Application of polyethylene glycol immobilized Clostridium sp. LS2 for continuous hydrogen production from palm oil mill effluent in upflow anaerobic sludge blanket reactor

A novel polyethylene glycol (PEG) gel was fabricated and used as a carrier to immobilize Clostridium sp. LS2 for continuous hydrogen production in an upflow anaerobic sludge blanket (UASB) reactor. Palm oil mill effluent (POME) was used as the substrate carbon source. The optimal amount of PEG-immobilized cells for anaerobic hydrogen production was 12% (w/v) in the UASB reactor. The UASB reactor containing immobilized cells was operated at varying hydraulic retention times (HRT) that ranged from 24 to 6 h at 3.3 g chemical oxygen demand (COD)/L/h organic loading rate (OLR), or at OLRs that ranged from 1.6 to 6.6 at 12 h HRT. The best volumetric hydrogen production rate of 336 mL H₂/L/h (or 15.0 mmol/L/h) with a hydrogen yield of 0.35 L H₂/g COD_removed was obtained at a HRT of 12 h and an OLR of 5.0 g COD/L/h. The average hydrogen content of biogas and COD reduction were 52% and 62%, respectively. The major soluble metabolites during hydrogen fermentation were butyric acid followed by acetic acid. It is concluded that the PEG-immobilized cell system developed in this work has great potential for continuous hydrogen production from real wastewater (POME) using the UASB reactor.     

Batch and continuous fermentative production of hydrogen with anaerobic sludge entrapped in a composite polymeric matrix

Cell immobilization techniques were adopted to biohydrogen production using immobilized anaerobic sludge as the seed culture. Sucrose-based synthetic wastewater was converted to H₂ using batch and continuous cultures. A novel composite polymeric material comprising polymethyl methacrylate (PMMA), collagen, and activated carbon was used to entrap biomass for H₂ production. Using the PMMA immobilized cells, the favorable conditions for batch H₂ fermentation were 35 °C, pH 6.0, and an 20 g COD l⁻¹ of sucrose, giving a H₂ production rate of 238 ml h⁻¹ l⁻¹ and a H₂ yield of 2.25 mol H₂ mol sucrose⁻¹. Under these optimal conditions, continuous H₂ fermentation was conducted at a hydraulic retention time (HRT) of 4–8 h, giving the best H₂-producing rate of 1.8 l h⁻¹ l⁻¹ (over seven-fold of the best batch result) at a HRT of 6 h and a H₂ yield of 2.0 mol H₂ mol sucrose⁻¹. The sucrose conversion was essentially over 90% in all runs. The biogas consisted of only H₂ and CO₂. The major soluble metabolites were butyric acid, acetic acid, and 2,3-butandiol, while a small amount of ethanol also detected. The PMMA-immobilized-cell system developed in this work seems to be a promising H₂-producing process due to the high stability in continuous operations and the capability of achieving a competitively high H₂ production rate under a relatively low organic loading rate.     

Chemical characterization,in vitro dry matter and ruminal crude protein degradability and microbial protein synthesis of some cowpea (Vigna unguiculata L. Walp) haulm varieties

