Fed-batch culture of Candida guilliermondii FTI 20037 for xylitol production from sugar cane bagasse hydrolysate

A fed-batch culture system was used to study xylitol production by Candida guilliermondii FTI 20037 in a synthetic and a sugar cane bagasse hydrolysate medium. The values achieved for xylitol yield and volumetric productivity were, respectively, 0 · 84 g g⁻¹ and 0 · 64 g l⁻¹ h⁻¹ using the synthetic medium and 0 · 78 g g⁻¹ and 0 · 62 g l⁻¹ h⁻¹ using the hydrolysate medium.     

Itaconic acid production by Aspergillus terreus on raw starchy materials

Starchy materials such as corn starch, soft wheat flour, potato flour, cassava flour, sorghum starch, sweet potato and industrial potato flours, either acid or enzymatically hydrolysed, were used as substrates for itaconic acid production by Aspergillus terreus NRRL 1960. Both production and yield were highest on corn starch (18·4 g l⁻¹ and 34·0%, respectively). The degree of hydrolysis had a great influence on acid production which was highest when corn starch was saccharified at 85 DE (dextrose equivalent). In a 3 litre benchtop fermenter, itaconic acid production and productivity were 19·8 g l⁻¹ and 0·13 g l⁻¹ h⁻¹, respectively.     

Effects of feeding and induction strategy on the production of BmR1 antigen in recombinant E. coli

Aim:  To investigate the effects of feeding and induction strategies on the production of Bm R1 recombinant antigen.
Methods and Results:  Fed-batch fermentation was studied with respect to the specific growth rate and mode of induction to assess the growth potential of the bacteria in a bioreactor and to produce high yield of Bm R1 recombinant antigen. Cells were grown at a controlled specific growth rate (μ_set) during pre-induction, followed by constant feeding postinduction. The highest biomass (24·3 g l⁻¹) was obtained during fed-batch process operated at μ_set of 0·15 h⁻¹, whereby lower μ_set (0·075 h⁻¹) gave the highest protein production (9·82 mg l⁻¹). The yield of Bm R1 was increased by 1·2-fold upon induction with 1 mmol l⁻¹ IPTG (isopropyl-β- d -thiogalactoside) compared to using 5 mmol l⁻¹ and showed a further 3·5-fold increase when the culture was induced twice at the late log phase.
Conclusions:  Combination of feeding at a lower μ_set and twice induction with 1 mmol l⁻¹ IPTG yielded the best result of all variables tested, promising an improved method for Bm R1 production .
Significance and Impact of the Study:  This method can be used to increase the production scale of the Bm R1 recombinant antigen to meet the increasing demand for Brugia Rapid^™, a commercial diagnostic test for detection of brugian filariasis.     

Structural stability of Sclerotium rolfsii ATCC 201126 β-glucan with fermentation time: a chemical, infrared spectroscopic and enzymatic approach

Aims:  Sclerotium rolfsii ATCC 201126 exopolysaccharides (EPSs) recovered at 48 h (EPS I) and 72 h (EPS II) of fermentation, with differences in rheological parameters, hydrogel topography, salt tolerance, antisyneresis, emulsifying and suspending properties, were subjected to a polyphasic characterization in order to detect structural divergences.
Methods and Results:  Fermenter-scale production led to productivity ( P _r) and yield ( Y _P/C) values higher at 48 h ( P _r = 0·542 g l⁻¹ h⁻¹; Y _P/C = 0·74) than at 72 h ( P _r = 0·336 g l⁻¹ h⁻¹; Y _P/C = 0·50). Both EPSs were neutral glucose-homopolysaccharides with a β-(1,3)-glycosidic backbone and single β-(1,6)-glucopyranosyl sidechains regularly attached every three residues in the main chain, as revealed by chemical analyses. The infra-red diagnostic peak at 890 cm⁻¹ confirmed β-glycosidic linkages, while gentiobiose released by β-(1,3)-glucanases confirmed single β-1,6-glycosidic branching for both EPSs.
Conclusions:  The true modular repeating unit of S. rolfsii ATCC 201126 scleroglucan could be resolved. Structural stability was corroborated and no structural differences could be detected as to account for the variations in EPSs behaviour.
Significance and Impact of the Study:  Recovery of S. rolfsii ATCC 201126 scleroglucan at 48 h might be considered based on better fermentation kinetic parameters and no detrimental effects on EPS structural features.     

