Optimization of parameters for improvement of phenol degradation by Rhodococcus UKMP-5M using response surface methodology

Phenol is a toxic compound and is one of the major pollutants contained in the waste water from petroleum and its downstream industries. Response surface methodology (RSM) was used to optimize medium composition and culture condition for enhancement of growth of Rhodococcus UKMP-5M and phenol degradation rate in shake flask cultures. Phenol and (NH₄)₂SO₄ concentrations as well as temperature were the most significant factors that influenced growth and phenol degradation. Central composite design (CCD) was used for optimization of these parameters with growth, and degradation rates were used as the responses. Cultivation with 0.5 g/L phenol and 0.3 g/L (NH₄)₂SO₄ and incubation at 36 °C greatly enhanced growth of Rhodococcus UKMP-5M, where the final cell concentration increased from 0.117 g/L to 0.376 g/L. On the other hand, the degradation rate was greatly increased in cultivation with 0.7 g/L phenol and 0.4 g/L (NH₄)₂SO₄ and incubation at 37 °C. In this cultivation, the time taken to degrade 1 g/L phenol in the culture was reduced from 48 h to 27 h. The model for both responses was found significant and the predicted values were found to be in a good agreement with experimental values and subsequently validated. Increases in phenol degradation rate during Rhodococcus UKMP-5M cultivation corresponded well with increasing phenol hydroxylase activity.     

<Emphasis Type="Italic">Lactococcus lactis</Emphasis> M4, a potential host for the expression of heterologous proteins

Background  

Many plasmid-harbouring strains of Lactococcus lactis have been isolated from milk and other sources. Plasmids of Lactococcus have been shown to harbour antibiotic resistance genes and those that express some important proteins. The generally regarded as safe (GRAS) status of L. lactis also makes it an attractive host for the production of proteins that are beneficial in numerous applications such as the production of biopharmaceutical and nutraceutical. In the present work, strains of L. lactis were isolated from cow's milk, plasmids were isolated and characterised and one of the strains was identified as a potential new lactococcal host for the expression of heterologous proteins.     

Intracellular production of IFN-alpha 2b in Lactococcus lactis

Human interferon alpha (IFN-α) was expressed in two strains of Lactococcus lactis by aid of two promoters (P32 and Pnis) giving rise to two recombinant strains: MG:IFN and NZ:IFN, respectively. The expression of IFN was confirmed by ELISA and western blotting. Highest production was achieved using glucose for growth of both recombinant strains with nisin, used for induction of the recombinant strain with Pnis promoter, at 30 ng/ml. The optimum time for MG:IFN was 9 h and for NZ:IFN was 4.5 h. The highest productions by MG:IFN and NZ:IFN were 1.9 and 2.4 μg IFN/l, respectively. Both of the expressed IFNs showed bioactivities of 1.9 × 10⁶ IU/mg that were acceptable for further clinical studies.     

Kojic acid production byAspergillus flavus using gelatinized and hydrolyzed sago starch as carbon sources

Direct conversion of gelatinized sago starch into kojic acid byAspergillus flavus strain having amylolytic enzymes was carried out at two different scales of submerged batch fermentation in a 250-mL shake flask and in a 50-L stirred-tank fermentor. For comparison, fermentations were also carried out using glucose and glucose hydrolyzate from enzymic hydrolysis of sago starch as carbon sources. During kojic acid fermentation of starch, starch was first hydrolyzed to glucose by the action of α-amylase and glucoamylase during active growth phase. The glucose remaining during the production phase (non-growing phase) was then converted to kojic acid. Kojic acid production (23.5g/L) using 100 g/L sago starch in a shake flask was comparable to fermentation of glucose (31.5 g/L) and glucose hydrolyzate (27.9 g/L) but in the 50-L fermentor was greatly reduced due to non-optimal aeration conditions. Kojic acid production using glucose was higher in the 50-L fermentor than in the shake flask.     