A study was carried out to evaluate the chemical composition, in vitro apparently and truly degraded dry matter (DM), utilizable crude protein at the duodenum (uCP) (total CP at the duodenum minus endogenous CP), methane production, and short chain fatty acid production of haulms of six cowpea varieties. The study was arranged in a 2 × 2 × 2 factorial design, with three replicates. Three improved (ITA2, ITA6 and ITA8) and three commercial (Oloyin, Peu and Sokoto) cowpea varieties harvested during wet and dry seasons were used for the study. After an initial gas test to evaluate 96 h gas production profiles of haulms with and without polyethylene glycol (PEG), the time to half maximal gas production was calculated and a second incubation conducted with fermentation stopped at substrate specific half time (t_1/2) and 24 h for each substrate. True DM degradability was measured from incubated residues and combined with gas volume to estimate the partitioning factor. Crude protein flow to the duodenum was estimated by combining gas volume with the measured ammonia nitrogen in the incubated fluid. Addition of PEG did not have any effect (P>0.05) on all the variables determined. Interaction between group (improved vs. commercial) and season was observed for CP (P=0.002), lignin (P=0.003) and hemicellulose (P=0.030) contents of the haulms. A group × season interaction was also observed for some of the variables at both substrate specific t_1/2 and 24 h. Commercial cowpea haulms had greater (P=0.002) microbial mass and produced less (P<0.05) methane than the improved cowpea haulms. The improved cowpea haulms were less (P<0.001) degraded in the rumen and as a result ensured greater (P<0.001) amount of uCP. The results validated that cowpea haulm is an important agro-based by-product that is adequate in protein and energy to sustain ruminant animal production in Nigeria and other Sub-Saharan African countries during the extended dry season.     

Efficient production of (R)-(−)-mandelic acid with highly substrate/product tolerant and enantioselective nitrilase of recombinant Alcaligenes sp.

For efficient production of (R)-(−)-mandelic acid, a nitrilase gene from Alcaligenes sp. ECU0401 was cloned and overexpressed in Escherichia coli. After simple optimization of the culture conditions, the biocatalyst production was greatly increased from 500 to 7000 U/l. The recombinant E. coli whole cells showed strong tolerance against a high substrate concentration of up to 200 mM, and the concentration of (R)-(−)-mandelic acid after only 4 h of transformation reached 197 mM with an enantiomeric excess (ee_p) of 99%. In a fed-batch reaction with 600 mM mandelonitrile as the substrate, the cumulative production of (R)-(−)-mandelic acid after 17.5 h of conversion reached 520 mM. The recombinant E. coli cells could also be repeatedly used in the biotransformation, retaining 40% of the initial activity after 10 batches of reaction. The highly substrate/product tolerable and enantioselective nature of this recombinant nitrilase suggests that it is of great potential for the practical production of optically pure (R)-(−)-mandelic acid.     

Oxidative stress caused by pyocyanin impairs CFTR Cl− transport in human bronchial epithelial cells

Pyocyanin (N-methyl-1-hydroxyphenazine), a redox-active virulence factor produced by the human pathogen Pseudomonas aeruginosa, is known to compromise mucociliary clearance. Exposure of human bronchial epithelial cells to pyocyanin increased the rate of cellular release of H₂O₂ threefold above the endogenous H₂O₂ production. Real-time measurements of the redox potential of the cytosolic compartment using the redox sensor roGFP1 showed that pyocyanin (100 μM) oxidized the cytosol from a resting value of − 318 ± 5 mV by 48.0 ± 4.6 mV within 2 h; a comparable oxidation was induced by 100 μM H₂O₂. Whereas resting Cl⁻ secretion was slightly activated by pyocyanin (to 10% of maximal currents), forskolin-stimulated Cl⁻ secretion was inhibited by 86%. The decline was linearly related to the cytosolic redox potential (1.8% inhibition/mV oxidation). Cystic fibrosis bronchial epithelial cells homozygous for ΔF508 CFTR failed to secrete Cl⁻ in response to pyocyanin or H₂O₂, indicating that these oxidants specifically target the CFTR and not other Cl⁻ conductances. Treatment with pyocyanin also decreased total cellular glutathione levels to 62% and cellular ATP levels to 46% after 24 h. We conclude that pyocyanin is a key factor that redox cycles in the cytosol, generates H₂O₂, depletes glutathione and ATP, and impairs CFTR function in Pseudomonas-infected lungs.     

