Rich Complex Medium Research Articles

Biorefinery concept for Cylindrospermum alatosporum CCALA 988 extracting multiple high-value compounds and residue utilization by P. pastoris fermentation producing phytase

Economically feasible biorefinery concepts rely on the production of multiple high-value products and valorization even of residual side streams. This generally holds true for biomass from agriculture, forestry, and aquaculture. Cyanobacteria are seen as promising organisms for this purpose. The potential for commercialization, however, strongly depends on the strain being used and the range of products it offers. The cyanobacterial strain Cylindrospermum alatosporum CCALA 988 was reported to produce high amounts of the cyclic lipopeptides puwainaphycins (PUWs) and minutisamides (MINs) as high-value products. Hence, this study investigated the biomass of the cyanobacterial strain as a potential feedstock for a biorefinery concept. The study also explored the enzymatic hydrolysis of the residual biomass and its utilization as a medium supplement for appA E. coli phytase expression with Pichia pastoris. Analysis of the cyanobacterial biomass revealed the following composition: ash 1.9 %, lipids 4.1 %, starch 2.4 %, structural saccharides 8.7 %, and protein 52.4 %. Further, a sequential extraction concept and a bench-scale mass balance were investigated, and products were proposed with 56.2 % of biomass being utilized and 43.8 % being left as residue. PUWs (21.6 mg/g), MINs (101.5 mg/g), phycobiliproteins (34.5 mg/g), and pigments (6.4 mg/g) were selected as products and quantified. The utilization of hydrolysate by fermentation of P. pastoris for appA E. coli phytase expression was studied in a 1 L system. In batch mode, the hydrolysate allowed to achieve comparable results to a rich complex medium, with over 500 U/mL of appA E. coli phytase. The obtained results provide a basis for further development of this biorefinery concept.

Impact ofMtrAon phosphate metabolism genes and the response to altered phosphate conditions inStreptomyces

Phosphate metabolism is known to be regulated by the PhoPR regulatory system in Streptomyces and some other bacteria. In this study, we report that MtrA also regulates phosphate metabolism in Streptomyces. Our data showed that, in Streptomyces coelicolor, MtrA regulates not only phosphate metabolism genes such as phoA but also phoP under different phosphate conditions, including growth on rich complex media without added inorganic phosphate and on defined media with low or high concentrations of inorganic phosphate. Cross-regulation was also observed among mtrA, phoP and glnR under these conditions. We demonstrated both in vitro and in vivo binding of MtrA to the promoter regions of genes associated with phosphate metabolism and to the intergenic region between phoR and phoU, indicating that these phosphate metabolism genes are targets of MtrA. We further showed that MtrA in S. lividans and S. venezuelae has detectable regulatory effects on expression of phosphate metabolism genes. Additionally, the MtrA homologue from Corynebacterium glutamicum bound predicted MtrA sites of multiple phosphate metabolism genes, implying its potential for regulating phosphate metabolism in this species. Overall, our findings support MtrA as a major regulator for phosphate metabolism in Streptomyces and also potentially in other actinobacteria.

Novel auto-selection systems for transformation selection of Saccharomyces cerevisiae in rich complex media.

The most widely used strategy for selection of yeast transformed with episomal plasmids comprises the use of auxotrophic yeast strains in combination with vectors containing complementing prototrophic marker genes. Another approach uses heterologous genes or cassettes which, if present in the vector, render the otherwise sensitive yeast strain resistant to antibiotics. In addition, auto-selection systems for Saccharomyces cerevisiae have been developed that eliminate the requirement for synthetic drop-out media or the use of antibiotics for transformation selection and subsequent plasmid maintenance in expression cultures. Here we describe a combination of host strain and vector system introducing a novel concept of auto-selection systems that allows for easy and robust propagation of host cells deleted in essential genes in supplemented media before being transformed with rescuing plasmids. With that, our approach is favorable over commonly used selection strategies and has major advantage over other auto-selection systems. Our approach complements the auto-selection toolbox already available for S. cerevisiae, thus contributing a novel system that enables the use of complex peptone-based media for protein expression and metabolic engineering approaches. We therefore expect that this new strategy will be of general interest to the yeast research community in academia and industry.