Optimisation and Characterisation of Lipase-Catalysed Synthesis of a Kojic Monooleate Ester in a Solvent-Free System by Response Surface Methodology

Kojic acid is widely used to inhibit the browning effect of tyrosinase in cosmetic and food industries. In this work, synthesis of kojic monooleate ester (KMO) was carried out using lipase-catalysed esterification of kojic acid and oleic acid in a solvent-free system. Response Surface Methodology (RSM) based on central composite rotatable design (CCRD) was used to optimise the main important reaction variables, such as enzyme amount, reaction temperature, substrate molar ratio, and reaction time along with immobilised lipase from Candida Antarctica (Novozym 435) as a biocatalyst. The RSM data indicated that the reaction temperature was less significant in comparison to other factors for the production of a KMO ester. By using this statistical analysis, a quadratic model was developed in order to correlate the preparation variable to the response (reaction yield). The optimum conditions for the enzymatic synthesis of KMO were as follows: an enzyme amount of 2.0 wt%, reaction temperature of 83.69°C, substrate molar ratio of 1:2.37 (mmole kojic acid:oleic acid) and a reaction time of 300.0 min. Under these conditions, the actual yield percentage obtained was 42.09%, which is comparably well with the maximum predicted value of 44.46%. Under the optimal conditions, Novozym 435 could be reused for 5 cycles for KMO production percentage yield of at least 40%. The results demonstrated that statistical analysis using RSM can be used efficiently to optimise the production of a KMO ester. Moreover, the optimum conditions obtained can be applied to scale-up the process and minimise the cost.     

Kinetics of kojic acid fermentation byAspergillus flavus link S44-1 using sucrose as a carbon source under different pH conditions

Kojic acid production byAspergillus flavus strain S44-1 using sucrose as a carbon source was carried out in a 250-mL shake flask and a 2-L stirred tank fermenter. For comparison, production of kojic acid using glucose, fructose and its mixture was also carried out. Kojic acid production in shake flask fermentation was 25.8 g/L using glucose as the sole carbon source, 23.6 g/L with sucrose, and 6.4 g/L from fructose. Reduced kojic acid production (13.5 g/L) was observed when a combination of glucose and fructose was used as a carbon source. The highest production of kojic acid (40.2 g/L) was obtained from 150 g/L sucrose in a 2 L fermenter, while the lowest kojic acid production (10.3 g/L) was seen in fermentation using fructose as the sole carbon source. The experimental data from batch fermentation and resuspended cell system was analysed in order to form the basis for a kinetic model of the process. An unstructured model based on logistic and Luedeking-Piret equations was found suitable to describe the growth, substrate consumption, and efficiency of kojic acid production byA. flavus in batch fermentation using sucrose. From this model, it was found that kojic acid production byA. flavus was not a growth-associated process. Fermentation without pH control (from an initial culture pH of 3.0) showed higher kojic acid production than single-phase pH-controlled fermentation (pH 2.5, 2.75, and 3.0).     

Kinetics and modelling of cell growth and substrate uptake inCentella asiatica cell culture

In this study, we have conducted kinetics and modelling studies ofCentella asiatica cell growth and substrate uptake, in an attempt to evaluate cell growth for a better understanding and control of the process. In our bioreactor cultivation experiment, we observed a growth rate of 0.18/day, a value only 20% higher than was seen in the shake flask cultivation trial. However, the observed maximum cell dry weight in the shake flask, 10.5 g/L, was 14% higher than was achieved in the bioreactor. Ninety seven percentage confidence was achieved via the fitting of three unstructured growth models; the Monod, Logistic, and Gompertz equations, to the cell growth data. The Monod equation adequately described cell growth in both cultures. The specific growth rate, however, was not effectively predicted with the Logistic and Gompertz equations, which resulted in deviations of up to 73 and 393%, respectively. These deviations in the Logistic and Gompertz models may be attributable to the fact that these models were developed for substrate-independent growth and fungi growth, respectively.     

