Improvement of culture conditions forl-proline production by a recombinant strain ofSerratia marcescens

Serratia marcescens SP511 was previously reported to be anl-proline-producing strain that harbors a recombinant plasmid carrying the mutant type of the proline operon. This strain produced 65 g/L ofl-proline in a medium containing 22% sucrose and urea after 5 d of incubation under the conventional culture conditions. We searched for more suitable culture conditions for more abundantl-proline production by SP511. To improve the supply of a nitrogen source to cells, ammonium was used instead of urea and fed to a culture under control of the pH of the medium. The concentrations of MgSO₄ and K₂HPO₄ were increased, and in addition, sucrose was continuously added to the culture at a final concentration of 32%. Under these conditions, the cell amount was increased twofold over that under the previous conditions andl-proline production reached a maximum of more than 100 g/L after 4 d of incubation.     

Production of ω-3 polyunsaturated fatty acids from cull potato using an algae culture process

Algal cultivation for converting cull potato to docosahexaenoic acid (DHA) was studied. Schizochytrium limacinum SR21 was selected as the better producing strain, compared with Thraustochytrium aureum because of higher cell density and DHA content. Used as both carbon and nitrogen source, an optimal ratio of hydrolyzed potato broth in the culture medium was determined as 50%, with which the highest production of 21.7 g/L dry algae biomass and 5.35 g/L DHA was obtained, with extra glucose supplemented. Repeat culture further improved the cell density but not fed batch culture, suggesting limited growth was most likely caused by metabolites inhibition.     

Lactase Production by Solid-state Cultivation of Kluyveromyces marxianus CDBBL 278 on an Inert Support: Effect of Inoculum, Buffer, and Nitrogen Source

A study was carried out to select the conditions for cultivation of Kluyveromyces marxianus CDBBL 278 in solid-state culture (SSC) using polyurethane foam (PUF) as an inert support. PUF was impregnated with culture media containing lactose (50 g/L) as the carbon and energy source. Evaluation of culture parameters during different growth phases was carried out by respirometry. The effect of inoculum level, buffer capacity of the medium, and nitrogen source upon the yield of biomass on lactose (Yx/s) and production of lactase and inulinase was investigated. The highest lactase titre was achieved with an inoculum level of 1 x 10(7) cells per gram of wet matter (gwm) and 20% of the total nitrogen source provided as urea. The best biomass yield (0.37) was obtained when less than 40% of the total nitrogen was provided as urea. Using potassium phosphate allowed 90% substrate consumption in 30 h. In the best conditions, intracellular lactase and extracellular inulinase activities of 1147.7 IU/gX and 241.6 IU/gX were obtained, respectively, with a lag phase of 13.8 h and a rate of respiratory activity (microCO2) of 0.23 +/- 0.01 h(-1). To our knowledge, this is the first report on lactase production by K. marxianus CDBBL 278 in SSC. This study gives basic information about biomass yield and enzyme production using lactose as the sole carbon source in SSC on an inert support.     

Nutritional requirements of a strain of bacillus thuringiensis subsp. kurstaki and use of gruel hydrolysate for the formulation of a new medium for δ-endotoxin production

Nutritional requirements of a strain ofBacillus thuringiensis (Bt) subsp.kurstaki were elucidated for δ-endotoxin production. The effect of some principal nutrients was deeply investigated, showing several nutritional and metabolite limitations in Bt growth and δ-endotoxin synthesis. This led us to formulate a new medium based on the hydrolysate of gruel, a cheap and abundant byproduct of semolina factories, supporting growth and δ-endotoxin synthesis. After hydrolysis of gruel by α-amylase, followed by proteolysis using alcalase, the resultant soluble material substituted glucose very well for Bt δ-endotoxin production. Indeed, 15 g/L total sugars coming from that hydrolysate, supplemented by 5.4 g/L ammonium sulfate as nitrogen source and either 5 g/L yeast extract or 3 g/L peptone from casein or 3 g/L casaminoacids or 0.25 g/L cysteine or aspartic acid, were the principal components of this new medium in which almost 1 g/L of δ-endotoxin in 4.5 g/L total dry biomass was produced.     