The Secretome landscape of Escherichia coli O157:H7: Deciphering the cell-surface, outer membrane vesicle and extracellular subproteomes

Among diarrheagenic E. coli (DEC), enterohaemorrhagic E. coli (EHEC) are the most virulent anthropozoonotic agents. The ability of bacterial cells to functionally interact with their surrounding essentially relies on the secretion of different protein effectors. To experimentally determine the repertoire of extracytoproteins in E. coli O157:H7, a subproteomic analysis was performed not only considering the extracellular milieu but the cell surface and outer membrane vesicles. Following a secretome-based approach, the proteins trafficking from the interior to the exterior of the cell were depicted considering cognate protein transport systems and subcellular localisation. Label-free quantitative analysis of the proteosurfaceome, proteovesiculome and exoproteome from E. coli O157:H7 grown in three different nutrient media revealed differential protein expression profiles and allowed defining the core and variant subproteomes. Network analysis further revealed the higher abundance of some protein clusters in chemically defined medium over rich complex medium, especially related to some outer membrane proteins, ABC transport and Type III secretion systems. This first comprehensive study of the EHEC secretome unravels the profound influence of environmental conditions on the extracytoplasmic proteome, provides new insight in the physiology of E. coli O157:H7 and identifies potentially important molecular targets for the development of preventive strategies against EHEC/STEC. SignificanceEscherichia coli O157:H7 is responsible for severe diarrhoea especially in young children. Despite years of investigations, the global view of the extracytoplasmic proteins expressed in this microorganism was eluded. To provide the first comprehensive view of the secretome landscape of E. coli O157:H7, the exoproteome, proteosurfaceome and proteovesiculome were profiled using growth conditions most likely to induce changes in bacterial protein secretion. The profound influence of growth conditions on the extracytoplasmic proteome was unravelled and allowed identifying the core and variant subproteomes. Besides new insight in the physiology of enterohaemorrhagic E. coli, these proteins potentially constitute important molecular targets for the development of preventive strategies.

Dependence of the Staphylococcal Volatilome Composition on Microbial Nutrition.

In vitro cultivation of staphylococci is fundamental to both clinical and research microbiology, but few studies, to-date, have investigated how the differences in rich media can influence the volatilome of cultivated bacteria. The objective of this study was to determine the influence of rich media composition on the chemical characteristics of the volatilomes of Staphylococcus aureus and Staphylococcus epidermidis. S. aureus (ATCC 12600) and S. epidermidis (ATCC 12228) were cultured in triplicate in four rich complex media (brain heart infusion (BHI), lysogeny broth (LB), Mueller Hinton broth (MHB), and tryptic soy broth (TSB)), and the volatile metabolites produced by each culture were analyzed using headspace solid-phase microextraction combined with comprehensive two-dimensional gas chromatography—time-of-flight mass spectrometry (HS-SPME-GC×GC-TOFMS). When comparing the chemical compositions of the staph volatilomes by the presence versus absence of volatiles produced in each medium, we observed few differences. However, when the relative abundances of volatiles were included in the analyses, we observed that culturing staph in media containing free glucose (BHI and TSB) resulted in volatilomes dominated by acids and esters (67%). The low-glucose media (LB and MHB) produced ketones in greatest relative abundances, but the volatilome compositions in these two media were highly dissimilar. We conclude that the staphylococcal volatilome is strongly influenced by the nutritional composition of the growth medium, especially the availability of free glucose, which is much more evident when the relative abundances of the volatiles are analyzed, compared to the presence versus absence.

The Relationship Study of Biomass, Situation, and Artificial Control: the Degradation of NP Using Estuary-Derived Fungi

Cultural situation generally played a crucial role on biodegradation mechanism establishment of pollutant. Extensive studies had focused on the optimization of cultivation environment based on in situ conditions. However, there were still few reports on the effects of artificial control on microbial growth and degradation. In this work, the relationship of biomass, situation, and artificial control was explored through the biodegradation of nonylphenols as standard containments by four trains named A. niger, A. terreus SHPP01, A. terreus NIH2624, and T. aff. harzianum from the estuary sediment of Jiaozhou Bay. Various culture conditions covering mineral salt medium, glucose synergistic medium, and carbon rich complex medium had been used to quest the relationship. As a conclusion, different strains usually showed different mechanisms within the same media. The correlation ship between biomass and degradation and removal rate was positive, and the natural medium was usually the best choice for microbial study in situ simulation. Hence, our study provided a valuable reference for the realization of more efficient and rapid biodegradation of pollutants.

