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Applied Microbiology and Biotechnology (v.84, #6)

Perspectives and limits of engineering the isoprenoid metabolism in heterologous hosts by Remco Muntendam; Elena Melillo; Annamargareta Ryden; Oliver Kayser (pp. 1003-1019).

Terpenoids belong to the largest class of natural compounds and are produced in all living organisms. The isoprenoid skeleton is based on assembling of C5 building blocks, but the biosynthesis of a great variety of terpenoids ranging from monoterpenoids to polyterpenoids is not fully understood today. Terpenoids play a fundamental role in human nutrition, cosmetics, and medicine. In the past 10 years, many metabolic engineering efforts have been undertaken in plants but also in microorganisms to improve the production of various terpenoids like artemisinin and paclitaxel. Recently, inverse metabolic engineering and combinatorial biosynthesis as main strategies in synthetic biology have been applied to produce high-cost natural products like artemisinin and paclitaxel in heterologous microorganisms. This review describes the recent progresses made in metabolic engineering of the terpenoid pathway with particular focus on fundamental aspects of host selection, vector design, and system biotechnology.

Keywords: Metabolic engineering; Synthetic biology; Terpenoids; Artemisinin; Paclitaxel; Biosynthesis

Novel approaches and achievements in biosynthesis of functional isoprenoids in Escherichia coli by Hisashi Harada; Norihiko Misawa (pp. 1021-1031).

Isoprenoids, also referred to as terpenes, are the most diverse class of natural products appearing in a variety of natural sources, specifically in higher plants, and have a wide range of biological functions. This review describes novel or recent approaches and achievements in pathway engineering of Escherichia coli towards efficient biosynthesis of functional isoprenoids, specifically carotenoids and sesquiterpene, following description of “regularity and simplicity” in the biosynthesis of isoprenoid basic structures. The introduction of heterologous mevalonate pathway-based genes into E. coli has been shown to improve the productivity of carotenoids or sesquiterpenes that are synthesized from farnesyl diphosphate. This achievement also enables relevant researchers to efficiently analyze an isolated gene candidate for a terpene synthase (terpene cyclase).

Keywords: α-Humulene; Lycopene; Sesquiterpene; Triterpene; Carotenoids; Isoprenoid

Microbial fatty acid conversion within the rumen and the subsequent utilization of these fatty acids to improve the healthfulness of ruminant food products by Mamun M. Or-Rashid; Tom C. Wright; Brian W. McBride (pp. 1033-1043).

Consumers are aware of foods containing microcomponents that may have positive effects on health maintenance and disease prevention. In ruminant milk, meat, and milk products; these functional food components include eicosapentaenoic acid (20:5n3), docosahexaenoic acid (22:6n3), 9c11t-conjugated linoleic acid, and vaccenic acid (11t-18:1). Modifying ruminal microbial metabolism of fatty acid in rumen through animal diet formulation is an effective way to enhance these functional fatty acids in ruminant-derived food products. However, it requires an understanding of the interrelationship between supply of lipid through the diet and rumen fermentation. Lipids in ruminant diets undergo extensive hydrolysis and biohydrogenation in the rumen. Apparent transfer efficiency of eicosapentaenoic acid and docosahexaenoic acid from feed to milk is very low (1.9 to 3.3%), which is, to a large extent, related to their extensive biohydrogenation in the rumen. Therefore, feeding a rumen-protected supplement containing eicosapentaenoic acid and docosahexaenoic acid, can be used to bypass the rumen. Ruminant-derived foods also contain different types of conjugated linoleic acid isomers, which are intermediates of rumen biohydrogenation of linoleic acid (9c12c-18:2). The predominant isomer of conjugated linoleic acid is 9c11t, which has numerous health benefits in animal models. The concentration of conjugated linoleic acid in ruminant-derived food products can be significantly enhanced through animal diet modification. We conclude that most current functional food products from ruminants have potential for their health-supporting properties, and for this market to succeed, an evidence-based approach should be developed in humans.

Keywords: Conjugated linoleic acid; Docosahexaenoic acid; Eicosapentaenoic acid; Fatty acid conversion; Rumen microbes and ruminant food products; Trans fatty acids

Advances in molecular methods to alter chromosomes and genome in the yeast Saccharomyces cerevisiae by Minetaka Sugiyama; Kazuo Yamagishi; Yeon-Hee Kim; Yoshinobu Kaneko; Masafumi Nishizawa; Satoshi Harashima (pp. 1045-1052).

