Scale-up of biopesticide production processes using wastewater sludge as a raw material

Studies were conducted on the production of Bacillus thuringiensis (Bt)-based biopesticides to ascertain the performance of the process in shake flasks, and in two geometrically similar fermentors (15 and 150 l) utilizing wastewater sludge as a raw material. The results showed that it was possible to achieve better oxygen transfer in the larger capacity fermentor. Viable cell counts increased by 38–55% in the bioreactor compared to shake flasks. As for spore counts, an increase of 25% was observed when changing from shake flask to fermentor experiments. Spore counts were unchanged in bench (15 l) and pilot scale (5.3–5.5 e⁺⁰⁸ cfu/ml; 150 l). An improvement of 30% in the entomotoxicity potential was obtained at pilot scale. Protease activity increased by two to four times at bench and pilot scale, respectively, compared to the maximum activity obtained in shake flasks. The maximum protease activity (4.1 IU/ml) was obtained in pilot scale due to better oxygen transfer. The Bt fermentation process using sludge as raw material was successfully scaled up and resulted in high productivity for toxin protein yield and a high protease activity.     

In vitro production of pectolytic and cellulolytic enzymes byColletotrichum lindemuthianum isolated from soybean grown in Saudi Arabia

Colletotrichum lindemuthianum isolated from soybean in Saudi Arabia produced polygalacturonase, pectin methylesterase, pectin trans-eliminase and carboxymethylcellulasein vitro. Polygalacturonase showed maximaum activity at 30 to 35°C and pH 4.0 to 5.0. The absorption maximum for pectin trans-eliminase reaction products was at approximately 548 nm. The polygalacturonase and pectin trans-eliminase activities increased with culture age. The degradation of carboxymethylcellulose was also demonstrated.     

Pectolytic enzymes activity and pathogenicity ofGaeumannomyces graminis var.tritici and related Phialophora-like fungi

Gaeumannmyces graminis var.tritici (Ggt), Phialophora sp. (lobed hyphopodia) andPhialophora graminicola vere grown in a liquid medium with pectin and on autoclaved wheat roots (root media) and the activity of pectolytic enzymes in culture filtrates was measured. Most strains of the fungi exhibited polygalacturonate trans-eliminase activity but no pectin methylesterase activity was detected.Ggt polygalacturonase was found in culture filtrates from all the media used whilePhialophora sp. did not exhibit activity of this enzyme in the unbuffered root media. No polygalacturonase activity was demonstrated forP. graminicola. A correlation was found (r=0.548) betweenin vitro polygalacturonase activity and the pathogenicity ofGgt to wheat seedlings.     

Culture conditions for the production of pectinolytic enzymes by the white-rot fungus Trametes trogii on a laboratory scale

Different cultural parameters that regulate pectinolytic enzyme production in vitro by Trametes trogii were studied. When grown in a medium containing pectin, T. trogii produced extracellular polymethylgalacturonase, polygalacturonase and pectin lyase but no pectate lyase activity. No significant differences in the maximum enzyme activities measured were observed with the addition of xylan, carboxymethylcellulose or both to the medium containing pectin. The addition of glucose to that medium considerably decreases all the activities studied, and in a medium with glucose as the sole carbon source no galacturonase activity could be measured, and pectin lyase activity was at its minimum. The low synthesis of pectin lyase in cultures containing glucose suggests that this enzyme is constitutive in contrast to the polygalacturonases that were not detected. The increase in pectin concentration stimulated growth and enzyme production. The highest specific activities were attained with the greatest concentration tested (15 g/l). Casamino acids were the best nitrogen source for enzyme production. Maximum growth was measured at pH 3.3; pH values of around 4.5 stimulated enzyme production, but high pectinase activities were also detected in media with more alkaline initial pH values (6.2 for galacturonases and 6.6 for lyases), probably owing to the specific induction of particular isoforms. In the range of 23 to 28°C, good results were obtained in growth as well as in enzyme production. The addition of Tween 80 promoted growth and gave the highest yield of polymethylgalacturonase and pectin lyase (0.37 and 36.2 E.U./ml, respectively). The highest polygalacturonase activity (1.1 E.U/ml) was achieved with polyethylene glycol. Tween 20 and Triton X-100 inhibited growth and pectinase production.     

