct use in bioreactor systems (Ulber et al. in Anal Bioanal Chem 376:342–348, 2003) as well as various electrochemical methods (Joo and Brown in Chem Rev 108:638–651, 2008). This review shows the variety of modern in situ sensing principles in bioprocess monitoring with emphasis on spectroscopic and optical techniques and the progress in the adaption to latest reactor concepts.

Keywords: In situ sensor; Online monitoring; Noninvasive measurement

Impact of sulphate-reducing bacteria on the performance of engineering materials by Reza Javaherdashti (pp. 1507-1517).

Microbiologically Influenced Corrosion (MIC) is an electrochemical corrosion influenced by the presence/action of biological agents such as, but not limited to, bacteria. One of the key elements of MIC is sulphate-reducing bacteria (SRB). There are still many misunderstandings about these bacteria, their role in the deterioration of engineering materials and their importance over other types of corrosion-related micro-/macro-organisms. SRB do not require oxygen, yet they can be found in oxygenated environments; they are capable of tolerating a relative wide range of temperature, pH, chloride concentration and pressure values. Not only can SRB have deteriorating impact on engineering materials, they are also capable of inducing harm to health and agriculture. In this paper, after reviewing facts and figures regarding ecological and economical impacts of corrosion in general and MIC, in particular, the central concept of MIC, that is, biofilm formation and its deterioration mechanisms and the role of SRB in such mechanisms are described. Also, the possible enhancing role of SRB on stress corrosion cracking of steels and the controversial concept of no relationship between the number of SRB and corrosion rate are addressed and reviewed.

Keywords: Corrosion; Microbiologically influenced corrosion; Sulphate-reducing bacteria; Biofilm; Stress corrosion cracking

Hybrid thermochemical processing: fermentation of pyrolysis-derived bio-oil by Laura R. Jarboe; Zhiyou Wen; DongWon Choi; Robert C. Brown (pp. 1519-1523).

Thermochemical processing of biomass by fast pyrolysis provides a nonenzymatic route for depolymerization of biomass into sugars that can be used for the biological production of fuels and chemicals. Fermentative utilization of this bio-oil faces two formidable challenges. First is the fact that most bio-oil-associated sugars are present in the anhydrous form. Metabolic engineering has enabled utilization of the main anhydrosugar, levoglucosan, in workhorse biocatalysts. The second challenge is the fact that bio-oil is rich in microbial inhibitors. Collection of bio-oil in distinct fractions, detoxification of bio-oil prior to fermentation, and increased robustness of the biocatalyst have all proven effective methods for addressing this inhibition.

Keywords: Levoglucosan; Bio-oil; Ethanol; Lipids; Inhibition; Toxicity; Furfural; Acetic acid

Challenges of the utilization of wood polymers: how can they be overcome? by Yunqiao Pu; Matyas Kosa; Udaya C. Kalluri; Gerald A. Tuskan; Arthur J. Ragauskas (pp. 1525-1536).

Diminishing fossil fuel resources as well as growing environmental and energy security concerns, in parallel with growing demands on raw materials and energy, have intensified global efforts to utilize wood biopolymers as a renewable resource to produce biofuels and biomaterials. Wood is one of the most abundant biopolymer composites on earth that can be converted into biofuels as well as used as a platform to produce bio-based materials. The major biopolymers in wood are cellulose, hemicelluloses, and lignin which account for >90% of dry weight. These polymers are generally associated with each other in wood cell walls resulting in an intricate and dynamic cell wall structure. This mini-review provides an overview of major wood biopolymers, their structure, and recent developments in their utilization to develop biofuels. Advances in genetic modifications to overcome the recalcitrance of woody biomass for biofuels are discussed and point to a promising future.

Keywords: Wood biopolymer; Biofuels; Genetic modification; Biodiesel

Improved homo l-lactic acid fermentation from xylose by abolishment of the phosphoketolase pathway and enhancement of the pentose phosphate pathway in genetically modified xylose-assimilating Lactococcus lactis by Satoru Shinkawa; Kenji Okano; Shogo Yoshida; Tsutomu Tanaka; Chiaki Ogino; Hideki Fukuda; Akihiko Kondo (pp. 1537-1544).

