Relation between Citric Acid Production and Respiration Rate of Aspergillus niger in Solid‐State Fermentation

Among the organic acids produced industrially, citric acid is the most important in quantitative terms. Solid‐state fermentation (SSF) has been an alternative method for citric acid production using agro‐industrial residues such as cassava bagasse (CB). The use of CB as a substrate can avoid environmental problems caused by its disposal into the environment. This study was developed to verify the influence of the treated bagasse amount, and consequently, the influence of the gelatinization degree of CB starch on citric acid production by SSF in Erlenmeyer flasks, horizontal drums, and trays. The best results were obtained in a horizontal drum bioreactor using 100 % of treated CB. However, trays showed advantages and good perspectives for large‐scale citric acid production due to economic reasons such as energy costs. A kinetic study was also carried out in order to compare citric acid production in glass columns (laboratory scale) and horizontal drum bioreactors (semi‐pilot scale). This study was accomplished in order to follow the influence of aeration on citric acid accumulation. In addition, the production of CO₂ was evaluated as an indirect method of biomass estimation. Citric acid production was higher in glass columns (309.70 g/kg of dry CB) than in HD bioreactors (268.94 g/kg of dry CB). Finally, it was possible to show that citric acid production was favored by a limited biomass production, which occurred with low aeration rates. Biomass production is related to CO₂ production and as a result, a respirometry analysis could be used for biomass estimation.     

Conidia production ofBeauveria sp. by solid-state fermentation for biocontrol ofIlex paraguariensis caterpillars

Conidia production of Beauveria sp. strain LAG by solid-state fermentation (SSF) using blends of agro-industrial residues (residual potatoes and sugar-cane bagasse) was optimized with respect to cultivation conditions and the composition of substrate mixture in Erlenmeyer flasks and column-type bioreactor. With a blend of 60 % residual potatoes and 40 % sugar-cane bagasse the optimum conditions achieved were: incubation temperature 26 degrees C, initial substrate pH 6, inoculum concentration 10(7) conidia per g substrate; optimal initial moisture of the substrate was 70 % for Erlenmeyer flasks, in column-type bioreactor (with forced aeration) the optimal initial moisture of the substrate was 65 % with airflow of 60 mL/min. The highest production (1.07 x 10(10) conidia per g dry substrate) was achieved after a 10-d fermentation. The conidia were used in laboratory assays against Thelosia camina and Hylesia sp., caterpillars that are serious pests of mate plants. The mortality of T. camina was >90 % 10 d after spraying caterpillars with 1 mL conidia suspension at a concentration 10(5)-10(8)/mL. For Hylesia sp., the mortality was 70 %, 7 d after immersion in the conidia suspension containing 108 conidia per mL. Therefore, the Beauveria sp. LAG can be considered to be an important biocontrol instrument in the prospect of the Integrated Pest Management for mate plants.     

Influence of Dissolved Oxygen Levels on Production of l-Asparaginase and Prodigiosin by Serratia marcescens

The effect of dissolved oxygen concentrations on the behavior of Serratia marcescens and on yields of asparaginase and prodigiosin produced in shaken cultures and in a 55-liter stainless-steel fermentor was studied. A range of oxygen transfer rates was obtained in 500-ml Erlenmeyer flasks by using internal, stainless-steel baffles and by varying the volume of medium per flask, and in the fermentor by high speed agitation (375 rev/min) or low rates of aeration (1.5 volumes of air per volume of broth per min), or both. Dissolved oxygen levels in the fermentation medium were measured with a membrane-type electrode. Peak yields of asparaginase were obtained in unbaffled flasks (3.0 to 3.8 IU/ml) and in the fermentor (2.7 IU/ml) when the level of dissolved oxygen in the culture medium reached zero. A low rate of oxygen transfer was accomplished by limited aeration. Production of prodigiosin required a supply of dissolved oxygen that was obtainable in baffled flasks with a high rate of oxygen transfer and in the fermentor with a combination of high-speed agitation and low-rate aeration. The fermentation proceeded at a more rapid rate and changes in pH and cell populations were accelerated by maintaining high levels of dissolved oxygen in the growth medium.     

