Fed-batch production of a bioflocculant from <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

The constant-rate fed-batch production of the polygalacturonic acid bioflocculant REA-11 was studied. A controlled sucrose-feeding strategy resulted in a slight improvement in biomass and a 7% reduction in flocculating activity compared with the batch process. When fed with a 3 g l⁻¹ urea solution, the flocculating activity was enhanced to 720 U ml⁻¹ in 36 h. High cell density (2.12 g l⁻¹) and flocculating activity (820 U ml⁻¹) were obtained in a 10-l fermentor by feeding with a sucrose-urea solution, with values of nearly two times and 50% higher than those of the batch process, respectively. Moreover, the residual sucrose declined to 2.4 g l⁻¹, and residual urea decreased to 0.03 g l⁻¹. Even higher flocculating activity of 920 U ml⁻¹ and biomass of 3.26 g l⁻¹ were obtained by feeding with a sucrose-urea solution in a pilot scale fermentation process, indicating the potential industrial utility of this constant-rate feeding strategy in bioflocculant production by Corynebacterium glutamicum.     

Characteristics and flocculating mechanism of a novel bioflocculant HBF-3 produced by deep-sea bacterium mutant Halomonas sp. V3a’

A novel bioflocculant HBF-3 produced by deep-sea bacterium mutant Halomonas sp. V3a’ was investigated with regard to its flocculating characteristics and mechanism. 4.0 m g l⁻¹ HBF-3 showed the maximum flocculating activity of 96.9% in 5.0 g l⁻¹ Kaolin suspension containing 11.25 mM CaCl₂, and that its flocculating activity was more than 90% within 5–40°C and over 80% in a wide pH range (3.0–11.0). Chemical analyses indicated that the biopolymer HBF-3 was mainly a polysaccharide, including neutral sugar residues (20.6%), uronic acid residues (7.6%), amino sugar residues (1.6%) and sulfate groups (5.3%). Fourier transform infrared (FTIR) spectrum showed the presence of carboxyl and hydroxyl groups in HBF-3 molecular. The average molecular mass of HBF-3, as determined by gel filtration chromatography (GFC), was approximately 590 kDa. Flocculation of Kaolin suspension with HBF-3 acted as a model to explore the flocculating mechanism in which bridging mediated by Ca²⁺ was proposed as the primary action based upon the experimental observations.     

<Emphasis Type="Italic">Cellulomonas macrotermitis</Emphasis> sp. nov., a chitinolytic and cellulolytic bacterium isolated from the hindgut of a fungus-growing termite

To investigate the symbiotic roles of the gut microbiota in the fungus-growing termite Macrotermes barneyi, a novel strain with chitinolytic and cellulolytic activity, designated strain an-chi-1^T, was isolated from the hindgut of M. barneyi. Strain an-chi-1^T grows optimally at 28–30 °C, pH 8.0 in PYG medium. On the basis of 16S rRNA gene sequence analysis, this isolate belongs to the genus Cellulomonas with high sequence similarity to Cellulomonas iranensis (99.4%), followed by Cellulomonas flavigena (98.4%), Cellulomonas phragmiteti (97.4%), Cellulomonas oligotrophica (97.2%) and Cellulomonas terrae (97.0%). The DNA–DNA relatedness between an-chi-1^T and the type strains of C. iranensis and C. flavigena DSM20109^T are 35.4% and 23.7%, respectively. The major cellular fatty acids are anteiso-C_15:0 and C_14:0. The polar lipid profile consists of diphosphatidylglycerol, phosphatidylinositol mannosides, phosphatidylinositol dimannosides and one unidentified phospholipid. The cell-wall sugar is ribose. The peptidoglycan contains glutamic acid, aspartic acid and alanine. The DNA G+C content is 67.3 mol%. Based on its distinctive phenotypic, phylogenetic, and chemotaxonomic characteristics, an-chi-1^T represents a novel species of the genus Cellulomonas, for which the name Cellulomonas macrotermitis sp. nov. is proposed. The type strain is an-chi-1^T (= JCM 31923^T = CICC 24195^T).     