In order to achieve efficient homo L-lactic acid fermentation from xylose, we first carried out addition of xylose assimilation ability to Lactococcus lactis IL 1403 by introducing a plasmid carrying the xylRAB genes from L. lactis IO-1 (pXylRAB). Then modification of xylose assimilation pathway was carried out. L. lactis has two pathways for xylose assimilation called the phosphoketolase pathway (PK pathway) that produces both lactic acid and acetic acid and the pentose phosphate pathway (PP pathway) that produces only lactic acid as a final product. Thus a mutant strain that disrupted its phosphokeolase gene (ptk) was constructed. The Δptk mutant harboring pXylRAB lacked the PK pathway and produced predominantly lactic acid from xylose via the PP pathway, although its fermentation rate slightly decreased. Further introduction of the transketolase gene (tkt) to disrupted ptk locus led restoration of fermentation rate and this was attributed to enhancement of the PP pathway. As a result, ptk::tkt strain harboring pXylRAB produced 50.1 g/l of L-lactic acid from xylose with a high optical purity of 99.6% and a high yield of 1.58 (moles per mole xylose consumed) that is close to theoretical value of 1.67 from xylose.

Keywords: Homo l-lactic acid fermentation; Xylose; Lactic acid bacteria; Lactococcus lactis

Improved phloroglucinol production by metabolically engineered Escherichia coli by Yujin Cao; Xinglin Jiang; Rubing Zhang; Mo Xian (pp. 1545-1552).

Phloroglucinol is a valuable chemical which has been successfully produced by metabolically engineered Escherichia coli. However, the low productivity remains a bottleneck for large-scale application and cost-effective production. In the present work, we cloned the key biosynthetic gene, phlD (a type III polyketide synthase), into a bacterial expression vector to produce phloroglucinol in E. coli and developed different strategies to re-engineer the recombinant strain for robust synthesis of phloroglucinol. Overexpression of E. coli marA (multiple antibiotic resistance) gene enhanced phloroglucinol resistance and elevated phloroglucinol production to 0.27 g/g dry cell weight. Augmentation of the intracellular malonyl coenzyme A (malonyl-CoA) level through coordinated expression of four acetyl-CoA carboxylase (ACCase) subunits increased phloroglucinol production to around 0.27 g/g dry cell weight. Furthermore, the coexpression of ACCase and marA caused another marked improvement in phloroglucinol production 0.45 g/g dry cell weight, that is, 3.3-fold to the original strain. Under fed-batch conditions, this finally engineered strain accumulated phloroglucinol up to 3.8 g/L in the culture 12 h after induction, corresponding to a volumetric productivity of 0.32 g/L/h. This result was the highest phloroglucinol production to date and showed promising to make the bioprocess economically feasible.

Keywords: Acetyl-CoA carboxylase; marA ; Metabolically engineered Escherichia coli ; phlD ; Phloroglucinol

Co-fermentation of cellulose/xylan using engineered industrial yeast strain OC-2 displaying both β-glucosidase and β-xylosidase by Satoshi Saitoh; Tsutomu Tanaka; Akihiko Kondo (pp. 1553-1559).

We constructed a recombinant industrial Saccharomyces cerevisiae yeast strain OC2-AXYL2-ABGL2-Xyl2 by inserting two copies of the β-glucosidase (BGL) and β-xylosidase (XYL) genes, and a gene cassette for xylose assimilation in the genome of yeast strain OC-2HUT. Both BGL and XYL were expressed on the yeast cell surface with high enzyme activities. Using OC2-AXYL2-ABGL2-Xyl2, we performed ethanol fermentation from a mixture of powdered cellulose (KC-flock) and Birchwood xylan, with the additional supplementation of a 30-g/l Trichoderma reesei cellulase complex mixture. The ethanol yield (gram per gram of added cellulases) of the strain OC2-AXYL2-ABGL2-Xyl2 increased approximately 2.5-fold compared to that of strain OC2-Xyl2, which lacked β-glucosidase and β-xylosidase activities. Notably, the concentration of additional T. reesei cellulase was reduced from 30 to 24 g/l without affecting ethanol production. The BGL- and XYL-displaying industrial yeast of the strain OC2-AXYL2-ABGL2-Xyl2 represents a promising yeast for reducing cellulase consumption of ethanol fermentation from lignocellulosic biomass by compensating for the inherent weak BGL and XYL activities of T. reesei cellulase complexes.

Keywords: Saccharomyces cerevisiae ; β-Xylosidase; β-Glucosidase; Xylose fermentation; Cellulosic ethanol

Molecular cloning and characterization of a novel SGNH arylesterase from the goat rumen contents by Guozeng Wang; Kun Meng; Huiying Luo; Yaru Wang; Huoqing Huang; Pengjun Shi; Xia Pan; Peilong Yang; Bin Yao (pp. 1561-1570).