Time course analysis of ABA and non-ionic osmotic stress-induced changes in water status,chlorophyll fluorescence and osmotic adjustment in Arabidopsis thaliana wild-type (Columbia) and ABA-deficient mutant (aba2)

In the present study, we investigated time course changes of water status including relative water content (RWC), leaf osmotic potential (Ψ_Π), stomatal conductance (g_s), proline (Pro), chlorophyll fluorescence (F_v/F_m) and total chlorophyll content in the Arabidopsis thaliana under PEG-induced drought stress after exogenous ABA treatment. To a better explanation for the role of ABA in the water status of A. thaliana to drought stress, wild-type (Columbia) and ABA-deficient mutant (aba2) of A. thaliana were used in the present study. Moreover, three weeks old Arabidopsis seedlings were applied exogenously with 50 μM ABA and exposed to drought stress induced by 40% PEG8000 (−0.73 MPa) for 6 h, 12 h and 24 h (hours). Our findings indicate that RWC of wild-type and aba2 started to decrease in the first 12 h and 6 h of PEG-induced drought stress, respectively. However, exogenous treatment of 50 μM ABA increased their RWC under drought stress. On the other hand, while Ψ_Π of both genotypes started to decrease in the first 6 h of drought stress, these declines in Ψ_Π were prevented by ABA treatment under stress throughout the experiment; it was more pronounced in aba2 at 24 h. While the highest increase in g_s was obtained in aba2 after 24 h stress, ABA-induced highest decrease in g_s was obtained in the same genotype during 12 h, as compared to PEG-treated group alone. On the other hand, Pro content increased in all treatment groups of ABA-deficient mutant aba2 at 12 h and 24 h. However, Pro content in ABA + PEG treated aba2 plants was higher than in PEG- and ABA-treated plants alone at the end of the 24 h. Drought stress decreased F_v/F_m and total chlorophyll contents of both genotypes while 50 μM ABA alleviated these reductions during drought stress, as compared to PEG stressed plants. On the other hand, 50 μM ABA treatment alone did not create any remarkable effect on F_v/F_m and total chlorophyll contents.These findings indicate that exogenous ABA showed an alleviative effect against damage of drought stress on relative water content, osmotic potential, stomatal conductance, proline, chlorophyll fluorescence and total chlorophyll content of both genotypes during 24 h of drought stress treatment.     

Partitioning of energy and degradability of browse plants in vitro and the implications of blocking the effects of tannin by the addition of polyethylene glycol

The gas production in vitro method was used to evaluate the degradability and gas production of browse plants in the absence or presence of polyethylene glycol 8000 (PEG). Substrates (leguminous and browse plants; 500 mg) were incubated for 24 h and the accumulated gas produced recorded. The incubation contents of the syringes were transferred into nylon bags and the undegraded residues weighed after washing and drying to constant weight (syringe-nylon bag (SNB) method). Substrates were also incubated in the rumen in nylon bags for 24 h to determine in sacco degradability. Gas production ranged between 10.3 and 64.4 ml whereas dry matter degradation ranges between 27.3 and 70.9%. Addition of PEG, which minimised the inhibitory effects of tannin on microbial fermentation resulted in an increase in both gas production and degradability in vitro, which ranged from 25.7 to 64.2 ml and 34.2 to 75.0%, respectively. Correlation analysis of the DM degradability estimated by the SNB method and in sacco method was greater in the presence of PEG (y=0.71x+14.9; r²=0.92) compared with absence of PEG (y=0.59x+15.0; r²=0.72). Partitioning factor (PF) of substrate to gas, which was expressed as mg DM degraded/ml gas, reflects the variation in microbial biomass yield. The PF figures, which varied from 4.94–11.05 to PF_+PEG values of 4.74–6.84 upon the addition of PEG, indicate the inhibitory effects of tannins on gas production. This suggests the presence of tannin has a potentially beneficial effect to protein nutrition of the host animal by altering partitioning of nutrients towards higher microbial yield rather than short chain fatty acids. PF values of browse plants determined both in the absence and presence of PEG may indicate the relative importance of tannins in different plant species on substrate degradability and partitioning of nutrients.     