Production of polygalacturonase from <Emphasis Type="Italic">Coriolus versicolor</Emphasis> grown on tomato pomace and its chromatographic behaviour on immobilized metal chelates

Tomato pomace and pectin were used as the sole carbon sources for the production of polygalacturonase from a strain of Coriolus versicolor in submerged culture. The culture of C. versicolor grown on tomato pomace exhibited a peak of polygalacturonase activity (1,427 U/l) on the third day of culture with a specific activity of 14.5 U/mg protein. The production of polygalacturonase by C. versicolor grown on pectin as a sole carbon source increased with the time of cultivation, reaching a maximum activity of 3,207 U/l of fermentation broth with a specific activity of 248 U/mg protein. The levels of different isoenzymes of polygalacturonase produced during the culture growth were analysed by native PAGE. Differential chromatographic behaviour of lignocellulosic enzymes produced by C. versicolor (i.e. polygalacturonase, xylanase and laccase) was studied on immobilized metal chelates. The effect of ligand concentration, pH, the length of spacer arm and the nature of metal ion were studied for enzyme adsorption on immobilized metal affinity chromatography (IMAC). The adsorption of these lignocellulosic enzymes onto immobilized metal chelates was pH-dependent since an increase in protein adsorption was observed as the pH was increased from 6.0 to 8.0. The adsorption of polygalacturonase as well as other enzymes to immobilized metal chelates was due to coordination of histidine residues which are available at the protein surface since the presence of imidazole in the equilibration buffer abolished the adsorption of the enzyme to immobilized metal chelates. A one-step purification of polygalacturonase from C. versicolor was devised by using a column of Sepharose 6B-EPI 30-IDA-Cu(II) and purified enzyme exhibited a specific activity of about 150 U/mg protein, final recovery of enzyme activity of 100% and a purification factor of about 10. The use of short spacer arm and the presence of imidazole in equilibration buffer exhibited a higher selectivity for purification of polygalacturonase on this column with a high purification factor. The purified enzyme preparation was analysed by SDS-PAGE as well as by "in situ" detection of enzyme activity.     

Implication of <Emphasis Type="Italic">Bacillus</Emphasis> sp. in the production of pectinolytic enzymes during cocoa fermentation

The role of bacilli in cocoa fermentation is not well known. Their potential of production of pectinolytic enzymes during this process was evaluated. Bacillus growth was monitored and pectinolytic strains were screened for their use of pectin as sole carbon source. Effects of cocoa fermentation parameters susceptible to influence on enzyme production were analysed. Among 98 strains isolated, 90 were positive for pectin degradation and 80% of them presented detectable pectinolytic activities in submerged fermentation. Forty-eight strains produced polygalacturonase (PG), 47 yielded pectin lyase (PL) and 23 strains produced both enzymes. Bacilli growth was not significantly affected during fermentation. PL production was favoured by galactose, lactose, glucose as sugars, and arginine, glutamine, cysteine and ammonium sulphate as nitrogen compounds. Pectin at low concentration (0.05%) and iron stimulated PL production. It was strongly repressed by galacturonic acid (1%), and negatively affected by nitrogen starvation, zinc and temperatures above 45°C. PL yield was very weak below pH 4.0 and in anaerobic conditions. PG production was weakened by sucrose and cation depletion. It was increased slightly by cysteine, ammonium nitrate and nitrogen starvation and significantly above 40°C. PG synthesis was not affected by acidic pH (3.0–6.0) or oxygen availability. As fermentation products, lactate and acetate lowered the production of both enzymes while ethanol had no effect. The high proportion of pectinolytic producers among the strains studied and analysis of factors influencing pectinolytic enzymes production, suggest that Bacillus sp. is liable to produce at least one enzyme during cocoa fermentation.     