An esterase-encoding gene, estR5, was directly obtained from the genomic DNA of goat rumen contents. The 555-bp full-length gene encodes a 184-residue polypeptide (EstR5) without putative signal peptide. Deduced EstR5 shared the highest identity (50%) to a putative arylesterase from Ruminococcaceae bacterium D16. Phylogenetic analysis indicated that EstR5 was closely related with microbial esterases of gastrointestinal source. A comparison of the conserved motifs shared with GDSL proteins revealed that EstR5 could be grouped into the GDSL family and was further classified into the subfamily of SGNH hydrolases. The gene estR5 was expressed in Escherichia coli BL21 (DE3) and purified to electrophoretic homogeneity. Recombinant EstR5 exhibited highest catalytic efficiency towards α-naphthyl acetate followed by phenyl acetate and p-nitrophenyl acetate and had no activity towards PNP esters with acyl chains longer than C8. The enzyme exhibited optimal activity at around 60°C and pH 8.0, was stable at pH ranging from 6.0 to 11.0 and was slightly activated by detergent Tween, Nonidet P-40, and Triton X-100. These properties suggest that EstR5 has great potential for basic research and industrial applications. To our knowledge, this is the first arylesterase obtained from rumen microenvironment.

Keywords: Esterase; Arylesterase; SGNH family; Goat rumen; Detergent-activated

Production, purification, and characterization of a novel killer toxin from Kluyveromyces siamensis against a pathogenic yeast in crab by Muhammad Aslam Buzdar; Zhe Chi; Qi Wang; Ming-Xia Hua; Zhen-Ming Chi (pp. 1571-1579).

The yeast Kluyveromyces siamensis HN12-1 isolated from mangrove ecosystem was found to be able to produce killer toxin against the pathogenic yeast (Metschnikowia bicuspidata WCY) in crab. When the killer yeast was grown in the medium with pH 4.0 and 0.5% NaCl and at 25 °C, it could produce the highest amount of killer toxin against the pathogenic yeast M. bicuspidata WCY. The killing activity of the purified killer toxin against the pathogenic yeast M. bicuspidata WCY was the highest when it was incubated at 25 °C in the assay medium without added NaCl and pH 4.0. The molecular weight of the purified killer toxin was 66.4 kDa. The killer toxin produced by the yeast strain HN12-1 could kill only the whole cells of M. bicuspidata WCY among all the yeast species tested in this study. This is the first time to report that the killer toxin produced by the yeast K. siamensis HN12-1 isolated from the mangrove ecosystem only killed pathogenic yeast M. bicuspidata WCY.

Keywords: Killer toxin; Killer yeast; Mangrove ecosystem; Pathogenic yeast; Yeast relationship

Improvement of lentiviral transfer vectors using cis-acting regulatory elements for increased gene expression by Gonçalo Real; Francisca Monteiro; Christa Burger; Paula M. Alves (pp. 1581-1591).

Lentiviral vectors are an important tool for gene delivery in vivo and in vitro. The success of gene transfer approaches relies on high and stable levels of gene expression. To this end, several molecular strategies have been employed to manipulate these vectors towards improving gene expression in the targeted animal cells. Low gene expression can be accepted due to the weak transcription from the majority of available mammalian promoters; however, this obstacle can be in part overcome by the insertion of cis-acting elements that enhance gene expression in various expression contexts. In this work, we created different lentiviral vectors in which several posttranscriptional regulatory elements, namely the Woodchuck hepatitis posttranscriptional regulatory element (WPRE) and different specialized poly(A) termination sequences (BGH and SV40) were used to develop vectors leading to improved transgene expression. These vectors combine the advantages of restriction enzyme/ligation-independent cloning eliminating the instability and recombinogenic problems occurring from traditional cloning methods in lentiviral expression vectors and were tested by expressing GFP and the firefly Luciferase reporter gene from different cellular promoters in different cell lines. We show that the promoter activity varies between cell lines and is affected by the lentiviral genomic context. Moreover, we show that the combination of the WPRE element with the BGH poly(A) signal significantly enhances transgene expression. The vectors herein created can be easily modified and adapted without the need for extensive recloning making them a valuable tool for viral vector development.