Enzymatic hydrolysis of corncob and ethanol production from cellulosic hydrolysate

 Enzymatic hydrolysis of corncob and ethanol fermentation from cellulosic hydrolysate were investigated. After corncob was pretreated by 1% H₂SO₄ at 108 °C for 3 h, the cellulosic residue was hydrolyzed by cellulase from Trichoderma reesei ZU-02 and the hydrolysis yield was 67.5%. Poor cellobiase activity in T. reesei cellulase restricted the conversion of cellobiose to glucose, and the accumulation of cellobiose caused severe feedback inhibition to the activities of β-1,4-endoglucanase and β-1,4-exoglucanase in cellulase system. Supplementing cellobiase from Aspergillus niger ZU-07 greatly reduced the inhibitory effect caused by cellobiose, and the hydrolysis yield was improved to 83.9% with enhanced cellobiase activity of 6.5 CBU g⁻¹ substrate. Fed-batch hydrolysis process was started with a batch hydrolysis containing 100 g l⁻¹ substrate, with cellulosic residue added at 6 and 12 h twice to get a final substrate concentration of 200 g l⁻¹. After 60 h of reaction, the reducing sugar concentration reached 116.3 g l⁻¹ with a hydrolysis yield of 79.5%. Further fermentation of cellulosic hydrolysate containing 95.3 g l⁻¹ glucose was performed using Saccharomyces cerevisiae 316, and 45.7 g l⁻¹ ethanol was obtained within 18 h. The research results are meaningful in fuel ethanol production from agricultural residue instead of grain starch.     

Enhanced bioconversion rate and released substrate inhibition in (R)-phenylephrine whole-cell bioconversion via partial acetone treatment

An approach was developed to enhance the efficiency for the bioconversion of 1-(3-hydroxyphenyl)-2-(methyamino)-ethanone to (R)-phenylephrine. The strain Serratia marcescens N10612, giving the benefit of 99% enantiomeric excess in (R)-PE conversion, was used. The fermentation was devised to harvest cells with high hydrophobic prodigiosin content inside the cells. Then, the partial acetone extraction was applied to remove prodigiosin from the cells. The treatment was found to increase the cells conversion rate without loss of the cells NADPH redox system. When using 50% (v/v) acetone for 5 min, the processed cells can give a specific conversion rate of 16.03 μmol/h/g-cells. As compared the treated cells with cells under the basal medium, the maximum reaction rate (V_max) increased from 6.69 to 10.27 (μmol/h/g-cells), the dissociation constant (K_m) decreased from 0.236 to 0.167 mM and the substrate inhibition constant (K_Si) increased from 0.073 to 1.521 mM. The 20-fold increase in substrate inhibition constant referred to a great release from the substrate inhibition for the use of S. marcescens N10612 in the bioconversion, which would greatly benefit the bioconversion to be industrialized.     

Efficient production of S-(+)-2-chlorophenylglycine by immobilized penicillin G acylase in a recirculating packed bed reactor

(S)-(+)-2-Chlorophenylglycine 1 is an important intermediate in the synthesis of Clopidogrel. A recirculating packed bed reactor (RPBR) was constructed for efficient production of (S)-1 by kinetic resolution of racemic N-phenylacetyl-2- chlorophenylglycine 2 using immobilized penicillin G acylase (PGA). The immobilized PGA exhibited maximum activity at 50 °C and pH 8.0 with (R,S)-2 as substrate. The kinetic constants (K_m and v_max) of immobilized PGA were calculated to be 20.61 mM and 83.2 mM/min/g, respectively. The substrate displayed inhibitory effect on immobilized PGA with inhibition constant of 221.23 mM. The immobilized PGA showed a strict enantiospecificity for substrate at different temperature, pH and substrate concentration examined. The performance and productivity of RPBR were evaluated by several critical parameters, including immobilized PGA load, substrate feeding rate, height to diameter ratio and so on. The kinetic resolution process shows higher initial reaction rate and conversion by recycling 100 mL of substrate solution (80 mM) through RPBRs packed with 6.0 g immobilized PGA with a feeding rate of 1.5 mL/min while the H/D ratio was 4.0. The immobilized PGA-catalyzed kinetic resolution of (R,S)-2 was successfully operated in the RPBR for 60 batches, with an average productivity of 1.2 g/L/h for (S)-1 in high optical purity (>97% enantiomeric excess) in semi-continuous operation. The residual (R)-2 can be easily racemized and then used as substrate.     