Formation and microbial community analysis of chloroanilines-degrading aerobic granules in the sequencing airlift bioreactor

Aims:  This paper investigates a selection-based acclimation strategy for improving the performance and stability of aerobic granules at a high chloroanilines loading.
Methods and Results:  The experiments were conducted in a sequencing airlift bioreactor (SABR) to develop aerobic granules fed with chloroanilines (ClA). The evolution of aerobic granulation was monitored using image analysis and scanning electron microscopy, and PCR–DGGE analysis of microbial community was performed. The sludge granulation was apparently developed by decreased settling time and gradual increased ClA loading to 0·8 kg m⁻³ day⁻¹. A steady-state performance of the granular SABR was reached at last, as evidenced by biomass concentration of 6·3 g l⁻¹ and constant ClA removal efficiency of 99·9%. The mature granules had a mean size of 1·55 mm, minimal settling velocity of 68·4 m h⁻¹, specific ClA degradation rate of 0·181 g gVSS⁻¹ day⁻¹. Phylogenetic analysis of aerobic ClA-degrading granules confirmed the dominance of β - , γ -Proteobacteria and Flavobacteria.
Conclusions:  The chosen operating strategy involving step increase in ClA loading and enhancement of major selection pressures was successful in cultivating the aerobic ClA-degrading granules.
Significance and Impact of the Study:  This research could be helpful for improving the stability of aerobic granules via optimizing operating conditions and developing economic feasible full-scale granular bioreactor.     

Response surface methodology to optimize the nutritional parameters for enhanced production of jasmonic acid by Lasiodiplodia theobromae

Aims:  To find out the cumulative effect of the nutritional parameters and to enhance the production of jasmonic acid (JA) in static fermentation by Lasiodiplodia theobromae using response surface methodology (RSM).
Method and Results:  Malt extract, sucrose, NaNO₃ and MgSO₄.7H₂O were analysed by a 30-trial central composite design using RSM for optimizing their concentrations in the medium and the effect of their mutual interaction on JA production. Sucrose and NaNO₃ were found highly significant in influencing the JA production. Malt extract and MgSO₄.7H₂O showed an effect on the JA production in interaction with other variables. When the optimum values of the parameters obtained through RSM (19·95 g l⁻¹ malt extract, 50 g l⁻¹ sucrose, 7·5 g l⁻¹ NaNO₃ and 3·51 g l⁻¹ MgSO₄.7H₂O) were applied, 32% increase in JA production (299 mg l⁻¹) was observed in comparison with 225 mg l⁻¹ of JA produced with same media components not analysed by RSM and subsequently validated the statistical model.
Conclusions:  Increase in JA production was achieved by optimizing the nutritional parameters.
Significance and Impact of the Study:  This is the first report of using RSM for optimizing a medium for JA production. It resulted in an increase in JA production without augmentation of costly additives.     

The effect of carbon and nitrogen sources on bovicin HC5 production by Streptococcus bovis HC5

Aims:  To investigate the effect of media composition and agroindustrial residues on bovicin HC5 production by Streptococcus bovis HC5.
Methods and Results:  Batch cultures of S. bovis HC5 were grown in basal medium containing different carbon and nitrogen sources. The activity of cell-free and cell-associated bovicin HC5 was determined in culture supernatants and acidic extracts obtained from cell pellets, respectively. Streptococcus bovis HC5 produced bovicin using a variety of carbon and nitrogen sources. The highest specific activity was obtained in media containing 16 g l⁻¹ of glucose, after 16 h of incubation. The peak in cell-free and cell-associated bovicin HC5 activity was detected when S. bovis HC5 cultures reached stationary phase. The bovicin HC5 specific activity and bacterial cell mass increased approximately 3-fold when yeast extract and trypticase (0·5 and 1·0 g l⁻¹, respectively) were added together to the basal medium. Streptococcus bovis HC5 cultures produced bovicin HC5 in cheese whey and sugar cane juice and maximal volumetric productivity was obtained after 12 h of incubation.
Conclusions:  Streptococcus bovis HC5 is a versatile lactic acid bacterium that can utilize several carbon and nitrogen sources for bovicin HC5 production. This bacterium could be a useful model to study bacteriocin production in the rumen ecosystem.
Significance and Impact of the Study:  The use of agroindustrial residues as carbon sources could have an economical impact on bovicin HC5 production. To our knowledge, this is the first report to show the use of sugar cane juice for bacteriocin production by lactic acid bacteria.     

