We describe the simultaneous production of Bacillus subtilis based proteases and alpha amylase using a computer controlled laboratory scale 7.5 L batch bioreactor. The present strain is the first to be reported that concomitantly produces these two industrially important enzymes. The growth and sporulation of Bacillus subtilis was monitored and maximum production of alkaline protease and alpha amylase was found to coincide with maximum sporulation. Two types of proteases were detected in the fermentation broth; a neutral and an alkaline protease most active in a pH range of 7.0-8.0 and 8.0-10, respectively. Maximum production of proteases was observed at an incubation temperature of 37ºC while that of alpha amylase was observed at 40ºC. The optimum aeration and agitation levels for protease production were 0.6 L/L/min and 200rpm, respectively, and for alpha amylase were 0.6 L/L/min and 150 rpm. The kinetic parameters Yp/x and qp were also found to be significant at the given fermentation conditions.

The purpose of the current study was intended to obtain the enhanced production of bacitracin by Bacillus licheniformis through random mutagenesis and optimization of various parameters. Several isolates of Bacillus licheniformis were isolated from local habitat and isolate designated as GP-35 produced maximum bacitracin production (14±0.72 IU ml-1). Bacitracin production of Bacillus licheniformis GP-35 was increased to 23±0.69 IU ml-1 after treatment with ultraviolet (UV) radiations. Similarly, treatment of vegetative cells of GP-35 with chemicals like N-methyl N'-nitro N-nitroso guanidine (MNNG) and Nitrous acid (HNO2) increased the bacitracin production to a level of 31±1.35 IU ml-1 and 27±0.89 IU ml-1 respectively. Treatment of isolate GP-35 with combined effect of UV and chemical treatment yield significantly higher titers of bacitracin with maximum bacitracin production of 41.6±0.92 IU ml-1. Production of bacitracin was further enhanced (59.1±1.35 IU ml-1) by optimization of different parameters like phosphate sources, organic acids as well as temperature and pH. An increase of 4.22 fold in the production of bacitracin after mutagenesis and optimization of various parameters was achieved in comparison to wild type. Mutant strain was highly stable and produced consistent yield of bacitracin even after 15 generations. On the basis of kinetic variables, notably Yp/s (IU/g substrate), Yp/x (IU/g cells), Yx/s (g/g), Yp/s, mutant strain B. licheniformis UV-MN-HN-6 was found to be a hyperproducer of bacitracin.

The present investigation deals with the kinetics of submerged extracellular lipases fermentation by both wild and mutant strains of Rhizopus oligosporus var. microsporus in a laboratory scale stirred fermentor. Other parameters studied were inoculum size, pH, agitation and rate of aeration. It was found that the growth and lipases production was increased gradually and reached its maximum 9.07± 0.42ª U mL-1 (W) and 42.49 ± 3.91ª U mL-1 (M) after 30h of fermentation for both wild and mutant strain. There is overall increase of 109% (W) and 124% (M) in the production of extracellular lipases as compared to shake flask. Another significant finding of the present study is that the fermentation period is reduced to 30 h in case of wild and 23 h in case of mutant from 48 h in shake flask studies. The specific productivity of mutant strain (qp = 377.3 U/g cells/h) was several folds higher than wild strain. The specific production rate and growth coefficient revealed the hyperproducibility of extracellular lipases using mutant IIB-63NTG-7.

A produção de L-DOPA a partir de tirosina pela cepa mutante de Aspergillus orizae UV-7 foi melhorada através de mutação química. Diferentes cepas foram testadas quanto a produção de L-DOPA por fermentação submersa, observando-se que a cepa denominada SI-12 foi a melhor produtora (300 mg de L-DOPA por g de células). A produção de L-DOPA pela cepa mutante a partir de diferentes fontes de carbono foi testada em diferentes fontes de nitrogênio, pH inicial e temperatura. Em pH ótimo (5,0) e temperatura ótima (30ºC), todos os açúcares foram utilizados para formação de biomassa, com um rendimento de L-DOPA de 150 mg.g-1, e produtividade volumétrica máxima e especifica de 125 mg.l.h-1 e 150 mg.g-1.h-1, respectivamente. A velocidade de formação do produto aumentou 3 vezes, sendo esse aumento o maior já relatado na literatura. Para explicar o mecanismo cinético da formação de L-DOPA e a inativação térmica da tirosinase, os parâmetros termodinâmicos foram determinados aplicando-se o modelo de Arrhenius: no caso da cepa mutante, a entalpia de ativação e entropia foram 40kj/mol e 0,076 kj/mol.K para produção de L-DOPA e 116 kj/mol and 0,590 kj/mol.K para inativação térmica, respectivamente. Os valores para formação do produto foram mais baixos e os para desativação do produto foram mais elevados que os valores correspondentes à cultura parental, indicando que a cepa mutante foi termodinamicamente mais resistente à denaturação térmica.