Production of pectinesterase and polygalacturonase by Aspergillus niger in submerged and solid state systems

Production of pectinesterase and polygalacturonase by Aspergillus niger was studied in submerged and solid-state fermentation systems. With pectin as a sole carbon source, pectinesterase and polygalacturonase production were four and six times higher respectively in a solid state system than in a submerged fermentation system and required a shorter time for enzyme production. The addition of glucose increased pectinesterase and polygalacturonase production in the solid state system but in submerged fermentation the production was markedly inhibited. A comparison of enzyme productivities showed that those determined for pectinesterase and polygalacturonase with pectin as a carbon source were three and five times higher by using the solid state rather than the submerged fermentation system. The productivities of the two enzymes were affected by glucose in both fermentation systems. The membranes of cells from the solid state fermentation showed increased levels of C18:1, C16:0 and C18:0 fatty acids. Differences in the regulation of enzyme synthesis by Aspergillus niger depended on the fermentation system, favoring the solid state over the submerged fermentation for pectinase production. Received 12 May 1997/ Accepted in revised form 19 September 1997     

Influence of culture conditions on thermostable lipase production by a thermophilic alkalitolerant strain ofBacillus sp

The effect of different culture conditions on thermostable lipase production byBacillus sp. was studied in shake flasks. A maximum enzyme activity of 67–75 nkat/mL was observed in a medium consisting of 0.5% soybean flour and 0.1% stearyl glycerol esters or natural fats. A lipase activity of about 117 nkat/mL was established when the cultivation was carried out in laboratory fermentor at 20% minimal dissolved oxygen level, the enzyme production being increased 1.5 fold compared to that in a flask culture.     

Changes in alginate molecular mass distributions, broth viscosity and morphology of Azotobacter vinelandii cultured in shake flasks

The effect of different aeration conditions during the culture of Azotobacter vinelandii on the production and molecular mass of alginate was evaluated in shake flasks. In baffled flasks, the bacteria grew faster and produced less alginate (1.5 g/l) than in conventional (unbaffled) flasks (4.5 g/l). The viscosity of the culture broth was also influenced by the type of flask. Higher final viscosities were attained in unbaffled flasks [520 cP (520 mPa s)] as compared to baffled flasks (30 cP). This latter phenomenon was closely related to the changes in the molecular mass distribution. In either cases, the mean molecular mass increased with culture age; however, at the end of the fermentation, the mean molecular mass of the alginate obtained in unbaffled flasks was fivefold higher than that obtained in baffled flasks. As the culture proceeded, the cells of Azotobacter grown in unbaffled flasks increased in diameter, whereas those cultured in baffled flasks decreased in size. Received: 13 December 1996 / Received revision: 10 April 1997 / Accepted: 27 April 1997     

Purification and characterization of an exo-polygalacturonase from Pycnoporus sanguineus

The present work describes the purification and characterization of a novel extracellular polygalacturonase, PGase I, produced by Pycnoporus sanguineus when grown on citrus fruit pectin. This substrate gave enhanced enzyme production as compared to sucrose and lactose. PGase I is an exocellular enzyme releasing galacturonic acid as its principal hydrolysis product as determined by TLC and orcinol-sulphuric acid staining. Its capacity to hydrolyze digalacturonate identified PGase I as an exo-polygalacturonase. SDS-PAGE showed that PGase I is an N-glycosidated monomer. The enzyme has a molecular mass of 42 kDa, optimum pH 4.8 and stability between pH 3.8 and 8.0. A temperature optimum was observed at 50–60 °C, with some enzyme activity retained up to 80 °C. Its activation energy was 5.352 cal mol⁻¹. PGase I showed a higher affinity towards PGA than citric pectin (Km = 0.55 ± 0.02 and 0.72 ± 0.02 mg ml⁻¹, respectively). Consequently, PGase I is an exo-PGase, EC 3.2.1.82.     