POT1-mediated δ-integration strategy for high-copy, stable expression of heterologous proteins in Saccharomyces cerevisiae.

In biotechnological industry, increased expression cassette stability and copy number serve as important means of maintaining consistently high production levels of heterologous proteins in Saccharomyces cerevisiae. In this study, we combined δ sequences for site-specific integration with TPI1 gene from Schizosaccharomyces pombe (POT1) as a selection marker to realize high-copy integration and stable expression of heterologous proteins in S. cerevisiae. With the newly developed POT1 platform, a 32-copy integration of enhanced green fluorescent protein (EGFP) expression cassette was obtained in a single round and was stably maintained after 100 generations of growth in a rich complex medium. Talaromyces emersonii cellobiohydrolase I gene was synthesized with S. cerevisiae codon bias and expressed under the control of TPI1 promoter and terminator via POT1-mediated δ-integration; the highest specific activity yielded 238 mU g-1 of dry cell weight when p-nitrophenyl-β-D-cellobioside was used as substrate, whereas the highest activity in cellulose hydrolysis reached 67% Avicel conversion. POT1-mediated δ-integration produces high protein levels over a wide dynamic range and enables broad applications in metabolic engineering and synthetic biology.

Gamma-Glutamylpolyamine Synthetase GlnA3 Is Involved in the First Step of Polyamine Degradation Pathway in Streptomyces coelicolor M145.

Streptomyces coelicolor M145 was shown to be able to grow in the presence of high concentrations of polyamines, such as putrescine, cadaverine, spermidine, or spermine, as a sole nitrogen source. However, hardly anything is known about polyamine utilization and its regulation in streptomycetes. In this study, we demonstrated that only one of the three proteins annotated as glutamine synthetase-like protein, GlnA3 (SCO6962), was involved in the catabolism of polyamines. Transcriptional analysis revealed that the expression of glnA3 was strongly induced by exogenous polyamines and repressed in the presence of ammonium. The ΔglnA3 mutant was shown to be unable to grow on defined Evans agar supplemented with putrescine, cadaverine, spermidine, and spermine as sole nitrogen source. HPLC analysis demonstrated that the ΔglnA3 mutant accumulated polyamines intracellularly, but was unable to degrade them. In a rich complex medium supplemented with a mixture of the four different polyamines, the ΔglnA3 mutant grew poorly showing abnormal mycelium morphology and decreased life span in comparison to the parental strain. These observations indicated that the accumulation of polyamines was toxic for the cell. An in silico analysis of the GlnA3 protein model suggested that it might act as a gamma-glutamylpolyamine synthetase catalyzing the first step of polyamine degradation. GlnA3-catalyzed glutamylation of putrescine was confirmed in an enzymatic in vitro assay and the GlnA3 reaction product, gamma-glutamylputrescine, was detected by HPLC/ESI-MS. In this work, the first step of polyamine utilization in S. coelicolor has been elucidated and the putative polyamine utilization pathway has been deduced based on the sequence similarity and transcriptional analysis of homologous genes expressed in the presence of polyamines.

Enhancing oil accumulation of a mixed culture of Chlorella sp. and Saccharomyces cerevisiae using fish waste hydrolysate

Mixed culture of Chlorella sp. and Saccharomyces cerevisiae using fish waste hydrolysate has been proposed as a novel strategy for enhancing oil formation in this study. Fish waste hydrolysate (FWH) concentration ranging from 1.1 to 8.8g/l of the mixed culture was investigated, the optimal biomass concentration reached 3626mg/l at 4.4g/l FWH, however, the maximum oil concentration was obtained as 1219mg/l at 8.8g/l FWH. Results indicated biomass and oil content of the mixed culture in mixotrophic cultivation with FWH medium was better than those in heterotrophic cultivation and mixotrophic cultivation with a rich complex medium. Organic acid supplementation to the mixed culture was performed to explain the mechanism of oil accumulation in the mixed culture. Butyric acid significantly enhanced biomass formation by 20% and oil accumulation by 143% under mixotrophic cultivation. The calculated cetane numbers of oils based on the fatty acid analysis from mixed culture and Chlorella sp. monoculture were 61.79 and 61.46, respectively, and they all complied with the European standard for biodiesel. FWH medium is a potential alternative for economic biodiesel production.