Efficient production of poly-gamma-glutamic acid by Bacillus subtilis ZJU-7

A strain with high poly-gamma-glutamic acid (gamma-PGA) production was isolated from fermented bean curd, a traditional Chinese food. The strain was named Bacillus subtilis ZJU-7 according to 16s rDNA sequencing and its taxonomic characters. The culture conditions for gamma-PGA production were evaluated. The most suitable carbon and nitrogen sources were sucrose and tryptone, respectively. Exogenous L-glutamic acid was necessary for gamma-PGA production, and the production of gamma-PGA increased on the addition of L-glutamic acid to the medium. In the medium containing 60 g/L of sucrose, 60 g/L of tryptone, 80 g/L of L-glutamic acid, and 10 g/L of NaCl, the yield of gamma-PGA reached 54.4 g/L after cultivation at 37 degrees C for 24 h, which was the highest gamma-PGA production compared with values reported in the literature. The average molecular mass of gamma-PGA produced was about 1.24 x 106 Daltons. B. subtilis ZJU-7 is genetically stable and can synthesize levan instead of gamma-PGA without the addition of L-glutamic acid to the medium.     

Two-Stage Cultivation of Pseudomonas sp. F12 for the Production of Enzymes Converting dl-2-Amino-Δ2-thiazoline-4-carboxylic Acid to l-Cysteine

Pseudomonas sp. F12 isolated from soil could transform dl-2-amino-??²-thiazoline-4-carboxylic acid (DL-ATC) to l-cysteine. It could grow in minimal medium containing DL-ATC as the sole carbon and nitrogen source, and the apparent activity of l-cysteine synthesis (CS) achieved 122?U/mL in a 5-L bioreactor. Pseudomonas sp. F12 could utilize glucose as carbon source and ammonia as nitrogen source for growth, but no CS activity was formed. To reduce the cost of DL-ATC, the cultivation process was divided into a growth stage on glucose and ammonia and a production stage induced by DL-ATC. The excessive glucose led to the production of byproduct(s) which seriously inhibited cell growth and CS production. Ammonium was accumulated when DL-ATC was consumed, and ammonium did not inhibit CS activity formation until 60?mM. Based on the above features, fed-batch cultivation of the growth stage was developed by supplying glucose restrictively. The volumetric CS activity was enhanced more than two times that obtained under the initial conditions.     

Inulinase synthesis from a mesophilic culture in submerged cultivation

A newly isolated mesophilic bacterial strain from dahlia rhizosphere, identified as Staphylococcus sp. and designated as RRL-M-5, was evaluated for inulinase synthesis in submerged cultivation using different carbon sources individually or in combination with inulin as substrate. Inulin appeared as the most favorable substrate at a 0.5–1.0% concentration. Media pH influenced the enzyme synthesis by the bacterial strain, which showed an optimum pH at 7.0–7.5. Supplementation of fermentation medium with external nitrogen (organic and inorganic) showed a mixed impact on bacterial activity of enzyme synthesis. The addition of soybean meal and corn steep solid resulted in about an 11% increase in enzyme titers. Among inorganic nitrogen sources, ammonium sulfate was found to be the most suitable. Maximum enzyme activities (446 U/L) were obtained when fermentation was carried out at 30°C for 24 h with a medium containing 0.5% inulin as a sole carbon source and 0.5% soybean meal as the nitrogen source. Bacterial inulinase could be a good source for the hydrolysis of inulin for the production of d-fructose.     

Biosynthesis of poly-β-hydroxybutyrate and exopolysaccharides on azotobacter chroococcum strain 6B utilizing simple and complex carbon sources

Coproduction of poly-β-hydroxybutyrate (PHB) and exopolysaccharides (EPS) was investigated with Azotobacter chroococcum strain 6B isolated from soil samples. The bacterium was cultured using various carbon sources solely or with 0.1 g/L of ammonium sulfate. Ammonium addition resulted in reduced PHB and EPS production with glucose, fructose, and sucrose media, but cellular mass remained constant except for sucrose. Protein was nearly twofold higher in ammonium-grown cultures. Glucose and fructose alone biosynthesized high amounts of EPS (maximum 2.1 and 1.1 g/L, respectively, at 72 h), whereas PHB was accumulated only in glucose-grown cells. Sucrose almost did not produce EPS. Conversely, PHB content was the highest obtained from all experimented conditions (1.1 g/L at 48 h, 40% cell dry wt). When a complex carbon source such as sugar cane molasses was utilized, PHB was accumulated concomitant with EPS production from the initial time to 48 h (0.75 g/L, 37% cell dry wt and 0.6 g/L, respectively), and then PHB decayed at 72 h (0.2 g/L). On the other hand, EPS continued to be biosynthesized (1.1 g/L, 72 h). PHB fractions of total intra- and extracellular biopolymers were calculated. Sucrose-modified Burk’s medium without ammonium addition is suggested as a medium capable of diverting the carbon source for the production of intracellular PHB rather than EPS with A. chroococcum 6B.     