Growth and laccase production by Pleurotus ostreatus in submerged and solid-state fermentation

Pleurotus ostreatus showed atypical laccase production in submerged vs. solid-state fermentation. Cultures grown in submerged fermentation produced laccase at 13,000 U l⁻¹, with a biomass production of 5.6 g l⁻¹ and four laccase isoforms. However, cultures grown in solid-state fermentation had a much lower laccase activity of 2,430 U l⁻¹, biomass production of 4.5 g l⁻¹, and three laccase isoforms. These results show that P. ostreatus performs much better in submerged fermentation than in solid-state fermentation. This is the first report that shows such atypical behavior in the production of extracellular laccases by fungi.     

Exopolysaccharides production in Lactobacillus bulgaricus and Lactobacillus casei exploiting microfiltration

The physiology of Lactobacillus delbrueckii ssp. bulgaricus and Lactobacillus casei, extensively used in the dairy industry, was studied in order to evaluate key parameters in the synthesis of exopolysaccharides and to improve their production through novel fermentation processes. Selected strains were studied in shake flasks and in fermentor experiments using glucose and lactose as main carbon sources and bacto casitone as the only complex component, in a temperature range between 35 and 42°C. The production of exopolysaccharides was monitored and correlated to the growth conditions using both a colorimetric assay and chromatographic methods. Fermentor experiments in batch mode yielded 100 mg l⁻¹ of EPS from L. bulgaricus and 350 mg l⁻¹ from L. casei. Moreover, the use of a microfiltration (MF) bioreactor resulted in exopolysaccharides (EPS) concentrations threefold and sixfold those of batch experiments, respectively. The monosaccharidic composition of the two analyzed polymers differed from those previously reported. The optimization of the production of EPSs using the MF fermentation strategy could permit the use of these molecules produced by generally recognised as safe (GRAS) microorganisms in the place of other polysaccharides in the food industry.     

Growth conditions of Aspergillus sp. ATHUM-3482 for polygalacturonase production

A wild type of Aspergillus sp. ATHUM-3482 produced extracellular polygalacturonase when grown in liquid medium containing citrus pectin as sole carbon source. A number of factors affecting enzyme activity were investigated. Polygalacturonase activities as high as␣4.3 U␣ml⁻¹(reducing-group-releasing activity) and 17␣U␣ml⁻¹ (viscosity-diminishing activity) were obtained under optimum growth conditions. With sugar-beet as sole carbon source the respective activities were 6.5 U␣ml⁻¹ and 40 U ml⁻¹, the highest achieved in this work. Under these conditions no pectin lyase or pectinesterase activity was detected. The above yields of polygalacturonase activity compare favourably with those reported for fungi grown under similar growth conditions. Received: 5 March 1996 / Received last revision: 29 October 1996 / Accepted: 2 November 1996     

A study of oxygen transfer in shake flasks using a non-invasive oxygen sensor

We describe a study of oxygen transfer in shake flasks using a non-invasive optical sensor. This study investigates the effect of different plugs, presence of baffles, and the type of media on the dissolved oxygen profiles during Escherichia coli fermentation. We measured the volumetric mass transfer coefficient (k(L)a) under various conditions and also the resistances of the various plugs. Finally, we compared shake flask k(L)a with that from a stirred tank fermentor. By matching k(L)a's we were able to obtain similar growth and recombinant protein product formation kinetics in both a fermentor and a shake flask. These results provide a quantitative comparison of fermentations in a shake flask vs. a bench-scale fermentor and should be valuable in guiding scale-up efforts.     