A fundamental issue in biotechnology is how to breed useful strains of microorganisms for efficient production of valuable biomaterials. On-going and more recent developments in gene manipulation technologies and chromosomal and genomic modifications in particular have facilitated important contributions in this area. “Chromosome manipulation technology” as an outgrowth of “gene manipulation technology” may provide opportunities for creating novel strains of organisms with a variety of genomic constitutions. A simple and rapid chromosome splitting technology called “PCR-mediated chromosome splitting” (PCS) that we recently developed has made it possible to manipulate chromosomes and genomes on a large scale in an industrially important microorganism, Saccharomyces cerevisiae. This paper focuses on recent advances in molecular methods for altering chromosomes and genome in S. cerevisiae featuring chromosome splitting technology. These advances in introducing large-scale genomic modifications are expected to accelerate the breeding of novel strains for biotechnological purposes, and to reveal functions of presently uncharacterized chromosomal regions in S. cerevisiae and other organisms.

Keywords: Genome engineering; Chromosome manipulation; Genome reconstruction; Saccharomyces cerevisiae

Features and applications of microbial sugar epimerases by Susumu Ito (pp. 1053-1060).

Sugar (carbohydrate) epimerases catalyze the reversible conversion of a sugar epimer into its counterpart form. More than 20 types of sugar epimerase have been reported to date, and their biological properties, catalytic mechanisms, and 3D structures are very diverse among them. Recently, microbial sugar epimerases have been characterized in detail. This review surveys the catalytic aspects of microbial epimerases, which are relevant for production of bioactive mono- and oligosaccharides.

Keywords: Epimerase; Carbohydrate; Rare sugar; Application; Prebiotics

Effect of temperature on the development of Steinernema carpocapsae and Steinernema feltiae (Nematoda: Rhabditida) in liquid culture by A. Hirao; R.-U. Ehlers (pp. 1061-1067).

For commercial use of the entomopathogenic nematodes Steinernema carpocapsae and Steinernema feltiae in biological control of insect pests, they are produced in liquid culture on artificial media pre-incubated with their symbiotic bacteria Xenorhabdus nematophila and Xenorhabdus bovienii, respectively. After 1 day of the bacterial culture, nematode dauer juveniles (DJs) are inoculated, which recover development. The adult nematodes produce DJ offspring, which are harvested and can be sprayed. This study determined optimal temperatures to obtain high DJ progeny within a short process time. Temperatures assessed were 23°C, 25°C, 27°C, and 29°C for S. carpocapsae and 20°C, 23°C, 25°C, and 27°C for S. feltiae. The recovery of inoculated DJs was hardly affected and was reduced only in S. carpocapsae at 29°C. The fecundity (eggs in uterus) in S. carpocapsae reached a maximum at 27°C; whereas, maximum yields were recorded at 25°C. For both Steinernema spp., highest DJ densities were obtained after 15 days incubation at 25°C. Optimal culture temperature for both nematode species is 25°C. S. carpocapsae was more sensible to suboptimal temperature than S. feltiae. Results on total DJ density and DJ proportion of the total nematode population were more variable at non-optimal temperature condition for S. carpocapsae than for S. feltiae. Suboptimal culture temperature also reduced DJ infectivity.

Keywords: Biological control; Entomopathogenic nematodes; Process temperature; Dauer juvenile density; Reproduction; Liquid culture

A process for the production of ectoine and poly(3-hydroxybutyrate) by Halomonas boliviensis by Héctor Guzmán; Doan Van-Thuoc; Javier Martín; Rajni Hatti-Kaul; Jorge Quillaguamán (pp. 1069-1077).