Systematic production of inactivating and non-inactivating suppressor mutations at the relA locus that compensate the detrimental effects of complete spot loss and affect glycogen content in Escherichia coli.

In Escherichia coli, ppGpp is a major determinant of growth and glycogen accumulation. Levels of this signaling nucleotide are controlled by the balanced activities of the ppGpp RelA synthetase and the dual-function hydrolase/synthetase SpoT. Here we report the construction of spoT null (ΔspoT) mutants obtained by transducing a ΔspoT allele from ΔrelAΔspoT double mutants into relA+ cells. Iodine staining of randomly selected transductants cultured on a rich complex medium revealed differences in glycogen content among them. Sequence and biochemical analyses of 8 ΔspoT clones displaying glycogen-deficient phenotypes revealed different inactivating mutations in relA and no detectable ppGpp when cells were cultured on a rich complex medium. Remarkably, although the co-existence of ΔspoT with relA proficient alleles has generally been considered synthetically lethal, we found that 11 ΔspoT clones displaying high glycogen phenotypes possessed relA mutant alleles with non-inactivating mutations that encoded stable RelA proteins and ppGpp contents reaching 45–85% of those of wild type cells. None of the ΔspoT clones, however, could grow on M9-glucose minimal medium. Both Sanger sequencing of specific genes and high-throughput genome sequencing of the ΔspoT clones revealed that suppressor mutations were restricted to the relA locus. The overall results (a) defined in around 4 nmoles ppGpp/g dry weight the threshold cellular levels that suffice to trigger net glycogen accumulation, (b) showed that mutations in relA, but not necessarily inactivating mutations, can be selected to compensate total SpoT function(s) loss, and (c) provided useful tools for studies of the in vivo regulation of E. coli RelA ppGpp synthetase.

Engineering a repression-free catabolite-enhanced expression system for a thermophilic alpha-amylase from Bacillus licheniformis MSG

Most expression systems used for the over production of many enzymes employ carbon catabolite repressible promoters and hence must use sugar free, rich complex media. Use of expression systems to overcome carbon repression opens an avenue for exploiting cheap carbon sources for the production of recombinant enzymes. A self-inducible, catabolite repression free and above all a glucose-activated expression system has been developed using an industrially suitable thermophilic alpha-amylase as a model. The alpha-amylase gene of Bacillus licheniformis MSG without the 5' cre operator produced unimpeded glucose-enhanced expression when fused to the phosphate starvation-inducible strong pst promoter with optimum translation signals in a protease deficient Bacillus subtilis. A combination of high glucose with limited phosphate permitted sufficient biomass and fast transition to quiescent phase by phosphate starvation permitting 1250-fold induction for 70h. A ~300-fold high productivity (9070UmL-1) and 131-fold increase in specific expression in 2% glucose and a 100-fold high yield in 4% molasses were obtained compared to the production by the parent strain. The yield was 18.5-fold higher than that from the native promoter in an isogenic clone. This strategy of catabolite enhanced enzyme expression uncoupled from biomass formation can be applied for cost effective high production of proteins using starch or molasses.

Aqueous extracts of a Mars analogue regolith that mimics the Phoenix landing site do not inhibit spore germination or growth of model spacecraft contaminants Bacillus subtilis 168 and Bacillus pumilus SAFR-032