The curdlan-type exopolysaccharide produced by <Emphasis Type="Italic">Cellulomonas flavigena</Emphasis> KU forms part of an extracellular glycocalyx involved in cellulose degradation

The genus Cellulomonas is comprised of a group of Gram-positive, soil bacteria capable of utilizing cellulose as their sole source of carbon and energy. Cellulomonas flavigena KU was originally isolated from leaf litter and subsequently shown to produce large quantities of a curdlan-type (-1,3-glucan) exopolysaccharide (EPS) when provided with an excess of glucose or other soluble carbon-source. We report here that curdlan EPS is also produced by Cellulomonas flavigena KU when growing on microcrystalline cellulose in mineral salts-yeast extract media. Microscopic examination of such cultures shows an adherent biofilm matrix composed of cells, curdlan EPS, and numerous surface structures resembling cellulosome complexes. Those Cellulomonas species that produce curdlan EPS are all non-motile and adhere to cellulose as it is broken down into soluble sugars. These observations suggest two very different approaches towards the complex process of cellulose degradation within the genus Cellulomonas.     

Characterization and flocculating properties of a novel bioflocculant produced by <Emphasis Type="Italic">Bacillus circulans</Emphasis>

We studied a novel bioflocculant, PX, that is produced from Bacillus Bacillus circulans X3, and has excellent flocculating activity with regard to its characterization and flocculating properties. The bioflocculant was purified from supernatant by ethanol precipitation, dialysis and gel permeation chromatography (GPC). The major component of PX was an acid polysaccharide including uronic (19.8%), pyruvic (6.5%) and acetic acids (0.7%). It consisted of galactose, mannose, xylitol, rhamnose and galacturonic acid in an approximate molar ration of 5:4.1:3:2:1.2. The molecular weight of PX was about 4.85 × 10⁴ Da as determined by GPC. The infrared spectrum of the bioflocculant indicated the presence of carboxyl, hydroxyl, amino and methoxyl groups. Studies of the flocculating properties revealed that it was stable at 60–100°C and pH 4–10. Moreover, it could flocculate a kaolin suspension over a wide range of pH and temperature in the presence of CaCl₂.     

Assessment of poly‐l‐lysine dendrigrafts for virus concentration in water: use of MS2 bacteriophage as proof of concept

Aims

Virus detection has often been difficult due to a low concentration in water. In this study, we developed a new procedure based on concentration of virus particles on an innovative support: poly‐l ‐lysine dendrigrafts (DGL), coupled with directed nucleic acid extraction and real‐time PCR quantification.

Methods and Results

This method was evaluated using the bacteriophage MS2 as a model virus. This virus exhibited the size and structural properties of human pathogenic enteric viruses and has often been used to assess new supports of concentration. Moreover, this bacteriophage is also a faecal contamination indicator. In this study, many water filtration conditions were tested (volume of water, concentration, etc.), and more than 80% of bacteriophage were recovered after filtration on polymer, in most conditions. We demonstrated that the method was linear (slope = 0·99 ± 0·04 and Y intercept when x = ?0·02 ± 0·28), valid (as manipulators, tested concentrations, volumes of sample and batch of polymer did not have any influence on concentration) and sensitive (allowing to concentrate up to 16 600‐fold 1 l of sample and to detect and quantify down to 750 GC l^?1 and 7500 GC l^?1, respectively).

Conclusions

To conclude, this support exhibits high interest to retain viruses and to allow to detect low concentration of virus in water.

Significance and Impact of the Study

This study gives valuable advance in the methods of concentration and diagnosis of virus in water.     