Influence of phosphorus on biofilm formation in model drinking water distribution systems

Aims:  This study investigated the effects of phosphorus on biofilm formation via annular reactor systems in terms of biofilm cell growth, exopolysaccharide (EPS) production, biofilm structure and cell metabolic potential.
Methods and Results:  Drinking water biofilms were developed in annular reactors with supplement of carbon and different levels of phosphorus. The biofilm formation was monitored over a period of 30 days. Biofilm related parameters were examined by various methods, which included heterotrophic plate count, total carbohydrate content, confocal laser scanning microscopy and GN2 microplate assay. Our results showed that phosphorus addition can promote the biofilm cell growth (cell count increased about 1 log with addition of 30 and 300 μg l⁻¹ of phosphorus). However, the addition of 30 and 300 μg l⁻¹ of phosphorus caused 81% and 77% decrease in EPS production, respectively. The results of biofilm structure analysis showed that the addition of 30 and 300 μg l⁻¹ of phosphorus can induce thicker and less homogeneous biofilms with more biomass. Furthermore, the addition of 30 and 300 μg l⁻¹ of phosphorus dramatically increased the biofilm cell metabolic potential. The addition of 3 μg l⁻¹ of phosphorus was found to have minor effects on the parameters examined.
Conclusions:  The results indicate phosphorus addition to drinking water distribution system (DWDS) has a complicated effect on the biofilm formation.
Significance and Impact of the Study:  As the addition of phosphorus at certain levels can affect the biofilm growth in DWDS, care should be taken when phosphate-based corrosion inhibitors are used in the DWDS.     

Water‐based measurement of rainbow trout melatonin

Melatonin (MLT) was isolated from water samples using solid phase extraction cartridges and measured by radioimmunoassay. In tanks of rainbow trout Oncorhynchus mykiss , concentrations of MLT in water increased rapidly from <0·1 ng l⁻¹ in the light phase to 0·7 ng l⁻¹ in the dark giving calculated release rates of <1 ng kg⁻¹ h⁻¹ and 15 ng kg⁻¹ h⁻¹, respectively. This cycle in water MLT values corresponds to reported changes in plasma MLT concentrations. The colour of the tanks and a daytime acute handling stress did not affect concentrations of MLT in either the light or dark phase.     

Culture conditions affect the chemical composition of the exopolysaccharide synthesized by the fungus Aureobasidium pullulans

Aims:  To identify if culture conditions affect the chemical composition of exopolysaccharide (EPS) produced by Aureobasidium pullulans .
Methods and Results:  In batch airlift and continuously stirred tank (CSTR) reactors the EPS produced with low (0·13 g l⁻¹ N) initial NaNO₃ or (NH₄)₂SO₄ levels contained pullulan, with maltotriose as its major component, similar to that synthesized in the airlift reactor with high (0·78 g l⁻¹ N) initial NaNO₃ levels. EPS produced by CSTR grown cultures with high (NH₄)₂SO₄ levels contained little pullulan, possibly because of a population shift from unicells to mycelium. This chemical difference may explain why total EPS yields did not fall as they did with cultures grown under identical conditions with high NaNO₃ levels, where the pullulan component of the EPS disappeared. EPS synthesized in N-limiting chemostat cultures of A. pullulans changed little with growth rate or N source, being predominantly pullulan consisting of maltotriose units.
Conclusions:  While the EPS chemical composition changed little under N-limiting conditions, high initial medium N levels determined maltotriose content and/or pullulan content possibly by dictating culture morphology.
Significance and Impact of the Study:  These results emphasize the requirement of all studies to determine EPS chemical composition when examining the influence of culture conditions on EPS yields.     