The paper reports a study involving the use of Halomonas boliviensis, a moderate halophile, for co-production of compatible solute ectoine and biopolyester poly(3-hydroxybutyrate) (PHB) in a process comprising two fed-batch cultures. Initial investigations on the growth of the organism in a medium with varying NaCl concentrations showed the highest level of intracellular accumulation of ectoine (0.74 g L⁻¹) at 10–15% (w/v) NaCl, while at 15% (w/v) NaCl, the presence of hydroxyectoine (50 mg L⁻¹) was also noted. On the other hand, the maximum cell dry weight and PHB concentration of 10 and 5.8 g L⁻¹, respectively, were obtained at 5–7.5% (w/v) NaCl. A process comprising two fed-batch cultivations was developed—the first culture aimed at obtaining high cell mass and the second for achieving high yields of ectoine and PHB. In the first fed-batch culture, H. boliviensis was grown in a medium with 4.5% (w/v) NaCl and sufficient levels of monosodium glutamate, NH ₄ ⁺ , and PO ₄ ³⁻ . In the second fed-batch culture, the NaCl concentration was increased to 7.5% (w/v) to trigger ectoine synthesis, while nitrogen and phosphorus sources were fed only during the first 3 h and then stopped to favor PHB accumulation. The process resulted in PHB yield of 68.5 wt.% of cell dry weight and volumetric productivity of about 1 g L⁻¹ h⁻¹ and ectoine concentration, content, and volumetric productivity of 4.3 g L⁻¹, 7.2 wt.%, and 2.8 g L⁻¹ day⁻¹, respectively. At salt concentration of 12.5% (w/v) during the second cultivation, the ectoine content was increased to 17 wt.% and productivity to 3.4 g L⁻¹ day⁻¹.

Keywords: Moderate halophile; Halomonas boliviensis ; Compatible solutes; Poly(3-hydroxybutyrate); Fed-batch cultivation

An innovative consecutive batch fermentation process for very high gravity ethanol fermentation with self-flocculating yeast by F. Li; X. Q. Zhao; X. M. Ge; F. W. Bai (pp. 1079-1086).

An innovative consecutive batch fermentation process was developed for very high gravity (VHG) ethanol fermentation with the self-flocculating yeast under high biomass concentration conditions. On the one hand, the high biomass concentration significantly shortened the time required to complete the VHG fermentation and the duration of yeast cells suffering from strong ethanol inhibition, preventing them from losing viability and making them suitable for being repeatedly used in the process. On the other hand, the separation of yeast cells from the fermentation broth by sedimentation instead of centrifugation, making the process economically more competitive. The VHG medium composed of 255 g L⁻¹ glucose and 6.75 g L⁻¹ each of yeast extract and peptone was fed into the fermentation system for nine consecutive batch fermentations, which were completed within 8–14 h with an average ethanol concentration of 15% (v/v) and ethanol yield of 0.464, 90.8% of its theoretical value of 0.511. The average ethanol productivity that was calculated with the inclusion of the downstream time for the yeast flocs to settle from the fermentation broth and the supernatant to be removed from the fermentation system was 8.2 g L⁻¹ h⁻¹, much higher than those previously reported for VHG ethanol fermentation and regular ethanol fermentation with ethanol concentration around 12% (v/v) as well.

Keywords: Self-flocculating yeast; VHG; Consecutive ethanol fermentation

Enhancement of toxin- and virus-neutralizing capacity of single-domain antibody fragments by N-glycosylation by M. M. Harmsen; C. B. van Solt; H. P. D. Fijten (pp. 1087-1094).

Single-domain antibody fragments (VHHs) have several beneficial properties as compared to conventional antibody fragments. However, their small size complicates their toxin- and virus-neutralizing capacity. We isolated 27 VHHs binding Escherichia coli heat-labile toxin and expressed these in Saccharomyces cerevisiae. The most potent neutralizing VHH (LT109) was N-glycosylated, resulting in a large increase in molecular mass. This suggests that N-glycosylation of LT109 improves its neutralizing capacity. Indeed, deglycosylation of LT109 decreased its neutralizing capacity three- to fivefold. We also studied the effect of glycosylation of two previously isolated VHHs on their ability to neutralize foot-and-mouth disease virus. For this purpose, these VHHs that lacked potential N-glycosylation sites were genetically fused to another VHH that was known to be glycosylated. The resulting fusion proteins were also N-glycosylated. They neutralized the virus at at least fourfold-lower VHH concentrations as compared to the single, non-glycosylated VHHs and at at least 50-fold-lower VHH concentrations as compared to their deglycosylated counterparts. Thus, we have shown that N-glycosylation of VHHs contributes to toxin- and virus-neutralizing capacity.

