Effect of aeration and agitation on the production of mycelial biomass and exopolysaccharides in an enthomopathogenic fungus Paecilomyces sinclairii

AIMS: The objective of the present study was to investigate the influence of aeration rate and agitation intensity on the production of mycelial biomass and exopolysaccharide (EPS) in Paecilomyces sinclairii. METHODS AND RESULTS: The P. sinclairii was cultivated under various aeration and agitation conditions in a 5 l stirred-tank bioreactor. The highest mycelial biomass (30.5 g l-1) and EPS production (11.5 g l-1) were obtained at a high aeration rate (3.5 v.v.m.) and at a high agitation speed (250 rev min-1). The apparent viscosities (6000-8000 cP) of fermentation broth increased rapidly towards the end of fermentations at high aeration and agitation conditions. CONCLUSIONS: The high level of dissolved oxygen achieved at a high aeration rate (3.5 v.v.m.) associated with higher hyphal density eventually resulted in enhanced EPS production. Agitation intensity was also proved to be a critical factor influencing on both the mycelial biomass and EPS production: high agitation speeds up to 250 rev min-1 were preferred to the yields of biomass and EPS production. SIGNIFICANCE AND IMPACT OF THE STUDY: The critical effects of aeration and agitation in the culture process of P. sinclairii were found, which is widely applicable to other kinds of basidiomycetes or ascomycetes in their submerged culture processes.     

Violacein and biofilm production in Janthinobacterium lividum

AIMS: To analyse the environmental stimuli modulating violacein and biofilm production in Janthinobacterium lividum. METHODS AND RESULTS: Violacein and biofilm production by J. lividum DSM1522(T) was assayed in different growth conditions. Our data suggest that violacein and biofilm production is controlled by the carbon source, being inhibited by glucose and enhanced by glycerol. J. lividum produced violacein also in the presence of different sub-inhibitory concentrations of ampicillin. As opposite, the production of N-acylhomoserine lactone(s), quorum sensing regulators was shown to be positively regulated by glucose. Moreover, violacein-producing cultures of J. lividum showed higher CFU counts than violacein-nonproducing ones. CONCLUSIONS: Taken together, our results suggest that violacein and biofilm production could be regulated by a common metabolic pathway and that violacein as well as biofilm could represent a response to environmental stresses and a key factor in the survival mechanisms of J. lividum. SIGNIFICANCE AND IMPACT OF THE STUDY: Although several recent studies disclosed a number of interesting biological properties of violacein, few data are reported on the physiologic function of violacein in J. lividum. This paper adds new information on the complex mechanisms allowing and regulating bacterial life in hostile environments.     

Beijerinckia derxii releases plant growth regulators and amino acids in synthetic media independent of nitrogenase activity

AIMS: This study aims at evaluating the ability of Beijerinckia derxii, a free-living nitrogen (N)-fixing bacterium frequently isolated from tropical soils, to release certain plant growth regulators [indoleacetic acid (IAA), ethylene, polyamines] and amino acids into the growth medium. METHODS AND RESULTS: The production of those substances was compared using both cultures in which nitrogenase was active (N-free medium) and cultures in which nitrogenase was repressed (combined-N cultures). Those cultures were grown under agitation and in absence of agitation. Total IAA production was higher in agitated, N-free cultures but specific production was greater in combined-N cultures under agitation. Putrescine and spermidine were detected under all conditions tested. Ethylene was produced in both N-free and combined-N cultures. A greatest diversity of amino acids was released in N-free cultures. CONCLUSIONS: There was no inhibition of the production of the analysed substances under conditions where nitrogenase was inactive. SIGNIFICANCE AND IMPACT OF THE STUDY: Beijerinckia derxii is potentially a producer of plant-active substances; its presence in the natural environment suggests that this bacterium may contribute to the development of other living organisms.     