Kinetic modeling and implementation of superior process strategies for β-galactosidase production during submerged fermentation in a stirred tank bioreactor

This paper reports development and implementation of superior fermentation strategies for β-galactosidase production by Lactobacillus acidophilus in a stirred-tank bioreactor. Process parameters (aeration and agitation) were optimized for the process by application of Central Composite Design. Aeration rate of 0.5 vvm and agitation speed of 250 rpm were most suitable for β-galactosidase production (2001.2 U/L). Further improvement of the operation in pH controlled environment resulted in 2135 U/L of β-galactosidase with productivity of 142.39 U/L h. Kinetic modeling for biomass and enzyme production and substrate utilization were carried out at the aforementioned pH controlled conditions. The logistic regression model (X₀ = 0.01 g/L; X_max = 2.948 g/L; μ_max = 0.59/h; R² = 0.97) was used for mathematical interpretation of biomass production. Mercier's model proved to be better than Luedeking–Piret model in describing β-galactosidase production (P₀ = 0.7942 U/L; P_max = 2169.3 U/L; P_r = 0.696/h; R² = 0.99) whereas the latter was more efficient in mathematical illustration of lactose utilization (m = 0.187 g/g h; Y_x/s = 0.301 g/L; R² = 0.98) among the two used in this study. Strategies like fed-batch fermentation (3694.6 U/L) and semi-continuous fermentation (5551.9 U/L) further enhanced β-galactosidase production by 1.8 and 2.8 fold respectively.     

Enhanced exopolysaccharide production and biological activity of Lactobacillus rhamnosus ZY with calcium and hydrogen peroxide

Exopolysaccharides (EPS) are important food and drug additives with beneficial antioxidant, anticancer, and immune-related effects on human health. However, the EPS is limited by low yields and the need for complex culture conditions in fermentation. Here, we report that hydrogen peroxide and calcium stimulated probiotic activity and production of crude exopolysaccharide (c-EPS) by Lactobacillus rhamnosus ZY. Accordingly, supplementation with 3 mM H₂O₂ allowed c-EPS biosynthesis to reach 567 mg/L after 24 h. Addition of both CaCl₂ and H₂O₂ resulted in a c-EPS yield of 2498 mg/L after 12 h, over 9-fold higher than that of an anaerobic culture. We observed that exposure to calcium and hydrogen peroxide made the cells more hydrophobic and led to the over-expression of GroEL, NADH peroxidase, and glyceraldehyde 3-phosphate dehydrogenase, thus increasing energy storage and EPS production. Chromatographic analysis revealed c-EPS was composed mainly of mannose (5.1%), galactose (15.3%), glucose (20–30%), and rhamnose (50–60%). Preliminary in vitro tests revealed that H₂O₂ and CaCl₂ enhanced the 2,2-diphenyl-1-picrylhydrazyl and hydroxyl radical scavenging capacities, resulting in a notable protective effect against oxidative damage in NIH/3T3 cells. Our study provides a simple and cost-effective approach for achieving high yields of good quality EPS using Lactobacillus rhamnosus.     