Keywords: Nanobody; Recombinant antibody; Neutralization; N-glycosylation; Yeast

Biochemical and molecular genetic characterisation of a novel laccase produced by the aquatic ascomycete Phoma sp. UHH 5-1-03 by C. Junghanns; M. J. Pecyna; D. Böhm; N. Jehmlich; C. Martin; M. von Bergen; F. Schauer; M. Hofrichter; D. Schlosser (pp. 1095-1105).

A laccase from the aquatic ascomycete Phoma sp. UHH 5-1-03 (DSM 22425) was purified upon hydrophobic interaction and size exclusion chromatography (SEC). Mass spectrometric analysis of the laccase monomer yielded a molecular mass of 75.6 kDa. The enzyme possesses an unusual alkaline isoelectric point above 8.3. The Phoma sp. laccase undergoes pH-dependent dimerisation, with the dimer (∼150 kDa, as assessed by SEC) predominating in a pH range of 5.0 to 8.0. The enzyme oxidises common laccase substrates still at pH 7.0 and 8.0 and is remarkably stable at these pH values. The laccase is active at high concentrations of various organic solvents, all together indicating a considerable biotechnological potential. One laccase gene (lac1) identified at the genomic DNA level and transcribed in laccase-producing cultures was completely sequenced. The deduced molecular mass of the hypothetical protein and the predicted isoelectric point of 8.1 well agree with experimentally determined data. Tryptic peptides of electrophoretically separated laccase bands were analysed by nano-liquid chromatography–tandem mass spectrometry. By using the nucleotide sequence of lac1 as a template, eight different peptides were identified and yielded an overall sequence coverage of about 18%, thus confirming the link between lac1 and the expressed laccase protein.

Keywords: Alkaline isoelectric point; Aquatic ascomycete; Dimeric laccase; Enzyme stability; Laccase gene

Gene cloning, expression and characterization of a new cold-active and salt-tolerant endo-β-1,4-xylanase from marine Glaciecola mesophila KMM 241 by Bing Guo; Xiu-Lan Chen; Cai-Yun Sun; Bai-Cheng Zhou; Yu-Zhong Zhang (pp. 1107-1115).

Although a lot of xylanases are studied, only a few xylanases from marine microorganisms have been reported. A new xylanase gene, xynA, was cloned from marine bacterium Glaciecola mesophila KMM 241. Gene xynA contains 1,272 bp and encodes a 423-amino acid xylanase precursor. The recombinant xylanase, XynA, expressed in Escherichia coli BL21 is a monomer with a molecular mass of 43 kDa. Among the characterized xylanases, XynA shares the highest identity (46%) to the xylanase from Flavobacterium sp. strain MSY2. The optimum pH and temperature for XynA is 7.0 and 30 °C. XynA retains 23% activity and 27% catalytic efficiency at 4 °C. XynA has low thermostability, remaining 20% activity after 60-min incubation at 30 °C. Its apparent melting temperature (T _m) is 44.5 °C. These results indicate that XynA is a cold-active xylanase. XynA shows a high level of salt-tolerance, with the highest activity at 0.5 M NaCl and retaining 90% activity in 2.5 M NaCl. It may be the first salt-tolerant xylanase reported. XynA is a strict endo-β-1,4-xylanase with a demand of at least four sugar moieties for effective cleavage. It efficiently hydrolyzes xylo-oligosaccharides and xylan into xylobiose and xylotriose without producing xylose, suggesting its potential in xylo-oligosaccharides production.

Keywords: Xylanase; XynA; Cold-active; Salt-tolerant; Glaciecola mesophila ; Marine; Xylan

Engineering of polyhydroxyalkanoate synthase by Ser477X/Gln481X saturation mutagenesis for efficient production of 3-hydroxybutyrate-based copolyesters by Fumi Shozui; Ken’ichiro Matsumoto; Takahiro Sasaki; Seiichi Taguchi (pp. 1117-1124).