Identification of avermectin-high-producing strains by high-throughput screening methods

Avermectins produced by Streptomyces avermitilis are potent against a broad spectrum of nematode and arthropod parasites with low-level side effects on the host organisms. This study was designed to investigate a high-throughput screening strategy for the efficient identification of avermectin high-yield strains. The production protocol was miniaturized in 96 deep-well microplates. UV absorbance at 245 nm was used to monitor avermectin production. A good correlation between fermentation results in both 96 deep-well microplates and conventional Erlenmeyer flasks was observed. With this protocol, the production of avermectins was determined in less than 10 min for a full plate without compromising accuracy. The high-yield strain selected through this protocol was also tested in 360 m³ batch fermentation with 1.6-fold improved outcome. Thus, the development of this protocol is expected to accelerate the selection of superior avermectin-producing strains.     

Screening,production, and characterization of dextranase from Catenovulum sp.

A psychrotolerant dextranase-producing bacterium was isolated from the Gaogong island seacoast near Jiangsu, China. The bacterium, denoted as DP03, was identified as Catenovulum sp. based on its phenotype, biochemical characteristics, and 16S rRNA gene comparison. The optimal enzyme production time, initial pH, temperature, and aeration conditions of strain DP03 were found to be 28 h, 8.0, 30 °C, and 25 % volume of liquid in 100-ml Erlenmeyer flasks, respectively. The ability of 1 % dextran T20 to induce dextranase was investigated. Dextranase from strain DP03 displayed its maximum activity at pH 8.0 and 40 °C and was found to be stable at 30 °C and over a broad range of pH values (pH 6–11). Scanning electron microscopy showed that dextranase from the isolate DP03 could at least partially prevent Streptococcus mutans from forming biofilms on glass coverslips.     

Upstream and Downstream Processing of Lovastatin by Aspergillus terreus

The present study describes the enhanced production and purification of lovastatin by Aspergillus terreus in submerged batch fermentation. The enhancement of lovastatin production from A. terreus was attempted by random mutagenesis using ultraviolet radiations and nitrous acid. UV mutants exhibited increased efficiency for lovastatin production as compared with nitrous acid mutants. Among all the mutants developed, A. terreus UV-4 was found to be the hyper producer of lovastatin. This mutant gave 3.5-fold higher lovastatin production than the wild culture of A. terreus NRRL 265. Various cultural conditions were also optimized for hyper-producing mutant strain. 5 % glucose as carbon source, 1.5 % corn steep liquor as nitrogen source, initial pH value of 6, 120 h of incubation period, and 28 °C of incubation temperature were found as best parameters for higher lovastatin production in shake flasks. Production of lovastatin by wild and mutant strains of A. terreus was also scaled up to laboratory scale fermentor. The fermentation process was conducted at 28 °C, 200 rpm agitation, and 1vvm air flow rate without pH control. After the optimization of cultural conditions in 250 ml Erlenmeyer flasks and scaling up to laboratory scale fermentor, the mutant A. terreus UV-4 gave eightfold higher lovastatin production (3249.95 μg/ml) than its production by wild strain in shake flasks. Purification of lovastatin was carried out by solvent extraction method which yielded 977.1 mg/l of lovastatin with 98.99 % chromatographic purity and 26.76 % recovery. The crystal structure of lovastatin was determined using X-ray diffraction analysis which is first ever reported.     

Cost-effective production of Bacillus thuringiensis by solid-state fermentation

Production of Bacillus thuringiensis (Bt) was standardized on wheat bran based media in 250 ml Erlenmeyer flasks. Scale-up of Bt production on the best medium in plastic tubs with aeration at 8 h intervals starting 16 h after incubation yielded a significant increase in spore count and toxin content of the product. Maximum lysis of Bt cells was obtained by 60 h of incubation at 30 degrees C. This protocol was suitable for production of Bt strains and local isolates. The Bt produced proved highly effective at 0.1% concentration against larvae of castor semilooper, Achaea janata L, resulting in complete mortality by three days in laboratory bioassays. In field trials, the population of castor semilooper larvae on the castor bean crop was reduced significantly by three days after application. The cost for material production of 1 kg of Bt was approximately US dollars 0.70.     