Study on the flocculability of the Arthrobacter sp., an actinomycete resuscitated from the VBNC state

A bioflocculant with high flocculating activity, LC13-SF, produced by strain LC13^T which was in a viable but nonculturable (VBNC) state, and which was woken up by Rpf (resuscitation promoting factor), was systematically investigated with regard to its fermentation conditions and flocculating activity. The key parameters influencing the bioflocculant LC13-SF were investigated through measuring the optical density at 660 (OD₆₆₀) of the fermentation liquid and the optical density at 550 (OD₅₅₀) of the centrifugal supernatant. The flocculating efficiency and the Zeta potentials were chosen as the response variables for the study of the flocculating activity. The results showed that the optimal conditions for bioflocculant LC13-SF production were a fermentation time of 72 h, an initial pH of 7.0, a fermentation temperature of 30°C and a shaking speed of 150 r/min. The optimized flocculating process was as follows: a final volume percentage of bioflocculant LC13-SF and 0.5% (w/w) CaCl₂ were 1.5 and 5%, respectively in a 4 g/L Kaolin suspension, and the system pH was adjusted to 8.0. Under these conditions, the flocculating efficiency and the absolute value of the Zeta potential reached 94.83% and 4.37, respectively.     

In vitro assays on the susceptibility of four species of nematophagous fungi to anthelmintics and chemical fungicides/antifungal drug

Using nematophagous fungi for the biological control of animal parasitic nematodes will become one of the most promising strategies in the search for alternative chemical drugs. The purpose of this study was to check the in vitro activity of four anthelmintics, four chemical fungicides and two antifungal drugs on the spore germination of nematophagous fungi: Duddingtonia flagrans (SF170), Arthrobotrys oligospora (447), Arthrobotrys superba (435) and Arthrobotrys sp. (PS011). A modified 24-well cell culture plate assay was conducted to evaluate the susceptibility of nematophagous fungi against drugs tested by calculating the effective middle concentrations (EC₅₀) of each tested drug to inhibit the germination of fungal spores. EC₅₀ ranged between 0·7 and 47·2 μg ml⁻¹ for fenbendazole, thiabendazole and ivermectin, except levamisole (546·5–4057·8 μg ml⁻¹). EC₅₀ of tested fungicides was 0·6–2·3 μg ml⁻¹ for carbendazim, 55·9–247·4 μg ml⁻¹ for metalaxyl, 24·4–45·2 μg ml⁻¹ for difenoconazole, and 555·9–1438·3 μg ml⁻¹ for pentachloronitrobenzene (PCNB). EC₅₀ of two antifungal drugs was 0·03–3·4 μg ml⁻¹ for amphotericin B and 0·3–10·9 μg ml⁻¹ for ketoconazole. The results showed that 10 tested drugs, except for levamisole and PCNB, had in vitro inhibitory effects on nematophagous fungi. The chlamydospores of D. flagrans had the highest sensitivity to nine tested drugs, except for ketoconazole.     

Increased synthesis of α‐tocopherol,paramylon and tyrosine by Euglena gracilis under conditions of high biomass production

Aims: To analyse the production of different metabolites by dark‐grown Euglena gracilis under conditions found to render high cell growth. Methods and Results: The combination of glutamate (5 g l^?1), malate (2 g l^?1) and ethanol (10 ml l^?1) (GM + EtOH); glutamate (7·15 g l^?1) and ethanol (10 ml l^?1); or malate (8·16 g l^?1), glucose (10·6 g l^?1) and NH₄Cl (1·8 g l^?1) as carbon and nitrogen sources, promoted an increase of 5·6, 3·7 and 2·6‐fold, respectively, in biomass concentration in comparison with glutamate and malate (GM). In turn, the production of α‐tocopherol after 120 h identified by LC‐MS was 3·7 ± 0·2, 2·4 ± 0·1 and 2 ± 0·1 mg [g dry weight (DW)]^?1, respectively, while in the control medium (GM) it was 0·72 ± 0·1 mg (g DW)^?1. For paramylon synthesis, the addition of EtOH or glucose induced a higher production. Amino acids were assayed by RP‐HPLC; Tyr a tocopherol precursor and Ala an amino acid with antioxidant activity were the amino acids synthesized at higher concentration. Conclusions: Dark‐grown E. gracilis Z is a suitable source for the generation of the biotechnologically relevant metabolites tyrosine, α‐tocopherol and paramylon. Significance and Impact of the Study: By combining different carbon and nitrogen sources and inducing a tolerable stress to the cell by adding ethanol, it was possible to increase the production of biomass, paramylon, α‐tocopherol and some amino acids. The concentrations of α‐tocopherol achieved in this study are higher than others reported previously for Euglena, plant and algal systems. This work helps to understand the effect of different carbon sources on the synthesis of bio‐molecules by E. gracilis and can be used as a basis for future works to improve the production of different metabolites of biotechnological importance by this organism.     