Effect of culturing processes and copper addition on laccase production by the white-rot fungus Fomes fomentarius MUCL 35117

Aim:  To produce high laccase activities from the white-rot fungus Fomes fomentarius .
Methods and Results:  Different culturing methods, viz, cell immobilization on stainless steel sponges and plastic material and solid-state fermentation (SSF) using wheat bran as substrate were used for laccase production by the white-rot fungus F. fomentarius . The SSF study expresses the highest laccase activities, nearly to 6400 U l⁻¹ after 13 days of laboratory flasks cultivation. When the wheat bran medium was supplemented with 2 mmol l⁻¹ copper sulfate, laccase activity increased by threefold in comparison to control cultures, reaching 27 864 U l⁻¹. With the medium thus optimized, further experiments were performed in a 3 l fixed-bed bioreactor (working volume 1·5 l) leading to a laccase activity of about 6230 U l⁻¹ on day 13.
Conclusions:  The results obtained clearly showed the superiority of wheat bran for laccase production over stainless steel sponges and plastic material. Supplementing the wheat bran solid medium with 2 mmol l⁻¹ copper sulfate allowed obtaining high activities at flask scale. The system was scaled to fixed-bed laboratory reactor.
Significance and Impact of the Study:  The high enzyme production along with the low-cost of the substrate, showed the suitability of the system F. fomentarius – SSF for industrial purposes.     

Inactivation of oenological lactic acid bacteria (Lactobacillus hilgardii and Pediococcus pentosaceus) by wine phenolic compounds

Aims:  To investigate the inactivation properties of different classes of phenolic compounds present in wine against two wine isolates of Lactobacillus hilgardii and Pediococcus pentosaceus , and to explore their inactivation mechanism.
Methods and Results:  After a first screening of the inactivation potency of 21 phenolic compounds (hydroxybenzoic and hydroxycinnamic acids, phenolic alcohols, stilbenes, flavan-3-ols and flavonols) at specific concentrations, the survival parameters (MIC and MBC) of the most active compounds were determined. For the L. hilgardii strain, the flavonols morin and kaempferol showed the strongest inactivation (MIC values of one and 5 mg l⁻¹, and MBC values of 7·5 and 50 mg l⁻¹, respectively). For the P. pentosaceus strain, flavonols also showed the strongest inactivation effects, with MIC values between one and 10 mg l⁻¹ and MBC values between 7·5 and 300 mg l⁻¹. Observations by epifluorescence and scanning electron microscopy revealed that the phenolics damaged the cell membrane and promoted the subsequent release of the cytoplasm material into the medium.
Conclusions:  The antibacterial activity of wine phenolics against L. hilgardii and P. pentosaceus was dependent on the phenolic compound tested, and led not only to bacteria inactivation, but also to the cell death.
Significance and Impact of the Study:  New information about the inactivation properties of wine lactic acid bacteria by phenolic compounds is presented. It opens up a new area of study for selecting/obtaining wine phenolic preparations with potential applications as a natural alternative to SO₂ in winemaking.     

Isolation and characterization of alginate-degrading bacteria for disposal of seaweed wastes

Aims:  Isolation of novel alginate degrading bacteria for the disposal of seaweed waste in composting process.
Methods and Results:  Decomposition of alginate polymers was checked by the 3,5-dinitrosalicylic acid (DNS) method for reducing sugar, and absorbance at 235 nm for unsaturated sugar. A bacterium A7 was isolated from wakame compost and confirmed to belong to the genus Gracilibacillus by partial 16S rDNA analysis. The optimum condition for the growth of A7 in a medium containing 5 g l⁻¹ of sodium alginate is as follows: pH, 8·5–9·5; NaCl, 0·5 mol l⁻¹; temperature, 30°C and polypeptone as nutrient content, 2–5 g l⁻¹. In a laboratory-scale composting experiment, the alginate content in wakame compost decreased to 14·3% after 72 h of composting from an initial value of 36%, indicating the effectiveness of alginate decomposition of A7 in wakame composting.
Conclusions:  The bacterium A7 was found to be alginate lyase-producing in genus Gracilibacillus and effective in degrading alginate to oligosaccharides in wakame during composting process.
Significance and Impact of the Study:  Development of new methods for the disposal of marine wastes and production of functional products.     