Effects of dietary crude protein and tannic acid on rumen fermentation,rumen microbiota and nutrient digestion in beef cattle

The objectives of the trial were to study the effects of dietary crude protein (CP) and tannic acid (TA) on rumen fermentation, microbiota and nutrient digestion in beef cattle. Eight growing beef cattle (live weight 350 ± 25 kg) were allocated in a 2 × 2 crossover design using two levels of dietary CP [111 g/kg dry matter (DM) and 136 g/kg DM] and two levels of TA (0 and 16.9 g/kg DM) as experimental treatments. Each experimental period lasted 19 d, consisting of 14-d adaptation and 5-d sampling. The impacts of dietary CP and TA on ruminal microbiota were analysed using high-throughput sequencing of 16S rRNA gene. Results indicated that no interactions between dietary CP and TA were found on rumen fermentation and nutrient digestibility. Increasing dietary CP level from 111 to 136 g/kg DM increased the ruminal concentrations of ammonia nitrogen (NH₃-N) (p < 0.01) and improved the CP digestibility (p < 0.001). Adding TA at 16.9 g/kg DM inhibited rumen fermentation and decreased the digestibility of dietary CP (p < 0.001), DM (p < 0.05) and organic matter (p < 0.01). Increasing the dietary CP level or adding TA did not affect the relative abundances of the major bacteria Firmicutes and Proteobacteria at the phylum level and Prevotella_1 and Christensenellaceae_R-7_group at the genus level, even though adding TA increased the Shannon index of the ruminal bacterial community. TA was partly hydrolysed to pyrogallol, gallic acid and resorcinol in rumen fluid and the inhibitory effects of TA on rumen fermentation and nutrient digestibility could have been resulted from the TA metabolites including pyrogallol, gallic acid and resorcinol as well as the protein-binding effect.     

Effect of oxygen supply on the suspension culture of genetically modified tobacco cells.

The effect of oxygen supply on the cultivation of the genetically modified tobacco cells and the formation of a foreign protein, beta-glucuronidase (GUS), was investigated in 250-mL Erlenmeyer flasks, a 5-L stirred tank fermenter, and a 7-L air-lift fermenter. The oxygen supply was varied by using different volumes of medium in the case of the 250-mL Erlenmeyer flask culture or by the different aeration rate in the case of the two types of fermenters tested. Higher oxygen supply stimulated cell growth and increased oxygen consumption rate, the level of phenolics, and GUS productions.     

Growth of myxococci in suspension in liquid media

Growth of different strains of myxococci in various liquid media was studied in Erlenmeyer flasks and in small fermentors. Medium containing a small concentration of agar allowed the myxobacteria to grow in the liquid phase in suspension. The dry weight of the cells increased about 100 to 200%. Substitution of other thickening substances for agar caused increased growth with all of eight tested agents.     

Note: Influence of hydrocarbons on some Claviceps cultures

Some Claviceps strains producing clavine alkaloids were cultured in shaken flasks in two-phase systems consisting of a water-immiscible organic solvent, n -alkanes and an aqueous culture medium. Culture behaviour was different with light and heavy alkanes but no changes in the proportions of individual alkaloids were found. n -Octane caused a stimulation of sclerotia-like growth and of specific biomass productivity. Increasing medium volumes in flasks brought about deterioration of aeration conditions and reduced production. Optimum fermentation results were restored by adding liquid paraffin to the culture. Heterogeneous systems of the above kinds appear to be suitable for optimization and scale-up of clavine fermentation.     

New Methods for Cultivating,Harvesting, and Purifying Mass Cultures of the Hypotrich Ciliate Euplotes aediculatus1

A new method is described for mass cultivation of Euplotes aediculatus, a hypotrich ciliate containing omikron-symbionts. The ciliate cultures, continuously aerated in Erlenmeyer flasks (5000 ml) with 4500 ml medium, yield densities of 2300 cells/ml which are four to five times higher than cell densities of cultures grown in unaerated Fernbach flasks. Harvesting such cultures involves the application of 25-μm mesh sieves. Cells so concentrated can be purified by using columns or special chambers in which Euplotes migrates towards the cathodes in an electric field (field strength 7 V/cm).     