Comparison of supplements* to enhance recovery of heat‐injured Salmonella from egg albumen

Aims: The purpose of this study was to determine the proficiency of supplements to enhance the recovery of Salmonella from heat‐treated liquid egg albumen on solid agar media. Methods and Results: Salmonella‐inoculated albumen, heated at 53·3°C for 4 min, was plated on 39 combinations of solid media with or without the addition of 12 supplements. Greater numbers of Salmonella (P < 0·05) recovered with the addition of 1·0 g l^?1 ferrous sulfate (FeSO₄) than with any other supplements, except for 0·5 or 1·0 g l^?1 3′3′‐thiodipropionic acid (TDP), which recovered equivalent populations. Addition of 1·0 g l^?1 sodium pyruvate or 6·0 g l^?1 yeast extract plus 1·0 g l^?1 sodium pyruvate supported greater resuscitation than unsupplemented tryptic soy agar (TSA) or supplementing with 0·01 or 0·1 g l^?1 N‐propyl gallate, 10 g l^?1 activated charcoal, 0·1 g l^?1 KMnO₄ or 50 mg l^?1 ethoxyquin. The remaining supplements supported recovery of equivalent numbers of Salmonella, which were fewer cells than recovered with 1·0 g l^?1 FeSO₄, yet greater populations than recovered with 50 mg l^?1 ethoxyquin. Conclusion: Supplementation of plating media with FeSO₄, TDP or sodium pyruvate enhanced recovery of sublethally injured Salmonella from albumen. Significance and Impact of the Study: Pasteurizing albumen impedes recovery of pathogens. These results suggest that the addition of supplements to plating media may assist resuscitation and colony development of heat‐injured salmonellae.     

Use chemometric techniques in the optimization of a specific bioassay for betalactams in milk

Aims: A microbiological bioassay using Geoacillus stearothermophilus was optimized to detect betalactams at concentrations near to the Maximum Residue Limits (MRLs), with low cross‐specificity for tetracycline. Methods and Results: A factorial design (3 × 4) was used to evaluate the effects of concentration of spores (2·0 × 10⁶, 4·0 × 10⁶ and 8·0 × 10⁶ spores ml^?1) and incubation time (3·0, 3·5, 4·0 and 4·5 h) on the response of the bioassay. Then, desirability function to raise the detection capabilities (CC_β) of tetracyclines and increase sensitivity to betalactams was implemented. Significant effects of Log[S] and incubation time [It] on the CC_β of betalactams and tetracyclines were observed. Finally, high value of global desirability (D = 0·853), adequate betalactams CC_β (3·8 μg l^?1 of penicillin ‘G’, 27 μg l^?1 of oxacillin, 8·1 μg l^?1 of ampicillin, 48 μg l^?1 of cloxacillin) and high tetracyclines CC_β (5260 μg l^?1 chlortetracycline, 1550 μg l^?1 of oxytetracycline, 1070 μg l^?1 of tetracycline) were calculated. Conclusions: The application of chemometric tools allows the optimization of a bioassay that detects betalactam residues in milk. The more robust conditions have been achieved in Log[S] = 6·30 and [It] = 4·20 h. Significance and Impact of the Study: The logistic regression model and the desirability function are adequate chemometric techniques to improve the properties of the methods, because it is possible to increase sensitivity and decrease cross‐specificity simultaneously.     

Neuraminidase Inhibitors from marine‐derived actinomycete Streptomyces seoulensis

Aims

This work was performed to characterize new secondary metabolites with neuraminidase (NA) inhibitory activity from marine actinomycete strains.