Class II polyhydroxyalkanoate synthase from Pseudomonas sp. 61-3 (PhaC1_Ps) synthesizes 3-hydroxybutyrate (3HB)-based copolyesters, P[3HB-co-3-hydroxyalkanoate (3HA)]. Four sites (130, 325, 477, and 481) in PhaC1_Ps that affect the cellular content and 3HB fraction of P(3HB-co-3HA) produced have been identified. Simple combination of beneficial mutations at the sites successfully increased 3HB fraction in the copolymers (62 mol.%). However, polymer content was often largely decreased (0.2 wt.%) regardless of an enhancement in 3HB fraction, compared to the wild-type enzyme (14 mol.% 3HB and 12 wt.%; Matsumoto et al. (2006) Biomacromolecules, 7:2436–2442). In the present study, we attempted to explore residues combination at the four sites to overcome the problem. Here, pairwise saturation mutagenesis at the neighboring sites 477 and 481 of PhaC1_Ps was performed using single and double mutations at sites 130 and 325 as templates to increase 3HB fraction in the copolymer without reducing the polymer content in recombinant Escherichia coli. These useful PhaC1_Ps mutants were screened based on enhanced P(3HB) content and were subsequently applied to P(3HB-co-3HA) production. Among the mutants tested, the Ser325Cys/Ser477Lys/Gln481Leu mutant exhibited increased 3HB fraction in copolymer (63 mol.%) and also polymer content (18 wt.%), indicating that mutation scrambling was effective for obtaining the desired mutants.

Keywords: PHA synthase; Evolutionary engineering; Monomer composition; P(3HB-co-3HA)

Novel microsatellite markers suitable for genetic studies in the white button mushroom Agaricus bisporus by Marie Foulongne-Oriol; Cathy Spataro; Jean-Michel Savoie (pp. 1125-1135).

Co-dominant microsatellite molecular markers were obtained from the Agaricus bisporus cultivated mushroom. Their potential for both the molecular characterisation of commercial strains and the monitoring of the intraspecific genetic variation was demonstrated. The analysis of 673 unique sequences issued from public database and 59 from an enriched A. bisporus genomic library resulted in the development of a total of 33 single sequence repeat or microsatellite (SSR) markers. Their usefulness for genetic analysis was assessed on 28 strains, which include six cultivars representative of traditional lineage, two hybrids and 20 strains originating from wild populations. A. bisporus SSR markers displayed each from two to ten alleles, with an average of 5.6 alleles per locus. The observed heterozygosity ranged from 0 to 0.88. Cluster analysis resulting from SSR fingerprintings was in agreement with published A. bisporus population structure. A combination of only three selected SSR markers was sufficient to discriminate unambiguously 27 out of 28 distinct genotypes. However, the two genetically related hybrids were not distinguishable. Multiplexing was tested, and up to seven loci could be genotyped simultaneously. We are therefore reporting the first development in A. bisporus of a set of microsatellite markers powerful and suitable for genetic analysis.

Keywords: Agaricus bisporus ; SSR; Genetic diversity; Strain typing

Quantification of live and dead probiotic bacteria in lyophilised product by real-time PCR and by flow cytometry by Mateja Kramer; Nataša Obermajer; Bojana Bogovič Matijašić; Irena Rogelj; Vojko Kmetec (pp. 1137-1147).

The basic requirement for probiotic bacteria to be able to exert expected positive effects is to be alive; therefore, appropriate quantification methods are crucial. Due to disadvantages of conventional microbiological methods, the bacterial quantification based on the nucleic acid detection is increasingly used. The objective of this study was to evaluate the possibility to use propidium monoazide (PMA) in combination with real-time polymerase chain reaction (PCR) method or LIVE/DEAD BacLight viability kit in combination with flow cytometry (FCM) for determination of probiotic bacteria in a lyophilised product containing Lactobacillus acidophilus LA-5 and Bifidobacterium animalis ssp. lactis BB-12. In addition, the viability of probiotic bacteria in lyophilised product during 3 months storage was investigated. In the product, the results of real-time PCR quantification of PMA-treated cells did not differ significantly from those of non-treated cells, which indicate that most of the bacterial cells retained the membrane integrity although they have lost the culturability. The results obtained by FCM analysis were comparable with those by PMA real-time PCR. In conclusion, the PMA real-time PCR and FCM determination of the viability of probiotic bacteria could complement the plate count method which considers only the culturable part of the population.