Xanthan gum production from cassava bagasse hydrolysate with Xanthomonas campestris using alternative sources of nitrogen

Cassava bagasse was hydrolyzed using HCl and the hydrolysate was used for the production of xanthan gum using a bacterial culture of Xanthomonas campestris. Cassava bagasse hydrolysate with an initial concentration of approx 20 g of glucose/L proved to be the best substrate concentration for xanthan gum production. Among the organic and inorganic nitrogen sources tested to supplement the medium—urea, yeast extract, peptone, potassium nitrate, and ammonium sulfate—potassium nitrate was most suitable. Ammonium sulfate was the least effective for xanthan gum production, and it affected sugar utilization by the bacterial culture. In media with an initial sugar concentration of 48.6 and 40.4 g/L, at the end of fermentation about 30 g/L of sugars was unused. Maximum xanthan gum (about 14 g/L) was produced when fermentation was carried out with a medium containing 19.8 g/L of initial reducing sugars supplemented with potassium nitrate and fermented for 72 h, and it remained almost the same until the end of fermentation (i.e., 96 h).     

Production of polyhydroxyalkanoate from sesame seed wastewater by sequencing batch reactor cultivation process of Haloferax mediterranei

Due to the high cost of bioplastic production, sesame wastewater, generated from the sesame seed hulling process, was investigated to be used as inexpensive and renewable carbon source for the production of biodegradable polyhydroxyalkanoate (PHA) by extreme Haloferax mediterranei. The sesame wastewater (SWW) was hydrolyzed using different concentrations of hydrochloric acid (0.4. 1.00 and 2.00 M) at different period of times (15, 60 and 90 min). The concentration of salt (NaCl) and nitrogen source (NH₄Cl and yeast) required for H. mediterranei cells growth and the accumulation of PHA biopolymer was optimized. A maximum 0.53 g/L concentration of PHA was achieved when the SWW extract media was supplemented with 100 g/L NaCl and 6.0 g/L yeast extract. The cultivation was scaled-up using sequencing batch reactor (SBR) fermentation under non-sterile conditions. The SBR results showed that SWW needs an auxiliary carbon source to obtain high PHA production. Consequently, the system fed with SWW and glucose produced higher PHA (20.9 g/L) than the system fed with SWW.     

Bioflocculant Exopolysaccharide Production by Azotobacter indicus Using Flower Extract of Madhuca latifolia L

Efficacy of Azotobacter indicus ATCC 9540 strain for production exopolysaccharide (EPS) bioflocculant was investigated. Mahua flower extract (Madhuca latifolia L), a natural substrate at the concentration of 20 g L^?1, gave maximum recovery of EPS followed by sucrose and mannitol as compared to other carbon sources after 172 h. Yeast extract was found to be the most effective nitrogen source as compared to beef extract, sodium nitrate, ammonium sulfate, casein hydrolysate, and urea for the production of EPS. EPS production was increased in presence of nitrogen (5.51 g L^?1) as compared to nitrogen-free medium (3.51 g L^?1), and fermentation time was also reduced by 28 h. Maximum EPS production (6.10 g L^?1) was found in the presence of 20 g L^?1 flower extract and 0.5 g L^?1 yeast extract containing Ashby’s media with 180 rpm at 30 °C at 144 h, under controlled conditions in 2.5 L fermenter using optimized medium. The isolated EPS showed cation-dependent flocculating activity. Concentration of EPS played an important role in bioflocculating activity which increased in a concentration-dependent manner up to a certain limit, with the maximum flocculation of 72% at 500 mg L^?1 concentration but remained almost static after this concentration. Extracted polymer was characterized by different chemical tests, FT-IR spectroscopy, and TLC which showed presence of uronic acids, O-acetyl groups, and Orcinol with suggestive indication of alginate like polymer. This study suggests that use of M. latifolia L. flowers can be a potential alternative bioresource for production of exopolysaccharide.     