Methods and Results

An actinomycete strain IFB‐A01, capable of producing new NA inhibitors, was isolated from the gut of shrimp Penasus orientalis and identified as Streptomyces seoulensis according to its 16S rRNA sequence (over 99% homology with that of the standard strain). Repeated chromatography of the methanol extract of the solid‐substrate culture of S. seoulensis IFB‐A01 led to the isolation of streptoseolactone ( 1 ), and limazepines G ( 2 ) and H ( 3 ). The structures of 1 – 3 were determined by a combination of IR, ESI‐MS, 1D (¹H and ¹³C NMR, and DEPT) and 2D NMR experiments (HMQC, HMBC, ¹H‐¹H COSY and NOESY). Compounds 1 – 3 showed significant inhibition on NA in a dose‐dependent manner with IC₅₀ values of 3·92, 7·50 and 7·37 μmol l^?1, respectively.

Conclusions

This is the first report of two new ( 1 and 2 ) and known ( 3 , recovered as a natural product for the first time in the work) NA inhibitors from the marine‐derived actinomycete S. seoulensis IFB‐A01.

Significance and Impact of the Study

The three natural NA inhibitors maybe of value for the development of drug(s) necessitated for the treatment of viral infections.     

Effect of plant secondary compounds on in vitro methane,ammonia production and ruminal protozoa population

Aims

The objective of this study was to evaluate the potential of secondary plant metabolites from 38 sources to serve as antimethanogenic additives in ruminant diets. The effect of leaf tannins from these different plant sources on rumen fermentation, protozoal populations and methanogenesis was also studied.

Methods and Results

Samples (200 mg dry matter, DM) were incubated without and with polyethylene glycol (PEG)‐6000 (400 mg DM) as a tannin binder during 24‐h incubation in the in vitro Hohenheim gas system. In the leaf samples, total phenol (g kg^?1 DM) was maximum in Pimenta officinalis (312) followed by Oenothera lamarckiana (185) and Lawsonia inermis (105). Of the 38 samples, condensed tannins exceeded 4·0 g kg^?1 in only Alpinia galanga (7·50), Cinnamomum verum (4·58), Pelargonium graveolens (18·7) and Pimenta officinalis (23·2) and were not detected in seven samples. When the bioactivity of the leaf samples was assessed using the tannin bioassay, the percentage increase in the amount of gas produced during incubation of samples with the tannin‐binding agent PEG‐6000 over the amount produced during incubation without the tannin binder ranged from nil (zero) to 367%, with the highest being recorded with A. galanga leaves. The ratio of methane reduction per ml of total gas reduction was maximum with Rauvolfia serpentina (131·8) leaves, followed by Indigofera tinctoria (16·8) and Withania somnifera (10·2) leaves. Total and differential protozoal counts increased with added PEG in twenty‐two samples, maximum being in Pimenta officinalis. Increased accumulation of total volatile fatty acids during incubation with added PEG‐6000 was recorded, and the values ranged from zero to 61%. However, the increase was significant in only 11 of the 38 tannin sources tested indicating noninterference of tannin on in vitro fermentation of carbohydrates by the majority of samples tested. Conversely, in 26 of 38 plant sources, the leaf tannins reduced N‐digestibility as evidenced by increased accumulation of NH₃‐N with added PEG.

Conclusions

Our study unequivocally demonstrated that plants containing secondary metabolites such as Rauvolfia serpentine, Indigofera tinctoria and Withania somnifera have great potential to suppress methanogenesis with minimal adverse effect of feedstuff fermentation.

Significance and Impact of the Study

It was established that methanogenesis was not essentially related to the density of protozoa population in vitro. The tannins contained in these plants could be of interest in the development of new additives in ruminant nutrition.     

Characterization and application of bioflocculant prepared by Rhodococcus erythropolis using sludge and livestock wastewater as cheap culture media