Solvent-free methanolysis of canola oil in a packed-bed reactor with use of Novozym 435 plus loofa

Enzymatic methanolysis of canola oil in the solvent-free system was studied in a packed-bed reactor (PBR) using small pieces of loofa plus Novozym 435. Response surface methodology (RSM) was applied to determine the effect of the transesterification conditions, namely flow rate of substrate (x₁), temperature (x₂) and methanol to canola oil molar ratio (x₃) as the regressors, on the methyl ester production. A central composite design (CCD) was employed to optimize the reaction. A second-order polynomial multiple regression model was chosen and analysis of variance (ANOVA) showed a high coefficient of determination (R²) value of 0.996, thus adjustment of the model with experimental data was ensured. The methyl ester yield increased as the flow rate of the reaction mixture in the PBR increased from its low to the middle level thereafter, increasing the flow rate corresponded to decreasing the yield. The same trends of changes were observed for the other two factors. The optimum process conditions for biodiesel production in the PBR were found to be: x₁ = 6.3 mL/min, x₂ = 38 °C and x₃ = 4.3. The same batch was successfully used repeatedly in the PBR for six enzymatic cycles (432 h), where the methyl ester yield was maintained above 97%.     

Purification and characterization of a new carbonyl reductase from Leifsonia xyli HS0904 involved in stereoselective reduction of 3,5-bis(trifluoromethyl) acetophenone

Leifsonia xyli HS0904 can stereoselectively catalyze the bioreduction of 3,5-bis(trifluoromethyl) acetophenone (BTAP) to its corresponding alcohol, which is a valuable chiral intermediate in the pharmaceuticals. In this study, a new carbonyl reductase derived from L. xyli HS0904 was purified and its biochemical properties were determined in detail. The carbonyl reductase was purified by 530-fold with a specific activity of 13.2 U mg⁻¹ and found to be a homodimer with a molecular mass of 49 kDa, in which the subunit molecular-weight was about 24 kDa. The purified enzyme exhibited a maximum enzyme activity at 34 °C and pH 7.2, and retained over 90% of its initial activity at 4 °C and pH 7.0 for 24 h. The addition of various additives, such as Ca²⁺, Mg²⁺, Mn²⁺, l-cysteine, l-glutathione, urea, PEG 1000 and PEG 4000, could enhance the enzyme activity. The maximal reaction rate (V_max) and apparent Michaelis–Menten constant (K_m) of the purified carbonyl reductase for BTAP and NADH were confirmed as 33.9 U mg⁻¹, 0.383 mM and 69.9 U mg⁻¹, 0.412 mM, respectively. Furthermore, this enzyme was found to have a broad spectrum of substrate specificity and can asymmetrically catalyze the reduction of a variety of ketones and keto esters.     

Inhibitory effect of novel tetrahydropyrimidine-2(1H)-thiones on melanogenesis

The series of imidazoldine-2-thiones 2 and tetrahydropyrimidine-2-thiones 3 were discovered as inhibitor of α-MSH-induced melanin production in melanoma B16 cells. The primary bioassay showed that 1-(4-ethylbenzyl)-tetrahydropyrimidine-2(1H)-thione 3e (>100% inhibition at 10 μM, IC₅₀ = 1.2 μM) and 1-(4-tert-butylbenzyl)-tetrahydropyrimidine-2(1H)-thione 3f (>100% inhibition at 10 μM, IC₅₀ = 0.76 μM) exhibited potent inhibitory effect against α-MSH-induced melanin production. Compounds 3 inhibit the biosynthesis of tyrosinase without affecting its catalytic activity in melanogenesis.     