Effects of Different Carbon and Nitrogen Sources on Naphthoquinone Production of Cordyceps unilateralis BCC 1869

The production of six naphthoquinone derivatives, erythrostominone, deoxyerythrostominone, 4-O-methyl erythrostominone, epierythrostominol, deoxyerythrostominol, and 3,5,8-trihydroxy-6-methoxy-2-(5-oxohexa- 1,3-dienyl)-1,4-naphthoquinone, was examined during the growth of Cordyceps unilateralis BCC 1869 on different carbon and nitrogen sources. Erythrostominone production by the fungus accounted for more than 50% of total naphthoquinones, but production of each of the other five derivatives accounted for less than 20% of total naphthoquinones. The highest volumetric production rate of erythrostominone and overall naphthoquinone production rate were obtained on mannose as a sole carbon source and ammonium sulfate as a sole nitrogen source (4922.4 +/- 118.8 mg/[L.d] and 5.03 g/[L.d], respectively). The highest growth rate was obtained on arabinose (0.043 h-1), whereas the maximum overall naphthoquinone concentration was obtained on lactose (2 g/L) at 237 h. These naphthoquinones were produced with no pH control and were first detected at a pH of about 3.0 to 4.0. These results suggest that carbon and nitrogen influenced directly the production of naphthoquinones.     

Evaluation of the Use of Elicitors for the Production of Antioxidant Compounds in Liquid Cultures of Ganoderma curtisii from Costa Rica

The use of substances or conditions as elicitors can significantly increase the production of secondary metabolites. In this research, the effects of different elicitors on the production of antioxidant secondary metabolites were evaluated in a strain of Ganoderma sp. The elicitors tested were pH changes in different growth phases of the fungus (pH 3, 5.5 and 8), different concentrations of peptone as a nitrogen source (1 g/L and 10 g/L), and the addition of chemical agents to the culture medium (ethanol, growth regulators, and salts). The alkaline pH during the stationary phase and the high availability of nitrogen were effective elicitors, producing cultures with higher antioxidant activity (37.87 g/L and 43.13 g/L dry biomass) although there were no significant differences with other treatments.     

Fed-Batch Strategies for Production of PHA Using a Native Isolate of <Emphasis Type="Italic">Halomonas venusta</Emphasis> KT832796 Strain

In this study, polyhydroxyalkanoates (PHA) accumulation by Halomonas venusta KT832796, a moderate halophilic bacteria isolated from marine source was studied. Both nutritional requirements and process parameters for submerged cultivation of the organism in bioreactor have been standardized. From the shake flask studies, glucose and ammonium citrate as carbon and nitrogen source produced maximum PHA at a ratio 20 with 3.52 g/L of dry cell weight and 70.56% of PHA content. However, ammonium sulfate as the nitrogen source was found to be more suitable for fed-batch cultivation. Several feeding strategies including pH-based fed-batch and variants of pulse feeding were studied to improve the PHA levels. pH-based feeding, although improved PHA level to 26 g/L, most of the carbon flux was diverted towards biomass formation; hence, the percent PHA was only 39.15% of the dry cell weight. Maximum PHA of 33.4 g/L, which corresponded to 88.12% of the dry cell, was obtained from high concentration single pulse method. There was a net 8.65-fold increase in PHA using this feeding strategy when compared to batch studies. According to our knowledge, this is the highest amount of PHA reported for a Halomonas venusta strain.     

Isolation of PHA–Producing Bacteria from Date Syrup Waste

Since the major problem associated with the industrial production of Polyhydroxyalkanoates (PHAs) is their high production cost, this study was carried out using date syrup as the major carbon source to decrease the production cost and also help to supply other nutrient requirements. To isolate PHA–producing bacteria for this purpose, microorganisms were isolated from the syrup waste of a local date factory. These purified colonies were screened for intracellular granules by staining with Sudan Black. The positive-staining strains were grown for production of PHAs in 5% date syrup as carbon source supplemented with mineral salt medium. The culture was incubated at 30 °C with shaking at 140 rpm for 60 h. Among positively stained bacteria, the best PHA producers were selected on the basis of cell growth, cell dry weight, PHA content and the monomer composition of PHA. One of them could utilize date syrup for growth and produce the homopolymer of Polyhydroxybutyrate (PHB) with a cell density of about 5.1 g/L and maximum concentration of PHB equal to 3.6 g/L which is 71% of cell dry weight. Another one produces copolymer of Poly (hydroxybutyrate-hydroxyvalerate) in date syrup media with a maximum concentration of 2.2 g/L containing 10 wt % valerate in shake flask cultivation.     

Isolation and characterization of a novel feather-degrading bacterial strain

Feather waste, generated in large quantities as a byproduct of commercial poultry processing, is almost pure keratin, which is not easily degradable by common proteolytic enzymes. Feather-degrading bacteria were isolated from a Brazilian poultry industrial waste. Among these isolates, a strain identified as kr2 was the best feather-degrading organism when grown on basal medium containing 10 g/L of native feather as a source of energy, carbon, and nitrogen. The isolate was characterized according to morphological characteristics and biochemical tests belonging to the Vibrionaceae family. Keratinolytic activity of this isolate was monitored throughout the cultivation of the bacterium on raw feather at different temperatures. The optimum temperature for growth was about 30°C, at which maximum enzyme and soluble protein production were achieved. The enzyme had a pH and temperature optima of 8.0 and 55°C, respectively.     