A new bioflocculant was produced by culturing Rhodococcus erythropolis in a cheap medium. When culture pH was 7.0, inoculum size was 2 % (v/v), Na₂HPO₄ concentration was 0.5 g L^?1, and the ratio of sludge/livestock wastewater was 7:1 (v/v), a maximum flocculating rate of 87.6 % could be achieved. Among 13 different kinds of pretreatments for sludge, the optimal one was the thermal-alkaline pretreatment. Different from a bioflocculant produced in a standard medium, this bioflocculant was effective over a wide pH range from 2 to 12 with flocculating rates higher than 98 %. Approximately, 1.6 g L^?1 of crude bioflocculant could be harvested using cold ethanol for extraction. This bioflocculant showed color removal rates up to 80 % when applied to direct and disperse dye solutions, but only 23.0 % for reactive dye solutions. Infrared spectrum showed that the bioflocculant contained functional groups such as –OH, –NH₂, and –CONH₂. Components in the bioflocculant consisted of 91.2 % of polysaccharides, 7.6 % of proteins, and 1.2 % of DNA. When the bioflocculant and copper sulfate (CuSO₄) were used together for decolorization in actual dye wastewater, the optimum decolorization conditions were specified by the response surface methodology as pH 11, bioflocculant dosage of 40 mg/L, and CuSO₄ 80 mg/L, under which a decolorization rate of 93.9 % could be reached.     

Competitive exclusion as a mode of action of a novel Bacillus cereus aquaculture biological agent

Aims: To determine the contribution of potential modes of action of a Bacillus cereus aquaculture biological control agent in inhibition of the fish pathogen, Aeromonas hydrophila. Methods and Results: When B. cereus was tested in plate well inhibition studies, no production of antimicrobial compounds was detected. Bacillus cereus had a high growth rate (0·96 h^?1), whereas Aer. hydrophila concentration decreased by c. 70% in co‐culture experiments. In nutrient limitation studies, B. cereus had a significantly higher growth rate when cultured under glucose (P < 0·05) and iron (P < 0·01) limitation in comparison with Aer. hydrophila. Bacillus cereus glucose (0·30 g l^?1 h^?1) and iron (0·60 mg l^?1 h^?1) uptake rates were also significantly higher (P < 0·01) than the Aer. hydrophila glucose (0·14 g l^?1 h^?1) and iron (0·43 mg l^?1 h^?1) uptake rates. Iron uptake was facilitated by siderophore production shown in time profile studies where relative siderophore production was c. 60% through the late exponential and sporulation phases. Conclusions: Competitive exclusion by higher growth rate, competition for organic carbon and iron, facilitated by siderophore production, could be identified as mechanisms of pathogen growth inhibition by B. cereus. Significance and Impact of the Study: This study is the first elucidation of the mechanism of action of our novel B. cereus biological agent in growth attenuation of pathogenic Aer. hydrophila. This study enhances the application knowledge and attractiveness for adoption of B. cereus NRRL 100132 for exploitation in aquaculture.     

Cloning and characterization of a thermostable and halo-tolerant endoglucanase from <Emphasis Type="Italic">Thermoanaerobacter tengcongensis</Emphasis> MB4

A β-1,4-endoglucanase (Cel5A) was cloned from the genomic DNA of saccharolytic thermophilic eubacterium Thermoanaerobacter tengcongensis MB4 and functionally expressed in Escherichia coli. Substrate specificity analysis revealed that Cel5A cleaves specifically the β-1,4-glycosidic linkage in cellulose with high activity (294 U mg⁻¹; carboxymethyl cellulose sodium (CMC)). On CMC, kinetics of Cel5A was determined (K _m 1.39 ± 0.12 g l⁻¹; k _cat/K _m 1.41 ± 0.13 g⁻¹ s⁻¹). Cel5A displays an activity optimum between 75 and 80 °C. Residues Glu187 and Glu289 were identified as key catalytic amino acids by sequence alignment. Interestingly, derived from a non-halophilic bacterium, Cel5A exhibits high residual activities in molar concentration of NaCl (3 M, 49.3%) and KCl (4 M, 48.6%). In 1 M NaCl, 82% of Cel5A activity is retained after 24 h incubation. Molecular Dynamics studies performed at 0 and 3 M NaCl, correlate the Cel5A stability to the formation of R-COO⁻···Na⁺ ···⁻OOC-R salt bridges within the Cel5A tertiary structure, while activity possibly relates to the number of Na⁺ ions trapped into the negatively charged active site, involving a competition mechanism between substrate and Na⁺. Additionally, Cel5A is remarkably resistant in ionic liquids 1-butyl-3-methyllimidazolium chloride (1 M, 54.4%) and 1-allyl-3-methylimidazolium chloride (1 M, 65.1%) which are promising solvents for cellulose degradation and making Cel5A an attractive candidate for industrial applications.     