Because Mars is a primary target for life detection and habitability assessment missions, its exploration is also by necessity a Planetary Protection issue. The recent finding of significant levels of perchlorate (ClO4-) in regolith sampled from the Phoenix landing site raises the question of its potential biotoxicity to putative indigenous martian life, microbial forward contaminants from Earth, or future human visitors. To address this issue, an analogue regolith was constructed based on regolith chemistry data from the Phoenix landing site. A Mars Aqueous Regolith Extract (MARE) was prepared from the Phoenix analogue regolith and analyzed by ion chromatography. The MARE contained (mg/L) the cations Na+ (1411±181), Mg2+ (1051±160), Ca2+ (832±125), and K+ (261±29), and the anions SO42-(5911±993), ClO4-(5316±1767), Cl-(171±25) and F− (2.0±0.4). Nitrogen-containing species NO3-(773±113) and NO2-(6.9±2.3) were also present as a result of regolith preparation procedures, but their relevance to Mars is at present unknown. The MARE was tested for potential toxic effects on two model spacecraft contaminants, the spore-forming bacteria Bacillus subtilis strain 168 and Bacillus pumilus strain SAFR-032. In B. subtilis, spore germination and initial vegetative growth (up to ∼5h) was not inhibited in a rich complex medium prepared with the MARE, but growth after 5h was significantly suppressed in medium prepared using the MARE. Both B. subtilis and B. pumilus exhibited significantly higher rates of spore germination and growth in the MARE vs. DW with no additions (likely due to endogenous spore nutrients), but germination and growth was further stimulated by addition of glucose and a combination of buffered inorganic salts (K2HPO4, KH2PO4, (NH4)2SO4, and MgSO4). The data indicate that the aqueous environment in the regolith from the Phoenix landing site containing high levels of perchlorate does not pose a significant barrier to growth of putative forward contaminants such as B. subtilis and B. pumilus under Earth laboratory conditions.

Biotransformation of Naringenin to Eriodictyol by Saccharomyces cerevisiea Functionally Expressing Flavonoid 3′ Hydroxylase

To increase the biological activities of flavonoids and to enhance their stability and solubility by functionalization reactions (polymerization, esterification, alkylation, glycosylation and acylation), an increase in the number of hydroxyl groups in these molecules is needed. Hydroxylation reactions may be achieved using either chemical or enzymatic methods, the latter being more highly specific than the former. In our study, the flavonoid 3' hydroxylase (F3'H) from Gerbera hybrid, functionally expressed in Saccharomyces cerevisiae, was used to hydroxylate naringenin (the first flavonoid core synthesized in plants). Furthermore, we studied factors that may affect naringenin hydroxylation by recombinant cell-like yeast growth on selective or rich media and plasmid stability. The whole recombinant cells hydroxylated naringenin at position 3' to give eriodictyol. In a selective media, the yeast failed to grow to high cell densities (maximum 5 g/L), but the plasmid stability was nearly 90%, and naringenin hydroxylation reached 100%. In a rich complex media, the biomass reached 10 g/L, but the yield of naringenin hydroxylation reached only 71%, and the plasmid stability decreased. When yeast functionally expressing F3'H from Gerbera hybrid was used, in a selective media, 200 mg/L of eriodictyol from naringenin was produced.

Expression of human interleukin-1β in Saccharomyces cerevisiae using PIR4 as fusion partner and production in aerated fed-batch reactor

To circumvent cell wall retention commonly associated to Saccharomyces cerevisiae when used as a host for heterologous protein production, we have created a translational fusion of human interleukin-1β (IL-1β) to the Pir4 cell wall protein, so as to drive the secretion of the recombinant product to the growth medium. The auxotrophic S. cerevisiae BY4741 was used as host to express the Pir4-IL1β fusion protein. Once it was ascertained that the fusion protein was secreted to the culture medium and behaved as a growth-linked product, S. cerevisiae BY4741 [PIR4-IL1β] was cultured in an aerated fed-batch reactor to achieve high cell density and, consequently, high product concentration in the medium. Two cultivation media were employed, a rich complex and a defined mineral medium, the latter suitably supplemented with bacto-casamino acids as ACA (auxotrophy-complementing amino acid) source. The rich complex medium allowed a good performance of the producer strain only during batch growth, but was revealed to be inadequate for long-term fed-batch operations. The defined mineral medium ensured a better performance, even though not yet satisfactory in spite of a proper ACA supplementation. The behaviour of BY4741 was attributed to an intrinsic sensitivity of the producer strain to long-term aerated fed-batch operations.