Keywords: Lentivirus; Polyadenylation; WPRE; Gateway; Viral vector

Efficient accumulation of oleic acid in Saccharomyces cerevisiae caused by expression of rat elongase 2 gene (rELO2) and its contribution to tolerance to alcohols by Hisashi Yazawa; Yasushi Kamisaka; Kazuyoshi Kimura; Masakazu Yamaoka; Hiroshi Uemura (pp. 1593-1600).

When the cells of Saccharomyces cerevisiae are exposed to high concentration of ethanol, the content of oleic acid (C18:1n-9) increased as the initial concentration of ethanol increased. Based on this observation, we attempted to confer ethanol tolerance to S. cerevisiae by manipulating fatty acid composition of the cells. Rather than altering OLE1 expression [the desaturase making both C16:1n-7 (palmitoleic acid) and C18:1n-9], we introduced elongase genes. Introduction of rat elongase 1 gene (rELO1) into S. cerevisiae gave cis-vaccenic acid (cis-C18:1n-7) by conversion from C16:1n-7, and the increase in this C18:1 fatty acid did not confer ethanol tolerance to the cells. On the other hand, the introduction of rat elongase 2 gene (rELO2), which elongates C16:0 to C18:0, drastically increased C18:1n-9 content, and the cells acquired ethanol tolerance, emphasizing the specific role of C18:1n-9. Furthermore, the transformant of rELO2 also conferred tolerance to n-butanol, n-propanol, and 2-propanol.

Keywords: Saccharomyces cerevisiae ; Alcohol resistance; rELO2 ; Unsaturated fatty acids

Quantitative expression analysis of mleP gene and two genes involved in the ABC transport system in Oenococcus oeni during rehydration by Antonella Costantini; Enrico Vaudano; Kalliopi Rantsiou; Luca Cocolin; Emilia Garcia-Moruno (pp. 1601-1609).

Oenococcus oeni is recognized as the principal microorganism responsible for malolactic fermentation, and the control of its activity is of primary importance in winemaking. The aim of this study was to quantify the levels of expression of the malate transporter gene (mleP) and of two genes putatively involved in the ATP-binding cassette transport system (oeoe_1651, oeoe_0550) to better understand the physiological response of bacteria during rehydration. These genes coding for transporters were studied in different rehydration media. Initially, three different statistical algorithms were used to identify suitable reference genes to be used for the normalization of expression data in O. oeni during rehydration, and to this purpose, the best genes found were ddl and gyrB. The results showed that the genes for transporters of malate and sugar (mleP, oeoe_1651) were activated immediately after a few minutes of rehydration, when specific medium compositions were used.

Keywords: Gene expression; Reference gene validation; RT-qPCR; Oenococcus oeni

β-Carotene production by Saccharomyces cerevisiae with regard to plasmid stability and culture media by Nicole Lange; Alexander Steinbüchel (pp. 1611-1622).

A recombinant Saccharomyces cerevisiae strain was used for the production of β-carotene. The episomal plasmid YEplac195YB/I/E was extended by a gene coding for the mevalonate kinase (mvaK1) from Staphylococcus aureus. The adh1 promoter was chosen for constitutive expression of mvaK1. The recombinant strain S. cerevisiae G175 (YEplac-CaroSA) synthesised β-carotene by expressing the carotenogenic genes of Xanthophyllomyces dendrorhous together with the mvaK1 gene. Cells of this strain were investigated for their carotenoid contents in YNB and YPD media. A corresponding mvaK1 transcript in the recombinant yeast host was verified. Growth experiments of a specific erg12 deletion mutant showed that the mevalonate kinase (MvaK1) was able to complement the function of the deleted native mevalonate kinase (Erg12) from S. cerevisiae in the MVA pathway under control of the constitutive adh1 promoter. Cells of S. cerevisiae G175 (YEplac-CaroSA) exhibited high plasmid stability under either selective or non-selective cultivation conditions. Time course experiments demonstrated high plasmid stability even over extended cultivation periods. Carotenoid production was therefore also stable in larger culture volumes. Due to the stability of the plasmid, cultivation of the cells in complex YPD medium was possible, and 14.3 mg β-carotene per litre and a cell density of 9 g cell dry matter (CDM) per litre were achieved. The highest amount of 3,897 μg β-carotene per gramme CDM at a cell density of 1 g CDM per litre was measured after cultivation of the cells in YNB medium with glucose as sole carbon source.