Effect of agitation and aeration on the production of nitrile hydratase by Rhodococcus erythropolis MTCC 1526 in a stirred tank reactor

Aims: To evaluate the effect of different physicochemical parameters such as agitation, aeration and pH on the growth and nitrile hydratase production by Rhodococcus erythropolis MTCC 1526 in a stirred tank reactor. Methods and Results: Rhodococcus erythropolis MTCC 1526 was grown in 7‐l reactor at different agitation, aeration and controlled pH. The optimum conditions for batch cultivation in the reactor were an agitation rate of 200 rev min^?1, aeration 0·5 v/v/m at controlled pH 8. In this condition, the increase in nitrile hydratase activity was almost threefold compared to that in the shake flask. Conclusion: Agitation and aeration rate affected the dissolved‐oxygen concentration in the reactor which in turn affected the growth and enzyme production. Significance and Impact of the Study: Cultivation of R. erythropolis MTCC 1526 in the reactor was found to have significant effect on the growth and nitrile hydratase production when compared to the shake flask.     

Occurrence of β-Aminoglutaric Acid in Marine Bacteria

β-Aminoglutaric acid, a nonprotein amino acid isomer of glutamic acid, was found in the free amino acid pool of a marine bacterium, Alteromonas luteoviolacea. It was also found in a mixed culture of fermenting bacteria enriched from an anoxic marine sediment.     

Effect of Agitation on Ligninase Activity and Ligninase Production by Phanerochaete chrysosporium

The white rot fungus Phanerochaete chrysosporium produces extracellular ligninases as part of its idiophasic ligninolytic system. Agitation has been widely reported to suppress both ligninase production and lignin degradation. Results show that mechanical inactivation of ligninase is possibly the reason why ligninase accumulation is low or absent in agitated shake-flask cultures. Agitation seems to affect the catalytic activity of ligninase and has no apparent effect on either the rate of ligninase production or the physiology of P. chrysosporium. The detergents Tween 20, Tween 40, Tween 60, Tween 80, and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) are able to protect both purified ligninase and extant ligninase in culture fluids (free of biomass) against mechanical inactivation due to agitation. Addition of Tween 80 at the end of primary growth to agitated shake flasks containing either pelleted or immobilized mycelial cultures results in production and maintenance of high levels of ligninase activity over several days under conditions of high agitation. Possible mechanisms by which the detergents could protect ligninase are discussed.     

Occurrence of beta-Aminoglutaric Acid in Marine Bacteria

beta-Aminoglutaric acid, a nonprotein amino acid isomer of glutamic acid, was found in the free amino acid pool of a marine bacterium, Alteromonas luteoviolacea. It was also found in a mixed culture of fermenting bacteria enriched from an anoxic marine sediment.     

Inhibition of bacterial quorum sensing by vanilla extract

AIMS: The purpose of this study was to search for a novel quorum sensing inhibitor and analyse its inhibitory activity. METHODS AND RESULTS: Quorum sensing inhibition was monitored using the Tn-5 mutant, Chromobacterium violaceum CV026. Vanilla beans (Vanilla planifolia Andrews) were extracted using 75% (v/v) aqueous methanol and added to C. violaceum CV026 cultures. Inhibitory activity was measured by quantifying violacein production using a spectrophotometer. The results have revealed that vanilla extract significantly reduced violacein production in a concentration-dependent manner, indicating inhibition of quorum sensing. CONCLUSIONS: Vanilla, a widely used spice and flavour, can inhibit bacterial quorum sensing. SIGNIFICANCE AND IMPACT OF THE STUDY: The results suggest that the intake of vanilla-containing food materials might promote human health by inhibiting quorum sensing and preventing bacterial pathogenesis. Further studies are required to isolate specific substances from vanilla extract acting as quorum sensing inhibitors.     