Phosphatidylethanolamine Synthesis Is Required for Optimal Virulence of Brucella abortus

The Brucella cell envelope contains the zwitterionic phospholipids phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Synthesis of PC occurs exclusively via the PC synthase pathway, implying that the pathogen depends on the choline synthesized by the host cell to form PC. Notably, PC is necessary to sustain a chronic infection process, which suggests that the membrane lipid content is relevant for Brucella virulence. In this study we investigated the first step of PE biosynthesis in B. abortus, which is catalyzed by phosphatidylserine synthase (PssA). Disruption of pssA abrogated the synthesis of PE without affecting the growth in rich complex medium. In minimal medium, however, the mutant required choline supplementation for growth, suggesting that at least PE or PC is necessary for Brucella viability. The absence of PE altered cell surface properties, but most importantly, it impaired several virulence traits of B. abortus, such as intracellular survival in both macrophages and HeLa cells, the maturation of the replicative Brucella-containing vacuole, and mouse colonization. These results suggest that membrane phospholipid composition is critical for the interaction of B. abortus with the host cell.

Regulatory role of RsgI in sigI expression in Bacillus subtilis

The sigma gene, sigI, of Bacillus subtilis belongs to the group IV heat-shock response genes and has many orthologues in the bacterial phylum Firmicutes. The B. subtilis sigI gene is considered to constitute an operon with rsgI (regulation of sigI, formerly ykrI). As little is known about either the structure and function of the sigI-rsgI operon or the SigI regulons, the role of RsgI in heat-inducible transcription of the sigI-rsgI operon was investigated, using Northern analysis and a heat-stable beta-galactosidase reporter assay. Heat-inducible, SigI-dependent transcription of the sigI-rsgI operon was stimulated greatly by disrupting rsgI. Yeast two-hybrid analysis showed direct interaction between the N-terminal portion of the presumed RsgI protein and SigI. Without RsgI function, induction of transcription of the sigI-rsgI operon upon transient heat stress depended on dnaK activity. However, transcription of the operon was induced during growth at prolonged higher temperature even without DnaK function. Without RsgI function, sigI-rsgI operon transcription was induced after the end of growth independent of any temperature shift in a sporulation medium and toward the end of growth in a rich complex medium. Furthermore, glucose addition resulted in a strong suppression of sigI-rsgI transcription. Therefore it is hypothesized that transcription of the sigI-rsgI operon of B. subtilis is negatively regulated by the putative transmembrane protein RsgI, which moderates SigI's sensitivity to heat shock or nutritional stress.

New insights into the cell cycle profile of Paracoccidioides brasiliensis

The present work focuses on the analysis of cell cycle progression of Paracoccidioides brasiliensis yeast cells under different environmental conditions. We optimized a flow cytometric technique for cell cycle profile analysis based on high resolution measurements of nuclear DNA. Exponentially growing cells in poor-defined or rich-complex nutritional environments showed an increased percentage of daughter cells in accordance with the fungus’ multiple budding and high growth rate. During the stationary growth-phase cell cycle progression in rich-complex medium was characterized by an accumulation of cells with higher DNA content or pseudohyphae-like structures, whereas in poor-defined medium arrested cells mainly displayed two DNA contents. Furthermore, the fungicide benomyl induced an arrest of the cell cycle with accumulation of cells presenting high and varying DNA contents, consistent with this fungus’ unique pattern of cellular division. Altogether, our findings seem to indicate that P. brasiliensis may possess alternative control mechanisms during cell growth to manage multiple budding and its multinucleate nature.

Geobacillus toebii subsp. decanicus subsp. nov., a hydrocarbon-degrading, heavy metal resistant bacterium from hot compost