High-cell-density fermentation studies of a recombinantE. coli that expresses atrial natriuretic factor

Summary Studies are presented on the fermentation of recombinantEscherichia coli that express rat atrial natriuretic factor (ANF) as a fusion protein. Our objective was to achieve high cell density while maintaining ANF expression at the same level as observed in shake flasks. Improved fermentation conditions included: maintaining glucose concentrations at 1 g/l, using an enriched medium, adding concentrates of medium throughout the fermentation, and blending oxygen for adequate aeration. Cell densities of 12 g/l (dry weight) were achieved, which represented a 10-fold increase over non-improved conditions, while maintaining ANF levels at 7 mg/g of dry cell mass. When galactose was used as an initial carbon source or as a feed supplement, there was a 2-3-fold increase in the expression of ANF from these high-cell-density fermentations. The recombinant ANF was biologically active.     

CO2 accumulation in bioreactor suspension cultures of Cyclamen persicum Mill. and its effect on cell growth and regeneration of somatic embryos

CO₂ accumulation in different culture systems containing embryogenic cell suspension cultures of cyclamen (Cyclamen persicum Mill.) was analyzed. In bioreactors equipped with a bubble-free or a bubble aeration system, CO₂ mole fractions in the gas phase of more than 10% were determined whereas in Erlenmeyer flasks, CO₂ mole fractions were below 2%. CO₂ accumulation in bioreactors was severely growth inhibiting in comparison to the flasks. By removing CO₂ in the aeration gas of a bubble-free aerated bioreactor, cell growth comparable to that in flasks was achieved. The regeneration ability of cell suspensions after being cultured in bioreactors with CO₂ accumulation was better than those after culture in bioreactors without CO₂ accumulation or in flasks. Received: 16 June 1998 / Revision received: 13 August 1998 / Accepted: 1 December 1998     

Kinetics of Gibberella fujikuroi growth and gibberellic acid production by solid-state fermentation in a packed-bed column bioreactor

In this work the growth of Gibberella fujikuroi and gibberellic acid (GA3) production were studied using coffee husk and cassava bagasse as substrates in a packed-bed column bioreactor connected to a gas chromatograph for exit gas analysis. With the respirometric data, a logarithmic correlation between accumulated CO2 and biomass production was determined, and the kinetics of the fungal growth was compared for estimated and experimental data. The solid medium consisted of coffee husk (pretreated with alkali solution), mixed with cassava bagasse (7:3 dry weight basis), with a substrate initial pH of 5.2 and moisture of 77%. Cultivation was carried out in glass columns, which were packed with preinoculated substrate and with forced aeration of 0.24 L of air/[h (g of substrate)] for the first 3 days, and 0.72 L of air/[h (g of substrate)] for the remaining period. The maximum specific growth rate (microm) obtained was 0.052 h(-1) (between 24 and 48 h of fermentation). A production of 0.925 g of GA3/kg of substrate was achieved after 6 days of fermentation.     

Production of carotenoids and lipids by <Emphasis Type="Italic">Rhodococcus opacus</Emphasis> PD630 in batch and fed-batch culture

Production of carotenoids by Rhodococcus opacus PD630 is reported. A modified mineral salt medium formulated with glycerol as an inexpensive carbon source was used for the fermentation. Ammonium acetate was the nitrogen source. A dry cell mass concentration of nearly 5.4 g/L could be produced in shake flasks with a carotenoid concentration of 0.54 mg/L. In batch culture in a 5 L bioreactor, without pH control, the maximum dry biomass concentration was ~30 % lower than in shake flasks and the carotenoids concentration was 0.09 mg/L. Both the biomass concentration and the carotenoids concentration could be raised using a fed-batch operation with a feed mixture of ammonium acetate and acetic acid. With this strategy, the final biomass concentration was 8.2 g/L and the carotenoids concentration was 0.20 mg/L in a 10-day fermentation. A control of pH proved to be unnecessary for maximizing the production of carotenoids in this fermentation.     