Keywords: β-carotene; Carotenoids; Mevalonate kinase; Mevalonate (MVA) pathway; Plasmid stability; Saccharomyces cerevisiae

Effects of nitrogen sources on production and composition of sophorolipids by Wickerhamiella domercqiae var. sophorolipid CGMCC 1576 by Xiao-jing Ma; Hui Li; Ling-jian Shao; Jing Shen; Xin Song (pp. 1623-1632).

The effects of nitrogen sources on growth of sophorolipid-producing yeast, Wickerhamiella domercqiae var. sophorolipid CGMCC 1576 and on production and composition of sophorolipids were studied. Organic nitrogen sources are more favorable for accumulation of biomass than inorganic ones. Presence of ammonium ion from different inorganic nitrogen sources (except NH₄HCO₃) greatly inhibited the production of lactonic sophorolipids. However, when organic nitrogen sources were used, lactonic sophorolipid production was strongly increased. Production of crystalline lactonic sophorolipids from organic/inorganic nitrogen sources was enhanced with the increase of pH value adjusted by sodium hydroxide or sodium citrate solution. Fourier-transform infrared (FT-IR), gas chromatography mass spectrometry (GC-MS), high-performance liquid chromatography (HPLC), and mass spectra (MS) were employed to compare the composition of sophorolipid mixture obtained from different nitrogen sources. More than 15 acidic sophorolipid molecules and only 4 lactonic sophorolipid molecules were produced by using 1.27 g/l ammonium sulfate as nitrogen source; they were separated by preparative HPLC and their structures were elucidated by MS. These results suggest extraordinary regulatory effects of nitrogen source on growth and sophorolipid synthesis of W. domercqiae var. sophorolipid.

Keywords: Wickerhamiella domercqiae var. sophorolipid CGMCC 1576; Lactonic sophorolipid; Acidic sophorolipid; Organic nitrogen source; Inorganic nitrogen source

Evaluation of encapsulation stress and the effect of additives on viability and photosynthetic activity of Synechocystis sp. PCC 6803 encapsulated in silica gel by David J. Dickson; Roger L. Ely (pp. 1633-1646).

Stresses imposed on the cyanobacterium Synechocystis sp. PCC 6803 by various compounds present during silica sol–gel encapsulation, including salt, ethanol (EtOH), polyethylene glycol (PEG), glycerol, and glycine betaine, were investigated. Viability of encapsulated cells and photosynthetic activity of cells stressed by immediate (2 min) and 24-h exposure to the five stress-inducing compounds were monitored by pulse amplitude modulated fluorometry. Cells of Synechocystis sp. PCC 6803 readily survive encapsulation in both alkoxide-derived gels and gels from aqueous precursors and can remain active at least 8 weeks with slight degradation in PSII efficiency. Post-encapsulation survival was improved in gels containing no additive when compared with gels containing PEG or glycerol. Glycerol was shown to have a detrimental effect on Synechocystis sp. PCC 6803, reducing ϕPSII and F _v′/F _m′ by as much as 75%, possibly a result of disrupting excitation transfer between the phycobilisomes and photosystems. PEG was similarly deleterious, dramatically reducing the ability to carry out a state transition and adequately manage excitation energy distribution. EtOH stress also hindered state transitions, although less severely than PEG, and the cells were able to recover nearly all photosynthetic efficiency within 24 h after an initial drop. Betaine did not interfere with state transitions but did reduce quantum yield and photochemical quenching. Finally, Synechocystis sp. PCC 6803 was shown to recover from salt stress.

Keywords: Silica sol–gel encapsulation; Encapsulation stress; Fluorescence; Synechocystis sp. PCC 6803; Glycerol; Polyethylene glycol; Betaine

Dynamic microbial response of sulfidogenic wastewater biofilm to nitrate by Janani Mohanakrishnan; Michael Vedel Wegener Kofoed; Jeremy Barr; Zhiguo Yuan; Andreas Schramm; Rikke Louise Meyer (pp. 1647-1657).