Correlation between bacterial haemoglobin gene (vgb) and aeration: their effect on the growth and alpha-amylase activity in transformed Enterobacter aerogenes

AIMS: To evaluate the effects of bacterial haemoglobin on bacterial growth and alpha-amylase formation under different aeration conditions. METHODS AND RESULTS: Enterobacter aerogenes was transformed with the gene encoding Vitreoscilla (bacterial) haemoglobin, vgb. The growth kinetics and ability to synthesize alpha-amylase enzyme were investigated in this transformed Enterobacter strain as well as in two other Enterobacter control strains that do not harbour the vgb gene. Such comparison was made under variable aeration conditions, using the agitation rate as a measure of aeration. The expression of bacterial haemoglobin-supported cell growth determined as O.D.600 and cell viability in addition to the alpha-amylase production. These positive effects of bacterial haemoglobin were observed under both low and high aerations, but at different extents. CONCLUSIONS: In addition to improving cell growth under low aeration, the bacterial haemoglobin is able to promote bacterial cell tolerance during exposure to high oxygen tension. SIGNIFICANCE AND IMPACT OF THE STUDY: The expression of bacterial haemoglobin is advantageous in reducing the burden of certain toxic conditions such as high oxygen levels. It may have the same impact on some environmental toxic substances. This, haemoglobin biotechnology can be extended to induce enzymes of pollutants degradation or production of some useful industrial substances.     

Genetic analysis of violacein biosynthesis by Chromobacterium violaceum

Chromobacterium violaceum presents a distinctive phenotypic characteristic, the production of a deep violet pigment named violacein. Although the physiological function of this pigment is not well understood, the sequencing of the genome of this bacterium has given some insight into the mechanisms and control of violacein production. It was found that erythrose-4-phosphate (E4P), a precursor to aromatic amino acid biosynthesis, is produced by the non-oxidative portion of the hexose monophosphate pathway, since it lacks 6-phosphogluconate dehydrogenase. All genes leading from E4P plus phosphoenolpyruvate to tryptophan are present in the genome. Nevertheless, these genes are not organized in an operon, as in E. coli, indicating that other mechanisms are involved in expression. The sequencing data also indicated the presence and organization of an operon for violacein biosynthesis. Three of the four gene products of this operon presented similarity with nucleotide-dependent monooxygenases and one with a limiting enzyme polyketide synthase. As previously suggested, genes encoding proteins involved in quorum sensing control by N-hexanoyl-homoserine-lactone, an autoinducer signal molecule, are present in the bacterial genome. These data should help guide strategies to increase violacein biosynthesis, a potentially useful molecule.     

Antinematode Activity of Violacein and the Role of the Insulin/IGF-1 Pathway in Controlling Violacein Sensitivity in Caenorhabditis elegans

The purple pigment violacein is well known for its numerous biological activities including antibacterial, antiviral, antiprotozoan, and antitumor effects. In the current study we identify violacein as the antinematode agent produced by the marine bacterium Microbulbifer sp. D250, thereby extending the target range of this small molecule. Heterologous expression of the violacein biosynthetic pathway in E. coli and experiments using pure violacein demonstrated that this secondary metabolite facilitates bacterial accumulation in the nematode intestine, which is accompanied by tissue damage and apoptosis. Nematodes such as Caenorhabditis elegans utilise a well-defined innate immune system to defend against pathogens. Using C. elegans as a model we demonstrate the DAF-2/DAF-16 insulin/IGF-1 signalling (IIS) component of the innate immune pathway modulates sensitivity to violacein-mediated killing. Further analysis shows that resistance to violacein can occur due to a loss of DAF-2 function and/or an increased function of DAF-16 controlled genes involved in antimicrobial production (spp-1) and detoxification (sod-3). These data suggest that violacein is a novel candidate antinematode agent and that the IIS pathway is also involved in the defence against metabolites from non-pathogenic bacteria.     

Preliminary characterization of exopolysaccharides produced by a marine biofilm-forming bacterium Pseudoalteromonas ruthenica (SBT 033)