Keywords: Quantification; Real-time PCR; Propidium monoazide; Probiotic bacteria; Flow cytometry

Investigating the effectiveness of DNA microarray analysis for identifying the genes involved in l-lactate production by Saccharomyces cerevisiae by Takashi Hirasawa; Aki Ookubo; Katsunori Yoshikawa; Keisuke Nagahisa; Chikara Furusawa; Hideki Sawai; Hiroshi Shimizu (pp. 1149-1159).

In order to determine whether transcriptome data obtained by DNA microarray analysis could be used to identify the genes involved in target metabolite production, we tried to identify the genes involved in l-lactate production by l-lactate-producing recombinant Saccharomyces cerevisiae strains. We obtained DNA microarray data for these strains. Plasmids carrying lactic acid bacteria, bovine, and human l-lactate dehydrogenase (LDH) genes were introduced into PDC1-disrupted S. cerevisiae strains. l-Lactate productivity of the strains harboring the human and bovine LDH genes was higher than that of the strains harboring lactic acid bacteria LDH genes. DNA microarray analysis revealed that the expression of 388 genes was significantly altered in the strains with the human and bovine LDH genes. Of these, the l-lactate productivity of human LDH-harboring deletion strains of 289 genes was compared with that of the standard and 56 randomly selected deletion strains containing the same LDH gene to validate the effectiveness of DNA microarray analysis for identifying the genes responsible for l-lactate production in the recombinant strains. Only deletion strains of the genes selected on the basis of the DNA microarray data showed significantly altered l-lactate production as compared to the standard and the randomly selected deletion strains. Our results indicated that the genes related to l-lactate production could be successfully identified by selecting the genes that exhibited significantly altered expression on DNA microarray analysis, and the effectiveness of DNA microarray analysis for identifying the genes responsible for l-lactate production was discussed.

Keywords: Saccharomyces cerevisiae ; l-Lactate; DNA microarray; Knockout strain library

Sequence-specific bacterial growth inhibition by peptide nucleic acid targeted to the mRNA binding site of 16S rRNA by Masashi Hatamoto; Kazufumi Nakai; Akiyoshi Ohashi; Hiroyuki Imachi (pp. 1161-1168).

Peptide nucleic acid (PNA) targeted to the functional domains of 23S rRNA can inhibit translation and cell growth. However, effective inhibition of translation and cell growth using 16S rRNA-targeted PNA has still not been achieved. Here, we report that PNA targeted to the functional site of 16S rRNA could inhibit both gene expression in vitro and bacterial growth in pure culture with sequence specificity. We used 10-mer PNAs conjugated with a cell-penetrating peptide, which targeted the mRNA binding site at the 3′ end of 16S rRNA. Using 0.6 µM of the peptide–PNAs, cell-free ß-galactosidase production decreased by 50%, whereas peptide–PNAs with one or two mismatches to the target sequence showed much weaker inhibition effects. To determine the growth inhibition and bactericidal effects of the peptide–PNA conjugate, we performed OD measurement and viable cell counting. We observed dose- and sequence-dependent inhibition of cell growth and bactericidal effects. These growth inhibitory effects are observed both in the Gram-negative bacterium of Escherichia coli and the Gram-positive bacteria Bacillus subtilis and Corynebacterium efficiens, although inhibitory concentrations were different for each bacterial species. These results present possibilities for 16S rRNA sequence-based specific bacterial growth inhibition using a peptide–PNA conjugate.

Keywords: Peptide nucleic acid (PNA); 16S rRNA; mRNA binding site; Cell wall-permeablizing peptide

Regulation of hydantoin-hydrolyzing enzyme expression in Agrobacterium tumefaciens strain RU-AE01 by Meesbah Jiwaji; Rosemary Ann Dorrington (pp. 1169-1179).