Nitrate is one of the chemicals often added to wastewater to control hydrogen sulfide production by sulfate-reducing bacteria (SRB). While the effect of nitrate in various SRB pure cultures is well documented, the effect observed in mixed microbial communities is not consistent. This study investigates the response of mixed SRB communities to nitrate, by examining the changes in activity and community composition of sulfidogenic wastewater biofilm over a 10-day period with 10 mmol L⁻¹ nitrate exposure. Biofilms were enriched in SRB belonging to the Desulfobacter, Desulfobulbus, Desulfomicrobium, and Desulfovibrio genera. Nitrate exposure decreased dsrB transcription within 4 h, and sulfate consumption within 10 days, but it did not fully eliminate sulfide production in the biofilms. The effect of nitrate on SRB was genus specific; Desulfobacter and Desulfobulbus disappeared while Desulfovibrio and Desulfomicrobium persisted in the biofilms. Nitrate exposure also led to the rapid proliferation of nitrate-reducing bacteria within the biofilms, and increased the biofilm thickness. Nitrate consumption began within 2 h of nitrate exposure and gradually increased in rate over time. Transcription of the nitrate reductase napA, and the diversity of nitrate reductase genes narG and napA also increased concurrently. Our results demonstrate that some SRB, presumably those able to tolerate or detoxify nitrite, will persist in sulfidogenic wastewater biofilms despite continuous exposure to high levels of nitrate. Nitrate is therefore unlikely to provide lasting hydrogen sulfide suppression in wastewater biofilms harboring Desulfovibrio or Desulfomicrobium populations.

Keywords: Sulfide control; Nitrate addition; SRB; NRB; Community changes

Prokaryotic diversity, composition structure, and phylogenetic analysis of microbial communities in leachate sediment ecosystems by Jingjing Liu; Weixiang Wu; Chongjun Chen; Faqian Sun; Yingxu Chen (pp. 1659-1675).

In order to obtain insight into the prokaryotic diversity and community in leachate sediment, a culture-independent DNA-based molecular phylogenetic approach was performed with archaeal and bacterial 16S rRNA gene clone libraries derived from leachate sediment of an aged landfill. A total of 59 archaeal and 283 bacterial rDNA phylotypes were identified in 425 archaeal and 375 bacterial analyzed clones. All archaeal clones distributed within two archaeal phyla of the Euryarchaeota and Crenarchaeota, and well-defined methanogen lineages, especially Methanosaeta spp., are the most numerically dominant species of the archaeal community. Phylogenetic analysis of the bacterial library revealed a variety of pollutant-degrading and biotransforming microorganisms, including 18 distinct phyla. A substantial fraction of bacterial clones showed low levels of similarity with any previously documented sequences and thus might be taxonomically new. Chemical characteristics and phylogenetic inferences indicated that (1) ammonium-utilizing bacteria might form consortia to alleviate or avoid the negative influence of high ammonium concentration on other microorganisms, and (2) members of the Crenarchaeota found in the sediment might be involved in ammonium oxidation. This study is the first to report the composition of the microbial assemblages and phylogenetic characteristics of prokaryotic populations extant in leachate sediment. Additional work on microbial activity and contaminant biodegradation remains to be explored.

Keywords: Bacterial diversity; Archaeal diversity; 16S rRNA gene; Clone library; Leachate sediment

Kinetics of CO conversion into H₂ by Carboxydothermus hydrogenoformans by Ya Zhao; Ruxandra Cimpoia; Zhijun Liu; Serge R. Guiot (pp. 1677-1684).

The objective of this study was to improve the biological water–gas shift reaction for producing hydrogen (H₂) by conversion of carbon monoxide (CO) using an anaerobic thermophilic pure strain, Carboxydothermus hydrogenoformans. Specific hydrogen production rates and yields were investigated at initial biomass densities varying from 5 to 20 mg volatile suspended solid (VSS) L⁻¹. Results showed that the gas–liquid mass transfer limits the CO conversion rate at high biomass concentrations. At 100-rpm agitation and at CO partial pressure of 1 atm, the optimal substrate/biomass ratio must exceed 5 mol CO g⁻¹ biomass VSS in order to avoid gas–liquid substrate transfer limitation. An average H₂ yield of 94 ± 3% and a specific hydrogen production rate of ca. 3 mol g⁻¹ VSS day⁻¹ were obtained at initial biomass densities between 5 and 8 mg VSS⁻¹. In addition, CO bioconversion kinetics was assessed at CO partial pressure from 0.16 to 2 atm, corresponding to a dissolved CO concentration at 70°C from 0.09 to 1.1 mM. Specific bioactivity was maximal at 3.5 mol CO g⁻¹ VSS day⁻¹ for a dissolved CO concentration of 0.55 mM in the culture. This optimal concentration is higher than with most other hydrogenogenic carboxydotrophic species.

Keywords: Syngas; Carbon monoxide; Hydrogen; Carboxydothermus hydrogenoformans ; Kinetics; Inhibition; Gas–liquid mass transfer

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Applied Microbiology and Biotechnology (v.91, #6)

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Applied Microbiology and Biotechnology (v.91, #6)