AIM: The major objective of the present study was the partial characterization of the exopolysaccharides (EPS) produced by a marine biofilm-forming bacterium Pseudoalteromonas ruthenica under shake culture conditions. METHODS AND RESULTS: EPS-producing bacterial cultures were isolated from the sea water collected from the vicinity of coastal electric power station. Zobell marine broth medium was used for growth of the cultures and the EPS produced was quantified using phenol sulfuric acid method. Chemical characterization of the EPS was carried out using Fourier transform infrared spectroscopy (FTIR), and capillary gas chromatography (GC). Further, viscosity and rheological properties of the purified EPS were studied. The FTIR spectrum revealed prominent peaks of various groups of OH and CH(3) bending. GC analysis showed the presence of eight individual sugars. Rheological studies of the aqueous EPS showed good shearing property. CONCLUSIONS: Pseudoalteromonas ruthenica isolated from marine environment produced copious amount of EPS under shake culture conditions. GC analysis of the EPS revealed the presence of eight individual sugars and the EPS had good shearing property. SIGNIFICANCE AND IMPACT OF THE STUDY: The EPS produced by P. ruthenica is pseudoplastic in nature and is stable at higher pH levels. These properties suggest that the EPS may have potential applications in the oil, textiles and food industries.     

Production of <Emphasis Type="SmallCaps">l</Emphasis>-asparaginase in<Emphasis Type="Italic"> Enterobacter aerogenes</Emphasis> expressing<Emphasis Type="Italic"> Vitreoscilla</Emphasis> hemoglobin for efficient oxygen uptake

This study is the first utilizing Vitreoscilla hemoglobin in a heterologous bacterium, Enterobacter aerogenes, to determine the effect of such a highly efficient oxygen-uptake system on the production of l-asparaginase, an enzyme that has attracted considerable attention due to its anti-tumor activity. Here, we show that the Vitreoscilla hemoglobin expressing strain has from 10-fold to more than two orders of magnitude lower l-asparaginase activity than the wild type or the control without the Vitreoscilla hemoglobin gene under different aeration conditions. Aeration and agitation were also determining factors for enzyme production. The enzyme activity was reduced considerably under both full aerobic and anaerobic conditions, while the highest enzyme activity was determined in cultures under low aeration and low agitation. Also, the effect of different concentrations of glucose on enzyme production showed catabolic repression. Glucose at 1% caused almost total inhibition of enzyme activity, while at 0.1% it showed a slightly stimulatory effect on enzyme production, compared with glucose-free medium.     

Quorum sensing signaling molecules involved in the production of violacein by Pseudoalteromonas

The production of violacein by Pseudoalteromonas sp. 520P1 has many features of quorum sensing. Signaling molecules were extracted from bacterial culture and subsequently identified as N-(3-oxooctanoyl)-homoserine lactone and N-tetradecanoyl-homoserine lactone. The former but not the latter induced the production of violacein in strain 520P1. We conclude that N-(3-oxooctanoyl)-homoserine lactone is a signaling molecule involved in the production of violacein.     

Effects of agitation on enzymatic saccharification of cellulose

Summary Crystalline cellulose Avicel has been hydrolyzed byTrichoderma viride cellulase (Meicelase CEPB) under vaned agitation conditions and the effect of agitation on the adsorption of cellulase on cellulose has been studied. Agitation was found to enhance the hydrolysis pf crystalline cellulose; possibly the agitation enhances the adsorption of exoglucanase to shift the adsorption balance of exoglucanase and endoglucanase to a direction favorable for their synergistic action on the surface of cellulose.     

Statistical optimization of fermentation conditions and comparison of their influences on production of cellulases by a psychrophilic marine bacterium, <Emphasis Type="Italic">Psychrobacter aquimaris</Emphasis> LBH-10 using orthogonal array method

The optimal conditions for the production of cellulases by a marine bacterium, Psychrobacter aquimaris LBH-10, were established and their effects were compared using orthogonal array experiments based on the Taguchi method. The optimal conditions of rice bran, peptone and initial pH for the production of avicelase and CMCase by P. aquimaris LBH-10 were 50.0, 3.0, and 8.0 g/L, respectively, whereas those for filter paperase (FPase) were 100.0, 3.0, and 8.0 g/L, respectively. Rice bran was found to be the most important factor for the production of cellulases based on the calculated percentage of participation P (%) from an analysis of the variance (ANOVA). The optimal temperature for the cell growth of P. aquimaris LBH-10 was 25°C, whereas that for the production of avicelase, CMCase and FPase was 30°C. The optimal agitation speed and aeration rate for cell growth was 400 rpm and 1.5 vvm, respectively, whereas those for the production of CMCase were 300 rpm and 1.0 vvm, respectively. Aeration was found to be more important for cell growth and CMCase production than agitation. The maximum production of avicelase, CMCase and FPase in a 100 L bioreactor for 72 h under optimized conditions was 83.2, 388.7, and 75.4 U/mL, respectively.     