Evaluation of commercial soy sauce <Emphasis Type="Italic">koji</Emphasis> strains of <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> for γ-aminobutyric acid (GABA) production

In this study, four selected commercial strains of Aspergillus oryzae were collected from soy sauce koji. These A. oryzae strains designated as NSK, NSZ, NSJ and NST shared similar morphological characteristics with the reference strain (A. oryzae FRR 1675) which confirmed them as A. oryzae species. They were further evaluated for their ability to produce γ-aminobutyric acid (GABA) by cultivating the spore suspension in a broth medium containing 0.4 % (w/v) of glutamic acid as a substrate for GABA production. The results showed that these strains were capable of producing GABA; however, the concentrations differed significantly (P < 0.05) among themselves. Based on the A. oryzae strains, highest GABA concentration was obtained from NSK (194 mg/L) followed by NSZ (63 mg/L), NSJ (51.53 mg/L) and NST (31.66 mg/L). Therefore, A. oryzae NSK was characterized and the sequence was found to be similar to A. oryzae and A. flavus with 99 % similarity. The evolutionary distance (K _nuc) between sequences of identical fungal species was calculated and a phylogenetic tree prepared from the K _nuc data showed that the isolate belonged to the A. oryzae species. This finding may allow the development of GABA-rich ingredients using A. oryzae NSK as a starter culture for soy sauce production.     

Improved production of live cells of Lactobacillus rhamnosus by continuous cultivation using glucose-yeast extract medium

In this study, the growth kinetics of Lactobacillus rhamnosus and lactic acid production in continuous culture were assessed at a range of dilution rates (0.05 h(-1) to 0.40 h(-1)) using a 2 L stirred tank fermenter with a working volume of 600 ml. Unstructured models, predicated on the Monod and Luedeking-Piret equations, were employed to simulate the growth of the bacterium, glucose consumption, and lactic acid production at different dilution rates in continuous cultures. The maximum specific growth rate of L. rhamnosus, mu-max, was estimated at 0.40 h(-1), and the Monod cell growth saturation constant, Ks, at approximately 0.25 g/L. Maximum cell viability (1.3 x 10(10) CFU/ml) was achieved in the dilution rate range of D = 0.28 h(-1) to 0.35 h(-1). Both maximum viable cell yield and productivity were achieved at D = 0.35 h(-1). The continuous cultivation of L. rhamnosus at D = 0.35 h(-1) resulted in substantial improvements in cell productivity, of 267% (viable cell count) that achieved via batch cultivation.     

Improved mannan-degrading enzymes' production by Aspergillus niger through medium optimization

The effect of different carbon and nitrogen sources on the production of mannan-degrading enzymes, focussing on β-mannanase, by Aspergillus niger was investigated using shake flask culture. The β-mannanase activity obtained during growth of A. niger on guar gum (GG, 1495 nkat mL(-1)) was much higher than those observed on other carbon substrates, locust bean gum (1148 nkat mL(-1)), α-cellulose (10.7 nkat mL(-1)), glucose (8.8 nkat mL(-1)) and carboxymethylcellulose (4.6 nkat mL(-1)). For fermentation using GG as a carbon source, bacteriological peptone gave the highest β-mannanase activity (1744 nkat mL(-1)) followed by peptone from meat (1168 nkat mL(-1)), yeast extract (817 nkat mL(-1)), ammonium sulphate (241 nkat mL(-1)), ammonium nitrate (113 nkat mL(-1)) and ammonium chloride (99 nkat mL(-1)) when used as a nitrogen source. The composition of bacteriological peptone and initial pH of the medium were further optimized using response surface methodology (RSM). Medium consisted of 21.3 g L(-1) GG and 57 g L(-1) peptone with initial culture pH of 5.5 was optimum for β-mannanase production (2063 nkat mL(-1)) by A. niger. The β-mannanase production obtained in this study using A. niger was significantly higher than those reported in the literature.