Aspergillus oryzae mutant strain UV-7 was further improved for the production of L-DOPA from L-tyrosine using chemical mutation. Different putative mutant strains of organism were tested for the production of L-DOPA in submerged fermentation. Among these putative mutant strains, mutant designated SI-12 gave maximum production of L-DOPA (300 mg L-DOPA.g-1 cells). The production of L-DOPA from different carbon source solutions (So= 30 g.l-1) by mutant culture was investigated at different nitrogen sources, initial pH and temperature values. At optimum pH (pHo= 5.0), and temperature (t=30ºC), 100% sugars were utilized for production and cell mass formation, corresponding to final L-DOPA product yield of 150 mg.g-1 substrate utilized, and maximum volumetric and specific productivities of 125 mg.l-1.h-1, and 150 mg.g-1 cells. h-1, respectively. There was up to 3-fold enhancement in product formation rate. This enhancement is the highest reported in literature. To explain the kinetic mechanism of L-DOPA formation and thermal inactivation of tyrosinase, the thermodynamic parameters were determined with the application of Arrhenius model: activation enthalpy and entropy for product formation, in case of mutant derivative, were 40 k j/mol and 0.076 k j/mol. K for L-DOPA production and 116 k j/mol and 0.590 k j/mol. K for thermal inactivation, respectively. The respective values for product formation were lower while those for product deactivation were higher than the respective values for the parental culture. Therefore, the mutant strain was thermodynamically more resistant to thermal denaturation.

The present investigation deals with the kinetics of submerged citric acid fermentation by Aspergillus niger using blackstrap molasses as the basal fermentation media. A laboratory scale stirred fermentor of 15-L capacity having working volume of 9-L was used for cultivation process and nutritional analysis. Among the 10 stock cultures of Aspergillus niger, the strain GCBT7 was found to enhance citric acid production. This strain was subjected to parametric studies. Major effects were caused due to oxygen tension (1.0 l/l/min), pH value (6.0) and incubation temperature (30ºC). All fermentations were carried out following the growth on 150 g/l raw molasses sugars for 144 hours. Ferrocyanide (200 ppm) was used to control the trace metals present in the molasses medium. Ammonium nitrate (0.2%) was added as nitrogen source. Maximum citric acid production (99.56 ± 3.5a g/l) was achieved by Aspergillus niger GCBT7. The dry cell mass and sugar consumption were 18.5 and 96.55 g/l, respectively. The mycelia were intermediate round pellets in their morphology. The specific productivity of GCBT7 (qp = 0.074 ± 0.02a g/g cells/h) was several folds higher than other strains. The specific production rate and growth coefficient revealed the hyperproducibility of citric acid using mutant GCBT7.

Citric acid fermentation of cane-molasses by submerged fermentation in 15 L stirred fermentor (working volume 9 L) was carried out. A hyper mutant strain of Aspergillus niger GCMC-7 was used in the present study which was obtained from the culture collection of our own labs. Ferrocyanide treated molasses [K4Fe(CN)6 200 ppm] medium containing sugar 150 g/l was employed as the basal fermentation medium. Different cultural conditions such as incubation temperature (30ºC), initial pH (6.0), air supply (1.0 l-1l-1min), agitation intensity (200 rpm) and time profile (144 h after inoculation) were optimised for enhanced citric acid production. Maximum amount of anhydrous citric acid obtained during the course of study was 106.65 g/l, with a sugar consumption of 107 g/l. Final pH, ferrocyanide concentration and dry cell mass were 2.1, 60 ppm and 16.5 g/l, respectively.

L-DOPA is a useful drug for Parkinson’s disease. This investigation deals with the biosynthesis of L-DOPA by parental (GCB-6) and mutant (UV-7) strains of Aspergillus oryzae. There was a marked difference between the mycelial morphology and pellet type of the parental and UV-irradiated mutant cultures. The mutant strain of Aspergillus oryzae UV-6 showed pellet-like mycelial morphology and improved tyrosinase activity. Mould mycelium was used for biochemical conversion of L-tyrosine to L-DOPA since tyrosinase is an intracellular enzyme. The mutant was found to give 3.72 folds higher production of L-DOPA than the parental strain. The comparison of kinetic parameters was also done which showed greater ability of the mutant to yield L-DOPA (i.e. Yp/x 32.73 mg/mg with parent and 95.71 mg/mg with mutant). When cultures grown for various incubation periods, were monitored for Qp, Qs and q p, there was significant enhancement (p<0.0025-0.005) in these variables by the mutant strain of Aspergillus oryzae UV-7 over GCB-6 on all the rates.

The present investigation deals with a novel technique for high and consistent yield of citric acid in a fed-batch bioreactor of 9-L working volume. Five different potent cultures of Aspergillus niger were evaluated for citrate production using clarified cane-molasses as basal substrate. Among the cultures, NG GCB101 (developed by UV/chemical mutation in our labs) gave maximum production of citric acid i.e., 87.98 g/l after 6 days of mycelial inoculation. The values of kinetic parameters such as substrate consumption and product formation rates revealed the hyperproducibility of citric acid by selected Aspergillus niger NG GCB101 (LSD = 0.456, HS). The addition of MnSO4 to the culture medium promoted the formation of small rounded fluffy pellets (1.55 mm², dia.), which are desirable for maximal citric acid productivity. During recycling fed-batch cultivation, high and consistent yield of citric acid monohydrate (128.68 g/l) was obtained when the sampling vs. substrate feeding was maintained at 4-L (44.50% working volume). The incubation period was reduced from 6 to only 2 days, making the process highly economical for citric acid production. MnSO4 at a level of 2.0 x 10-6 M, added during inoculation time optimised the commercial exploitation of molasses in recycling fed-batch cultivation system. The technique developed in the current studies is a new addition in fermentation technology due to high yields of product, recycling of the fermentation medium and fungal mycelia, low manpower and energy consumption and the use of low cost raw substrate without any additional supplementation.