A thermophilic, spore-forming bacterial strain L1(T) was isolated from hot compost "Pomigliano Environment" s.p.a., Pomigliano, Naples, Italy. The strain was identified by using a polyphasic taxonomic approach. L1(T) resulted in an aerobic, gram-positive, rod-shaped, thermophilic with an optimum growth temperature of 68 degrees C chemorganotrophic bacterium which grew on hydrocarbons as unique carbon and energy sources and was resistant to heavy metals. The G+C DNA content was 43.5 mol%. Phylogenetic analysis of 16S rRNA gene sequence and Random Amplified Polymorphic DNA-PCR (RAPD-PCR) analysis of L1(T) and related strains showed that it forms within Geobacillus toebii, a separate cluster in the Geobacillus genus. The composition of cellular fatty acids analyses by Gas-Mass Spectroscopy differed from that typical for the genus Geobacillus in that it is lacking in iso-C15 fatty acid, while iso-C16 and iso-C17 were predominant. Isolates grew on a rich complex medium at temperatures between 55-75 degrees C and presented a doubling time (t(d)) of 2 h and 6 h using complex media and hydrocarbon media, respectively. Among hydrocarbons tested, n-decane (2%) was the more effective to support the growth (1 g/L of wet cells). The microorganism showed resistance to heavy metal tested during the growth. Furthermore, intracellular alpha-galactosidase and alpha-glucosidase enzymatic activities were detectable in the L1(T) strain. Based on phenotypic, phylogenetic, fatty acid analysis and results from DNA-DNA hybridization, we propose assigning a novel subspecies of Geobacillus toebii, to be named Geobacillus toebii subsp. decanicus subsp. nov., with the type strain L1(T) (=DSM 17041=ATCC BAA 1004).

Liquid-culture production of blastospores of the bioinsecticidal fungus Paecilomyces fumosoroseus using portable fermentation equipment.

The production of fungal spores using on-site, non-sterile, portable fermentation equipment is technically constrained. Very little information is available on the production requirements, such as medium concentration, inoculum stabilization, required fermentation times, and maintenance of axenic growth. In this study, we developed a two-part, liquid concentrate of the production medium that remains stable and soluble at room temperature. We also examined inoculum stability and showed that freeze- or air-dried blastospore preparations were stable for 7 days after rehydration when stored at 4 degrees C. The use of a low-pH (pH 4), relatively rich complex medium provided a growth environment deleterious to bacterial growth yet conducive to rapid sporulation by Paecilomyces fumosoroseus. High concentrations of blastospores (7.9 x 10(8)/ml) of P. fumosoroseus were produced in a 40-h fermentation with very low levels of bacterial contamination when the fermentor was charged with a blastospore production medium with a starting pH of 4 and inoculated with blastospore concentrations greater than 1 x 10(6) spores/ml. These studies demonstrate that the use of disinfected, portable fermentation equipment has potential for on-site production of high concentrations of blastospores of the bioinsecticidal fungus P. fumosoroseus.

Control of glycolytic flux inZymomonas mobilis by glucose 6-phosphate dehydrogenase activity

Glycolytic genes in Zymomonas mobilis are highly expressed and constitute half of the cytoplasmic protein. The first four genes (glf, zwf, edd, glk) in this pathway form an operon encoding a glucose permease, glucose 6-phosphate dehydrogenase (G6-P dehydrogenase), 6-phosphogluconate dehydratase, and glucokinase, respectively. Each gene was overexpressed from a tac promoter to investigate the control of glycolysis during the early stages of batch fermentation when flux (qCO(2)) is highest. Almost half of flux control appears to reside with G6-P dehydrogenase (C(J) (G6-P dehydrogenase) = 0.4). Although Z. mobilis exhibits one of the highest rates of glycolysis known, recombinants with elevated G6-P dehydrogenase had a 10% to 13% higher glycolytic flux than the native organism. A small increase in flux was also observed for recombinants expressing glf. Results obtained did not allow a critical evaluation of glucokinase and this enzyme may also represent an important control point. 6-Phosphogluconate dehydratase appears to be saturating at native levels. With constructs containing the full operon, growth rate and flux were both reduced, complicating interpretations. However, results obtained were also consistent with G6-P dehydrogenase as a primary site of control. Flux was 17% higher in operon constructs which exhibited a 17% increase in G6-P dehydrogenase specific activity, relative to the average of other operon constructs which contain a frameshift mutation in zwf. It is unlikely that all flux control residues solely in G6-P dehydrogenase (calculated C(J) (G6-P dehydrogenase) = 1.0) although these results further support the importance of this enzyme. As reported in previous studies, changes in flux were not accompanied by changes in growth rate providing further evidence that ATP production does not limit biosynthesis in rich complex medium.