Ethanol production by coupled saccharification and fermenation of sugar cane bagasse

Summary As initial studies showed that enzymatic saccharification of sugar cane bagasse in columns with recycling of eluate was slightly more efficient than in agitated flasks, ethanol production by fermentation of the eluates with fast-decanting yeast and recycling of the fermentate through the bagasse columns was studied. The alcohol yield from these coupled columns after 24 or 48 h was more than 10% more than that in a simultaneous saccharification and fermentation in agitated flasks at 40°.     

Ethanol fermentation of mahula (Madhuca latifolia L.) flowers using free and immobilized yeast Saccharomyces cerevisiae

There is a growing interest to find alternate bioresources for production of ethanol, apart from cane/sugar beet molasses and starchy crops like sweet sorghum, cassava and sweet potato. Mahula (Madhuca latifolia L.) is a forest tree abundantly available in the Indian subcontinent and its flowers are very rich in fermentable sugars (28.1-36.3 g 100 g(-1)). Batch fermentation of fresh and 12-month-stored flowers with free (whole cells) and immobilized cells of Saccharomyces cerevisiae (strain CTCRI) was carried out in 2-l Erlenmeyer flasks. The ethanol yields were 193 and 148 g kg(-1) (using free cells) and 205 and 152 g kg(-1) (using immobilized cells) from fresh and 12-month-stored mahula flowers, respectively.     

Scaled-up proliferation and regeneration of Nerine in liquid cultures Part I. The induction and maintenance of proliferating meristematic clusters by paclobutrazol in bioreactors

The natural propagation rate of bulb forming Amaryllidaceae including Nerine is low. Conventional micropropagation techniques are labor intensive and therefore expensive. Liquid cultures facilitate: scaling up, automation and cost reduction of micropropagation. Inflorescence-derived explants of Nerine were cultured on 2,4-dichlorophenoxyacetic acid (2,4-d) and 6-benzyladenine (BA) supplemented Murashige & Skoog (MS) medium. Callus-like tissue interspersed with nodular tissue, as well as direct organogenesis developed at the junction between flower pedicel and peduncle. Subculture of nodular tissue to 1-naphthalene acetic acid (NAA), BA and paclobutrazol (PAC) supplemented liquid medium in Erlenmeyer flasks or bubble bioreactors resulted in proliferation of rounded, compact, easily crumbled meristematic clusters. Growth and proliferation in bioreactors were higher than in shaken flasks and were affected differently by the inoculum to medium ratio in the two types of culture vessel. Nerine cultures showed low sensitivity to high aeration rates in bubble bioreactors despite the accumulation of debris. It was therefore possible to increase aeration rates without reducing the proliferation rate or damaging the quality of the meristematic aggregates. The conditions in semi-continuous culture in flasks and bioreactors were more favorable and increased the growth value by 100% and 140%, respectively. The total protein content increased by 180% in flasks and 90% in bioreactors. Although the presence of PAC throughout the culture period decreased growth and proliferation, it was a promotive bioregulator for meristernatic cluster formation. Proembryogenic clusters developed upon the removal of PAC. The use of meristematic clusters for micropropagation in scaled-up liquid cultures is discussed.     

Improved production of pristinamycin coupled with an adsorbent resin in fermentation by Streptomyces pristinaespiralis

Batch fermentation by Streptomyces pristinaespiralis with the addition of adsorbent resins was used to increase the production of pristinamycin. In consideration of the adsorption capacity and the desorption ability, a polymeric resin, JD-1, was finally selected. The maximum production of pristinamycin in Erlenmeyer flasks went up to 1.13 from 0.4 g l⁻¹, by adding 12% (w/v) resin JD-1 into the culture broth at 20 h after inoculation. In a 3 l bioreactor, pristinamycin fermentation with the addition of 12% (w/v) resin JD-1 at 20 h after inoculation reached 0.8 g l⁻¹, which was a 1.25-fold increase over fermentation without resin.