Effect of Lactobacillus gasseri BNR17 on blood glucose levels and body weight in a mouse model of type 2 diabetes

Aims:  To investigate the effect of Lactobacillus gasseri BNR17 isolated from human breast milk on blood glucose and body weight in type 2 diabetic animals.
Methods and results:  db/db mice were divided into one control group and five sample groups; the sample groups received BNR17 (10⁷, 10⁸, 10⁹ and 10¹⁰ CFU) or rosiglitazone (8 mg kg⁻¹) orally twice a day for 12 weeks. BNR17 groups had a dose-dependent reduction in food, water intake and amount of excrement. Body weight loss was not seen in the BNR17 groups. Fasting and postprandial 2 h blood glucose levels were significantly lower in the BNR17 (10¹⁰ CFU) group compared with the control group. HbA1c decreased in the BNR17 group, although it was not statistically significant. During the oral glucose tolerance test, the BNR17 groups exhibited dose-dependent improvement in glucose sensitivity.
Conclusions:  Lactobacillus gasseri BNR17 has a suppressing effect on blood glucose levels and improved diabetic symptoms in db/db mice.
Significance and Impact of the Study:  Blood glucose-lowering lactic acid bacteria are expected to be useful as a therapeutic for treating type 2 diabetes in humans.     

Stimulation of biomethanation by Clostridium sp. PXYL1 in coculture with a Methanosarcina strain PMET1 at psychrophilic temperatures

Aim:  Bioaugumentation of low temperature biogas production was attempted by addition of cold-adapted Clostridium and a methanogen.
Methods and Results:  A psychrotrophic xylanolytic acetogenic strain Clostridium sp. PXYL1 growing optimally at 20°C and pH 5·3 and a Methanosarcina strain, PMET1, growing optimally on acetate and producing methane at 15°C were isolated from a cattle manure digester. Anaerobic conversion of xylose at 15°C with the coculture of the two strains was performed, and batch culture methane production characteristics indicated that methanogenesis occurred via acetate through 'acetoclastic' pathway. Stimulation studies were also undertaken to evaluate the effect of exogenous addition of the coculture on biogas yields at 15°C. Addition of 3 ml of PXYL1 at the rate of 12 × 10² CFU ml⁻¹ increased the biogas 1·7-fold (33 l per kg cowdung) when compared to control (19·3 l per kg cowdung) as well as increased the volatile fatty acid (VFA) levels to 3210 mg l⁻¹ when compared to 1140 mg l⁻¹ in controls. Exogenous of addition of 10 ml PMET1 inoculum at the rate of 6·8 ± 10² CFU ml⁻¹ in addition to PXYL1 served to further improve the biogas yields to 46 l kg⁻¹ as well as significantly brought down the VFA levels to 1350 mg l⁻¹.
Conclusions:  Our results suggest that the rate-limiting methanogenic step at low temperatures could be overcome and that biogas yields improved by manipulating the population of the acetoclastic methanogens.
Significance and Impact of the Study:  Stimulation of biomethanation at low temperature by coculture.     

Optimization and biochemical characterization of a bacteriocin from a newly isolated Bacillus subtilis strain 14B for biocontrol of Agrobacterium spp. strains

Aims:  The identification of a new compound active against Agrobacterium tumefaciens .
Methods and Results:  The culture conditions of a newly isolated Bacillus subtilis strain, designed 14B, were optimized, as a first step, to produce its bacteriocin (termed Bac 14B) for the biocontrol of Agrobacterium spp., the causal agents of the crown gall disease. Bac 14B was then partially purified and biochemically characterized. Bacillus subtilis 14B was observed to produce an antibacterial compound having a protinaceous nature. As estimated by sodium dodecyl sulfate-polyacrilamide gel electrophoresis (SDS-PAGE), the semi-purified bacteriocin substance was found to be a monomeric protein with a molecular weight of 21 kDa. While the latter's antimicrobial activity was completely stable during exposure to a temperature range of up to 100°C for 2 h, its initial activity was totally lost at 121°C for 20 min. The maximum bacteriocin production (4096 AU ml⁻¹) was recorded after 96 h-incubation in an optimized Luria Bertani medium supplemented with 10 g l⁻¹ glucose, 15 g l⁻¹ K₂HPO₄ and 5 g l⁻¹ MgSO₄ 7H₂O at 30°C in a shaking flask culture. Interestingly, the B. subtilis 14B culture supernatant that contained the bacteriocin under study was proved efficient in reducing both the percentage of galled plants and the number of galls in tomato.
Conclusion:  The findings revealed that B. subtilis 14B and its bacteriocin are efficient in reducing the percentage of infections in plants caused by Ag. tumefaciens .
Significance and Impact of the Study:  The results could be useful for the nurserymen who are particularly interested in the biocontrol of the crown gall disease.     