Agitation and pressure effects on acetone-butanol fermentation

Batch fermentations were run at varying agitation rates and were either pressurized to 1 bar (15.2 psig) or nonpressurized. Agitation and pressure both affect the level of dissolved hydrogen gas in the media, which in turn influences solvent production. In nonpressurized fermentations volumetric productivity of butanol increased as the agitation rate decreased. While agitation had no significant effect on butanol productivity under pressurized conditions, overall butanol productivity was increased over that obtained in the nonpressurized runs. Maximum butyric acid productivity, however, was found to occur earlier and increased as agitation increased. Peak hydrogen productivity occurred simultaneously with peak butyric acid productivity. The proporation of reducing equivalents used in forming the above products was determined using a redox balance based on the fermentation stoichiometry. An inverse relationship between the final concentrations of acetone and acetoin was found in all fermentations studied. The results show that agitation and pressure are important parameters for solvent productivity in acetone-butanol fermentation.     

Impact of cultivation conditions on haemolytic activity of Pseudoalteromonas issachenkonii KMM 3549T

AIMS: The present work aimed to study the effects of cultivation conditions on the haemolytic activities of Pseudoalteromonas issachenkonii. METHODS AND RESULTS: The kinetics of growth and haemolytic activities was investigated on sea-salts and NaCl-based nutrient media supplemented with either starch, or KBr over a period of 140 h. The first haemolytic activity occurred when bacterial cells reached the late stationary phase. The second haemolytic activity was observed in marine broth (MB) after 110 h of incubation. Addition of Fe to the culture medium neither affected bacterial growth nor reduced the haemolytic activity. However, the activity was enhanced in the presence of iron chelator. The second haemolytic activity was not affected by Ca2+, or inhibited by chymotrypsin or EDTA. CONCLUSIONS: The production of haemolysins by P. issachenkonii was greater on MB and was dependent on both the medium composition and time of incubation. The second haemolytic activity was heat stable, nonproteinaceous, calcium-independent and was regulated by Fe. SIGNIFICANCE AND IMPACT OF THE STUDY: The results demonstrated the importance of optimization of both the media composition and monitoring the haemolytic activity over a prolonged cultivation time to detect different types of haemolysins.     

Utilization of chicory roots for microbial endoinulinase production

AIMS: The optimal culture conditions for endoinulinase production using chicory roots were studied in shake-flask culture. METHODS AND RESULTS: Much higher enzyme production was achieved with Xanthomonas sp. (15 U ml(-1)) than with Pseudomonas sp. (3 U ml(-1)). Optimized culture conditions of Xanthomonas sp. for endoinulinase production in flask culture were: chicory powder, 5 g l(-1); temperature, 37 degrees C; pH, 7.0; agitation speed, 100 rev min(-1). CONCLUSION: Maximum bacterial growth and enzyme production were 6.2 g l(-1) and 20 U ml(-1) under optimal conditions, respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: Chicory roots could be used as a fermentation substrate for the production of enndoinulinase.     

Competition-Mediated Antibiotic Induction in the Marine Bacterium Streptomyces tenjimariensis

Microbial competition for limiting natural resources within a community is thought to be the selective force that promotes biosynthesis of antimicrobial compounds The marine bacterium Streptomyces tenjimariensis produces the antibiotics istamycin A and B under select laboratory culture conditions; presumably these compounds serve an, ecological role under natural conditions. Here we report results of a novel marine microbial competion experiment that examined the impact of co-culture of marine bacteria on istamycin production by S. tenjimariensis. Twelve of the 53 bacterial species tested (i.e., 22.6%) induced Istamycin production; this antibiotic also inhibited growth of the competitor colonies. These results suggest that marine bacterial metabolites, serve an ecological role in countering competitive species.