Induction of polygalacturonase and the formation of oxalic acid by pectin in brown-rot fungi

Extracellular polygalacturonase (PG) production was estimated in vitro, using liquid cultures of three species of brown-rot decay fungi (Postia placenta, Gloeophyllum trabeum and Serpula incrassata), by cup-plate assay, assay of reducing sugars, and decrease in viscosity. Although all three experimental assays demonstrated that PG was induced by pectin in all three fungi, decrease in viscosity gave the best correlation with decay capacity in soil block tests. PG activity, determined as an increase in reducing sugar activity, was greatest in G. trabeum and weakest in S. incrassata. The optimum pH for PG activity was between pH 2.5 and 4.5. Oxalic acid production was also enhanced by pectin and functioned synergistically with PG activity. We conclude that these fungi produce PG that is best induced by pectin and that PG activity exceeds production of xylanase and endoglucanase activity in vitro. Polygalacturonase is likely to act synergistically with oxalic acid to solubilize and hydrolyse the pectin in pit membranes and middle lamellae. Thus, production of PG and oxalic acid should facilitate early spread of hyphae and enhance the lateral flow of wood-decay enzymes and agents into adjacent tracheids and the wood cell wall, thus initiating the diffuse decay caused by brown-rot fungi.The Forest Products Laboratory is maintained in co-operation with the University of Wisconsin. This article was written and prepared by US Government employees on official time, and it is therefore in the public domain and not subject to copyright.     

Poly(β-hydroxybutyrate) production by a moderate halophile,Halomonas boliviensis LC1

The moderate halophile Halomonas boliviensis, isolated from a Bolivian saline soil sample, was able to accumulate poly(β-hydroxybutyrate) (PHB) when grown under conditions of nutrient limitation and excess carbon source. The concentration of sodium chloride in the medium influenced the cell-growth, -size, and rate of PHB accumulation. Cultivation in shake flasks led to a PHB accumulation of about 54 wt.% with respect to cell dry weight at 4.5% (w/v) NaCl in a medium with butyric acid and sodium acetate as carbon sources. The production of PHB was substantially improved to a maximum value of 88 wt.% during cultivation under controlled conditions of pH and oxygen concentration in a fermentor. The use of glucose and sucrose, respectively, as carbon source could also lead to the production of PHB at an average level of 55 wt.%.     

Optimization of process parameters for the production of carbonyl reductase by <Emphasis Type="Italic">Candida viswanathii</Emphasis> in a laboratory-scale fermentor

The effect of pH, aeration and mixing on the growth and production of carbonyl reductase by Candida viswanathii was investigated in a 6.6-l fermentor. Controlling the pH at 8.0 had a very significant effect on the enzyme production. Aeration and agitation influenced the dissolved oxygen concentration which in turn affected growth as well as enzyme production. A maximum carbonyl reductase activity (53 Umg⁻¹) was attained in 24 h under the optimal cultivation conditions of controlled pH at 8.0, aeration rate 1 vvm and an agitation speed of 250 rpm at 25°C. The enzyme activity was twice as high (56 Umg⁻¹) in the fermentor as compared to a shake flask. Further, the duration of growth and enzyme production in the fermentor was shortened. Cells cultivated under the optimized conditions were used for the preparative scale reduction of N, N-dimethyl-(3-keto)-2-thienyl-propanamine to (S)-N, N-dimethyl-(3-hydroxy)-2-thienyl-propanamine, a key intermediate in the production of the important antidepressant drug (S)-duloxetine.     

High cell density fermentation of <Emphasis Type="Italic">Gluconobacter oxydans</Emphasis> DSM 2003 for glycolic acid production

Gluconobacter oxydans has a lower biomass yield. Uniform design (UD) was applied to determine the optimum composition of the critical media and their mutual interactions for increased biomass yield of Gluconobacter oxydans DSM 2003 in shake flasks. Fed-batch fermentation process for biomass was optimized in a 3.7-l fermentor. By undertaking a preliminary and improved fed-batch fermentation-process strategy, a cell density of 6.0 g/l (DCW) was achieved in 22 h and 14.1 g/l (DCW) in 35 h, which is the highest cell density of G. oxydans produced thus far in a 3.7-l bioreactor. The biomass production was increased by 135% compared with that using the original cultivation strategy. Bioconversion of ethylene glycol to glycolic acid was catalyzed by the resting cells of G. oxydans DSM 2003, and conversion rate reached 86.7% in 48 h. In summary, the approach including high-density fermentation of G. oxydans DSM 2003 and bioconversion process was established and proved to be an effective method for glycolic acid production.