Determination of kinetic constants of hybrid textile wastewater treatment system

The present study is related to treatment of textile wastewater in microaerophilic–aerobic hybrid reactor. The study showed the effectiveness of biological treatment of wastewater involving appropriate microorganism and suitable reactors. COD and color were reduced to 82–94%, and 99% respectively for textile wastewater. The reactor was operated at highest loading of 16.4 g COD g l⁻¹ d⁻¹ and obtained 80% COD and 72% color removal. Biokinetic models were applied to data obtained from experimental studies in continuously operated hybrid reactor. Treatment efficiencies of the reactor were investigated at different hydraulic retention times (2.3–9.1 d) and organic loading rates (2.6–16.4 g COD l⁻¹ d⁻¹). Second-order and a Stover–Kincannon models were best fitted to the hybrid column reactor. The second-order substrate removal rate constant (k_2(S)) was found as 41.44 d⁻¹ for hybrid reactor. Applying the modified Stover–Kincannon model to the hybrid reactor, the maximum removal rate constant (U_max) and saturation value constant (K_B) were found to be 212 g l⁻¹ d⁻¹ and 22.89 g l⁻¹ d⁻¹, respectively.     

3-Methylindole production is regulated in Clostridium scatologenes ATCC 25775

Aims:  3-Methylindole (3-MI) is a degradation product of l -tryptophan and is both an animal waste malodorant and threat to ruminant health. Culture conditions influencing 3-MI production in Clostridium scatologenes ATCC 25775 were investigated.
Methods and Results:  Extracellular 3-MI levels in cells cultured in brain heart infusion (BHI) medium (pH 7·0) at 33°C and 37°C for 72 h were 907 ± 38 and 834 ± 121  μ mol l⁻¹, respectively. Cells cultured in tryptone-yeast (TY) extract medium at 37°C for 48 h produced 104 ± 86  μ mol l⁻¹ 3-MI; however, addition of 1 mmol l⁻¹  l -tryptophan failed to increase extracellular levels (113 ± 50  μ mol l⁻¹ 3-MI). Specific activity of indole acetic acid decarboxylase measured in BHI, TY and TY plus 1 mmol l⁻¹ tryptophan-grown cells displayed 35-, 33- and 76-fold higher levels than in semi-defined medium-grown cells.
Conclusions:  When cultured in rich medium, at 33°C or 37°C and pH 7·0, Cl. scatologenes ATCC 25775 optimally produced 3-MI. Addition of l- tryptophan to medium did not lead to significant increases in extracellular 3-MI levels. Whole cell assays indicate growth in rich medium significantly up-regulated 3-MI production.
Significance and Impact of the Study:  Information presented here may prove useful in understanding what factors influence 3-MI production in malodorous animal wastes.     

Seasonal heterotrophic flagellate and bacterial plankton dynamics in a large oligotrophic lake– Loch Ness, Scotland

1. Bacterial production in the 0–30 m water column of Loch Ness was measured using a dual labelling procedure with [³H] thymidine and [¹⁴C] leucine between May 1993 and June 1994. In most cases the uptake of the two labels did not covary, suggesting unbalanced growth. Rates of bacterial production varied from undetectable to 46.2 μg C l^–1 day^–1. Highest production coincided with the period of highest primary production, but carbon derived from this source was insufficient to meet the bacterial carbon demand, which was met by allochthonous humic inputs to the system.
2. Heterotrophic flagellate (HNAN) grazing rates, measured using fluorescently labelled bacteria, ranged between 10.3 and 24.5 bacteria cell^–1 day^–1 at temperatures between 5 and 15 °C. They removed up to 27% of the bacterial production per day.
3. Heterotrophic flagellate specific growth rates ranged from 0.043 to 0.093 h^–1 between 5 and 15 °C, giving generation times of 7.4–16.1 h.
4. bacterial and HNAN abundances were not coupled, but the highest HNAN grazing impact related to a time of high bacterial productivity.