Astaxanthinogenesis in the yeastPhaffia rhodozyma

Astaxanthin is a diketo-dihydroxy-carotenoid produced byPhaffia rhodozyma, a basidiomicetous yeast. A low-cost fermentation medium consisting of raw sugarcane juice and urea was developed to exploit the active sucrolytic/urelolytic enzyme apparatus inherent to the yeast. As compared to the beneficial effect of 0.1 g% urea, a ready nitrogen source, mild phosphoric pre inversion of juice sucrose to glucose and fructose, promptly fermentable carbon sources, resulted in smaller benefits. Corn steep liquor (CSL) was found to be a valuable supplement for both yeast biomass yield (9.2 g dry cells/L) and astaxanthin production (1.3 mg/g cells). Distillery effluent (vinace), despite only a slightly positive effect on yeast growth, allowed for the highest pigment productivity (1.9 mg/g cells). Trace amounts of Ni² (1 mg/L, as a cofactor for urease) resulted in controversial effects, namely, biomass decrease and astaxanthin increase, with no effect on the release (and uptake) of ammonium ion from urea. Since the synthesized astaxanthin is associated with the yeast cell and the pigment requires facilitated release for aquaculture uses (farmed fish meat staining), an investigation of the yeast cell wall was undertaken using detergent-treated cells. The composition of the rigid yeast envelope was found to be heterogeneous. Its partial acid or enzymatic depolymerization revealed glucose and xylose as common monomeric units of the cell-wall glycopolymers. Yeast cell-wall partial depolymerization with appropriate hydrolases may improve the pigment bioavailability for captive aquatic species and poultry.

     

Efficient production of poly-γ-glutamic acid by bacillus subtilis ZJU-7

A strain with high poly-γ-glutamic acid (γ-PGA) production was isolated from fermented bean curd, a traditional Chinese food. The strain was named Bacillus subtilis ZJU-7 according to 16s rDNA sequencing and its taxonomic characters. The culture conditions for γ-PGA production were evaluated. The most suitable carbon and nitrogen sources were sucrose and tryptone, respectively. Exogenous l-glutamic acid was necessary for γ-PGA production, and the production of γ-PGA increased on the addition of l-glutamic acid to the medium. In the medium containing 60 g/L of sucrose, 60 g/L of tryptone, 80 g/L of l-glutamic acid, and 10 g/L of NaCl, the yield of γ-PGA reached 54.4 g/L after cultivation at 37°C for 24h, which was the highest γ-PGA production compared with values reported in the literature. The average molecular mass of γ-PGA produced was about 1.24×10⁶ Daltons. B. subtilis ZJU-7 is genetically stable and can synthesize levan instead of γ-PGA without the addition of l-glutamic acid to the medium.     

Dielectric Barrier Discharge for Ammonia Production

The leading after-treatment technology for NOx removal process in Diesel engines for stationary and mobile applications is the selective catalytic reduction of oxides of nitrogen [NOx] by ammonia [NH₃]. A novel non-thermal plasma electrode with a needle array in a dielectric barrier discharge reactor, powered by a high frequency neon transformer, is used for the thermal decomposition of solid urea [(NH₂)CO(NH₂)] to produce ammonia. The thermolysis of urea produces iso-cyanic acid [HNCO] as a byproduct, besides ammonia, which can react with water in the gas phase, thus giving carbon dioxide and more ammonia. The presence of water fed before and/or after the plasma reactor was studied to assess its effect on the amount of produced ammonia. Results clearly showed that water fed to the entrance of the reactor can efficiently promote the reaction of iso-cyanic acid to produce ammonia and this result can be improved when air is used as carrier gas for 115 V of input voltage to a neon transformer and with a gas flow rate of 4 L/min.     

Biosynthesis of Unsaturated Fatty Acids via Mortierella Isabellina Cultivated in a Medium Containing Butanol

IntroductionThe biosynthesis of unsaturated fatty acidshasattracted more attention in recent years. Forexample,linoleic and linolenic acids are importantmaterials for pharmaceutical and food industries.Besides,they can be used to synthesize paint,printing ink and surfactant,etc. A number ofmicroorganisms have been studied as potentialcommercial sources to produce unsaturated fattyacids[1] .Agricultural and industrial products,by-products,etc. have been employed in thecultivation of those orga…