Transformation of protoplasts ofCellulomonas flavigena

Summary Protoplasts ofCellulomonas flavigena (Cm^s) were transformed with plasmid pC194. Transformation frequency was 2.72×10^–3 in MR-1 regeneration medium with 2 g/ml chloramphenicol. Transformation conditions are described.     

Effect of soybean oil on the production of mycelial biomass and pleuromutilin in the shake-flask culture of <Emphasis Type="Italic">Pleurotus mutilis</Emphasis>

Effect of soybean oil on mycelial biomass and pleuromutilin biosynthesis by Pleurotus mutilis-04 was investigated in shake flask culture. The maximum pleuromutilin production and mycelial biomass were 8.32 ± 0.02 g l⁻¹ and 49.10 ± 1.00 g l⁻¹ when 20 g l⁻¹ soybean oil was fed at 24 and 96 h respectively. A repeated fed-batch fermentation strategy with feeding 3 g l⁻¹ soybean oil from 96 to 144 h at 24 h intervals was developed successfully to maintain mycelial growth and provide abundant fatty acids for pleuromutilin biosynthesis. Compared with glucose as the sole carbon source, soybean oil was obviously beneficial for the production of pleuromutilin. The results suggested that manipulation of metabolic regulation by soybean oil was an effective way to enhance the production pleuromutilin.     

Curdlan-like exopolysaccharide production by <Emphasis Type="Italic">Cellulomonas flavigena</Emphasis> UNP3 during growth on hydrocarbon substrates

Cellulomonas flavigena UNP3, a natural isolate from vegetable oil contaminated soil sample has been studied for growth associated exopolysaccharide (EPS) production during growth on glucose, groundnut oil and naphthalene. The EPS showed matrix formation surrounding the cells during scanning electron microscopy. Cell surface hydrophobicity and emulsifying activity studies confirmed the role of EPS as bioemulsifier. Emulsifying activity was found to increase with time (0.2 U/mg for 10 min to 0.27 U/mg for 30 min). Emulsification index, E₂₄ value increased with the increase in EPS concentration. Degradation of polyaromatic hydrocarbons was confirmed using gas chromatography analysis. FTIR analysis showed presence of characteristic absorbance at 895.10 cm⁻¹ for β-configuration of glucan. NMR studies also revealed EPS produced by C. flavigena UNP3 as a linear β-1, 3-d-glucan, and a curdlan like polysaccharide.     

Characterization of a compound bioflocculant produced by mixed culture of <Emphasis Type="Italic">Rhizobium radiobacter</Emphasis> F2 and <Emphasis Type="Italic">Bacillus sphaeicus</Emphasis> F6

A compound bioflocculant CBF-F26, produced by mixed culture of Rhizobium radiobacter F2 and Bacillus sphaeicus F6, was investigated with regard to its physicochemical and flocculating properties. It was identified as a polysaccharide bioflocculant composed of rhamnose, mannose, glucose, and galactose, respectively, in a 1.3: 2.1: 10.0: 1.0 molar ratio. The average molecular weight was determined as 4.79 × 10⁵ Da by gel-permeation chromatography. Infrared spectrum and X-ray photoelectron spectroscopy revealed the presence of carboxyl, hydroxyl and amino groups in its structure. Thermostability test suggested that CBF-F26 was thermostable and high flocculating activity was maintained. Thermogravimetric property, intrinsic viscosity and surface morphology of CBF-F26 were also studied. CBF-F26 was effective under neutral and weak alkaline conditions (pH 7.0–9.0), and flocculating activities of higher than 90% were obtained in the concentration range of 8–24 mg l⁻¹ at pH 8.0. The flocculation could be stimulated by cations Ca²⁺, Zn²⁺, Fe²⁺, Al³⁺, and Fe³⁺. In addition, the probable flocculation mechanisms were proposed.