Optically pure d-amino acids, like d-hydroxyphenylglycine, are used in the semi-synthetic production of pharmaceuticals. They are synthesized industrially via the biocatalytic hydrolysis of p-hydroxyphenylhydantoin using enzymes derived from Agrobacterium tumefaciens strains. The reaction proceeds via a three-step pathway: (a) the ring-opening cleavage of the hydantoin ring by a d-hydantoinase (encoded by hyuH), (b) conversion of the resultant d- N-carbamylamino acid to the corresponding amino acid by a d- N-carbamoylase (encoded by hyuC), and (c) chemical or enzymatic racemization of the un-reacted hydantoin substrate. While the structure and biochemical properties of these enzymes are well understood, little is known about their origin, their function, and their regulation in the native host. We investigated the mechanisms involved in the regulation of expression of the hydantoinase and N-carbamoylase enzyme activity in A. tumefaciens strain RU-AE01. We present evidence for a complex regulatory network that responds to the growth status of the cells, the presence of inducer, and nitrogen catabolite repression. Deletion analysis and site-directed mutagenesis were used to identify regulatory elements involved in transcriptional regulation of hyuH and hyuC expression. Finally, a comparison between the hyu gene clusters in several Agrobacterium strains provides insight into the function of d-selective hydantoin-hydrolyzing enzyme systems in Agrobacterium species.

Keywords: Agrobacterium tumefaciens ; hyu gene regulation; hyuH ; hyuC

Biological nitrification–denitrification with alternating oxic and anoxic operations using aerobic granules by Sunil S. Adav; Duu-Jong Lee; Juin-Yih Lai (pp. 1181-1189).

Efficient nitrification and denitrification of wastewater containing 1,700 mgl⁻¹ of ammonium-nitrogen was achieved using aerobic granular sludge cultivated at medium-to-high organic loading rates. The cultivated granules were tested in a sequencing batch reactor (SBR) fed with 6.4 or 10.2 kg NH ₄ ⁺ -N m⁻³ day⁻¹, a loading significantly higher than that reported in literature. With alternating 2 h oxic and 2 h anoxic operation (OA) modes, removal rate was 45.5 mg NH ₄ ⁺ -N g⁻¹ volatile suspended solids⁻¹ h⁻¹ at 6.4 kg NH ₄ ⁺ -N m⁻³ day⁻¹ loading and 41.3 ± 2.0 at 10.2 kg NH ₄ ⁺ -N m⁻³ day⁻¹ loading. Following the 60 days SBR test, granules were intact. The fluorescence in situ hybridization and confocal laser scanning microscopy results indicate that the SBR-OA granules have a distribution with nitrifers outside and heterotrophs outside that can effectively expose functional strains to surrounding substrates at high concentrations with minimal mass transfer limit. This microbial alignment combined with the smooth granule surface achieved nitrification–denitrification of wastewaters containing high-strength ammonium using aerobic granules. Conversely, the SBR continuous aeration mode yielded a distribution with nitrifers outside and heterotrophs inside with an unsatisfactory denitrification rate and floating granules as gas likely accumulated deep in the granules.

Keywords: Nitrification–denitrification; Aerobic granules; Microbial distribution; OLR

Fluctuation of microbial activities after influent load variations in a full-scale SBR: recovery of the biomass after starvation by Angela Cabezas; Patricia Draper; Claudia Etchebehere (pp. 1191-1202).

Due to variations in the production levels, a full-scale sequencing batch reactor (SBR) for post-treatment of tannery wastewater was exposed to low and high ammonia load periods. In order to study how these changes affected the N-removal capacity, the microbiology of the reactor was studied by a diverse set of techniques including molecular tools, activity tests, and microbial counts in samples taken along 3 years. The recover capacity of the biomass was also studied in a lab-scale reactor operated with intermittent aeration without feeding for 36 days. The results showed that changes in the feeding negatively affected the nitrifying community, but the nitrogen removal efficiencies could be restored after the concentration stress. Species substitution was observed within the nitrifying bacteria, Nitrosomonas europaea and Nitrobacter predominated initially, and after an ammonia overload period, Nitrosomonas nitrosa and Nitrospira became dominant. Some denitrifiers, with nirS related to Alicycliphilus, Azospirillum, and Marinobacter nirS, persisted during long-term reactor operation, but the community fluctuated both in composition and in abundance. This fluctuating community may better resist the continuous changes in the feeding regime. Our results showed that a nitrifying–denitrifying SBR could be operated with low loads or even without feeding during production shut down periods.

Keywords: Nitrification; Denitrification; Full scale; Long term; Community analysis; SBR

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Applied Microbiology and Biotechnology (v.84, #6)

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Applied Microbiology and Biotechnology (v.84, #6)