Anaerobic treatment of saline wastewater by Halanaerobium lacusrosei

An upflow anaerobic packed bed reactor was operated continuously with synthetic saline wastewater at different initial COD concentrations (COD₀ = 1900–6300 mg/L), salt concentrations (0–5%, w/v) and hydraulic retention times (θ_H = 11–30 h) to investigate the effect of those operating parameters on COD removal from saline synthetic wastewater. Anaerobic salt tolerant bacteria, Halanaerobium lacusrosei, were used as dominant microbial culture in the process. The percent COD removal reached up to 94% at COD₀ = 1900 mg/L, 19 h hydraulic retention time and 3% salt concentration. No substrate inhibition effect was observed at high feed CODs. Increasing hydraulic retention time from 11 h to 30 h resulted in a substantial improvement in the COD removal from 60% to 84% at around COD₀ = 3400 mg/L and 3% salt concentration. Salt inhibition effect on COD utilization was observed at above 3% salt concentration. Modified Stover–Kincannon model was applied to the experimental data to determine the biokinetic coefficients. Saturation value constant, and maximum utilization rate constant of Stover–Kincannon model for COD were determined as K_B = 5.3 g/L day, U_max = 7.05 g/L day, respectively.     

Synthesis and PGE2 production inhibition of 1H-furan-2,5-dione and 1H-pyrrole-2,5-dione derivatives

3,4-Diphenyl-substituted 1H-furan-2,5-dione and 1H-pyrrole-2,5-dione derivatives were synthesized and evaluated for the inhibitory activities on LPS-induced PGE₂ production in RAW 264.7 macrophage cells. Both 1H-furan-2,5-dione and 1H-pyrrole-2,5-dione rings as main scaffolds were easily obtained using one of three synthetic methods. Among the compounds investigated, 1H-3-(4-sulfamoylphenyl)-4-phenyl-pyrrole-2,5-dione (6l) showed a strong inhibitory activity (IC₅₀ = 0.61 μM) of PGE₂ production.     

Alkaline lipase from a novel strain Burkholderia multivorans: Statistical medium optimization and production in a bioreactor

An alkaline lipase from Burkholderia multivorans was produced within 15 h of growth in a 14 L bioreactor. An overall 12-fold enhanced production (58 U mL⁻¹ and 36 U mg⁻¹ protein) was achieved after medium optimization following the “one-variable-at-a-time” and the statistical approaches. The optimal composition of the lipase production medium was determined to be (% w/v or v/v): KH₂PO₄ 0.1; K₂HPO₄ 0.3; NH₄Cl 0.5; MgSO₄·7H₂O 0.01; yeast extract 0.36; glucose 0.1; olive oil 3.0; CaCl₂ 0.4 mM; pH 7.0; inoculum density 3% (v/v) and incubation time 36 h in shake flasks. Lipase production was maximally influenced by olive oil/oleic acid as the inducer and yeast extract as the additive nitrogen. Plackett–Burman screening suggested catabolite repression by glucose. Amongst the divalent cations, Ca²⁺ was a positive signal while Mg²⁺ was a negative signal for lipase production. RSM predicted that incubation time, inoculum density and oil were required at their higher levels (36 h, 3% (v/v) and 3% (v/v), respectively) while glucose and yeast extract were required at their minimal levels for maximum lipase production in shake flasks. The production conditions were validated in a 14 L bioreactor where the incubation time was reduced to 15 h.     

Effect of process parameters on lipase production by Candida cylindracea in stirred tank bioreactor using renewable palm oil mill effluent based medium

A two-level full factorial design (FFD) was employed to determine the effects of process parameters on lipase production by Candida cylindracea ATCC 14830 in palm oil mill effluent (POME)-based medium. Ten experimental runs based on three parameters (temperature, agitation and aeration) as indicated by the FFD were carried out in a stirred-tank bioreactor. On statistical analysis of the results, the optimum temperature, aeration and agitation rates were found to be 30 °C, 1.0 vvm and 400 rpm respectively, with a maximum activity of 41.46 U/ml after 36 h of fermentation. Analysis of variance (ANOVA) showed a high coefficient of determination (R²) value of 0.999, indicating a satisfactory fit of the model with the experimental data. All the three parameters were statistically significant at p < 0.05. The validation experiment also confirmed that apart from lipase production, there was an increase in chemical oxygen demand (COD) removal throughout the fermentation period.