Accumulation of poly-beta-hydroxybutyrate in Nostoc muscorum: regulation by pH, light-dark cycles, N and P status and carbon sources

Accumulation of poly-beta-hydroxybutyrate (PHB) in Nostoc muscorum was studied. Cells harvested at stationary phase of growth depicted maximum accumulation i.e. 8.6% (w/w) of dry cells as compared to lag (4.1%) or logarithmic (6.1%) phases of cultures. In contrast to alkaline pH, acidic pH, continuous illumination and cells grown in presence of combined nitrogen sources, such as NH(4)Cl and KNO(3), were found to affect PHB accumulation negatively. However, P-deficiency and addition of exogenous carbon sources (acetate, glucose, maltose, fructose and ethanol) were found stimulatory for PHB accumulation. In this report PHB accumulation in N. muscorum was boosted up to 35% (w/w) of dry cells when cells supplemented with 0.2% acetate were subjected to dark incubation for 7 days. Further studies are needed at metabolic engineering level or to apply genetic engineering techniques to improve the expression level of PHB photoproduction in cyanobacteria.     

The influence of nutritional and environmental conditions on the accumulation of poly-beta-hydroxybutyrate in Bacillus mycoides RLJ B-017

AIMS: To optimize the nutritional and environmental conditions for growth of and poly-beta-hydroxybutyrate (PHB) accumulation in Bacillus mycoides RLJ B-017. METHODS AND RESULTS: An isolate, identified as B. mycoides, was grown on different sources of carbon and nitrogen. Among these, sucrose, beef extract and di-ammonium sulphate were found to be the most suitable for growth and PHB accumulation. The overall maximum value of PHB (%) in cells, PHB yield (Yp/s) and productivities (Qp and qp) were 69.4 +/- 0.4% dry cell weight (DCW), 0.21 gp gS(-1), 0.104 +/- 0.012 gp l(-1) h(-1) and 0.03 gp gx(-1) h(-1), respectively when grown in a medium containing 20 gs l(-1) sucrose, supplemented with di-ammonium sulphate. The addition of beef extract increased the value of PHB (%) in cells, PHB yield and productivities by 17.58 +/- 0,3, 23.8, 19.23 +/- 0.3 and 13.8 +/- 0.2% , respectively. The overall maximum values of PHB (% DCW), PHB yield and productivities were obtained at pH 7.0 +/- 0 .1, temperature 30 +/- 0.5 degrees C, agitation 650 rev min(-1) and oxygen transfer rate 3.8 mmol O(2) l(-1) h(-1). CONCLUSIONS: Sucrose, glucose and fructose were found to be more suitable for cell growth and PHB accumulation, but sucrose was less expensive than glucose. Among the nitrogen sources, beef extract and di-ammonium sulphate promoted PHB synthesis. The accumulation of PHB was observed to be growth associated. SIGNIFICANCE AND IMPACT OF THE STUDY: Gram-positive bacteria have not been reported to accumulate large amounts of polyhydroxyalkanoate and hence have not been considered as potent candidates for industrial production. A number of Bacillus spp. have been reported to accumulate 9-44.5% DCW PHB. By comparison, Bacillus RLJ B-017 contained 69.4 +/- 0.4% DCW PHB. Therefore, this strain has been considered as a potent organism for industrial interest. A relatively high yield of PHB was obtained in this wild strain and PHB synthesis was independent of nutrient limitation. The conditions for the higher PHB yield and productivity will be optimized in the next phase using fed-batch culture.     

Enhanced co-production of hydrogen and poly-(R)-3-hydroxybutyrate by recombinant PHB producing E. coli over-expressing hydrogenase 3 and acetyl-CoA synthetase

Recombinant Escherichia coli was constructed for co-production of hydrogen and polyhydroxybutyrate (PHB) due to its rapid growth and convenience of genetic manipulation. In particular, anaerobic metabolic pathways dedicated to co-production of hydrogen and PHB were established due to the advantages of directing fluxes away from toxic compounds such as formate and acetate to useful products. Here, recombinant E. coli expressing hydrogenase 3 and/or acetyl-CoA synthetase showed improved PHB and hydrogen production when grown with or without acetate as a carbon source. When hydrogenase 3 was over-expressed, hydrogen yield was increased from 14 to 153mmol H(2)/mol glucose in a mineral salt (MS) medium with glucose as carbon source, accompanied by an increased PHB yield from 0.55 to 5.34mg PHB/g glucose in MS medium with glucose and acetate as carbon source.     

根瘤菌4012a菌株产细胞分裂素发酵条件的研究

用酶标免疫检测法研究了根瘤菌4012a菌株细胞分裂素发酵的适宜培养基和培养条件。结果表明,其最佳培养基为（g／L）：葡萄糖10.0,（NH4）2SO41.0,K2HPO4·3H2O0．6,MgSO4·7H2O0.1,CaCl2·2H2O0.4,FeCI3·6H2O0．04,Na2MoO4·2H2O0．1mg／L,泛酸钙100μg／L,腺漂吟200mg／L。该菌株在150r／min的旋转摇床上27℃振荡培养96h,发酵液中细胞分裂素产量可达908μg／L,生物活性（萝卜子叶扩大法）为1mg／L激动素当量。     

Effects of phosphorus concentration and light intensity on the biomass composition of Arthrospira (Spirulina) platensis

This paper presents the effects of various phosphorus concentrations (10, 50, 250 and 500 mg l(-1) K(2)HPO(4)) on the biomass production and composition of Arthrospira (Spirulina) platensis in relation to light intensity (24, 42 and 60 μE m(-2) s(-1)). The maximum biomass production was 3,592 ± 392 mg l(-1) and this was observed in 250 mg l(-1) K(2)HPO(4) at 60 μE m(-2) s(-1) light intensity after 32 days of cultivation. A maximum specific growth rate (μ(max)) of 0.55 d(-1) was obtained in 500 mg l(-1) K(2)HPO(4) at 60 μE m(-2) s(-1). The protein, lipid and chlorophyll contents of the biomass varied from 33.59 to 60.57 %, 5.34 to 13.33 % and 0.78 to 2.00 %, respectively. The most significant finding was that phosphorus limitation (10 mg l(-1) K(2)HPO(4)) caused a drastic increase of the carbohydrate content (59.64 %). The effect of phosphorus limitation on the carbohydrate content was independent of the light intensity. The accumulated carbohydrates are proposed to be used as substrate for biofuel generation via one of the appropriate biomass energy conversion technologies. Also, it was observed that phosphorus removal is a function of biomass density, phosphorus concentration and light intensity.     

Effect of high nitrate concentration on PHB storage in sequencing batch reactor under anoxic conditions

The study investigated effect of high influent nitrate concentration on poly-beta-hydroxybutyrate, (PHB), storage in a sequencing batch reactor, (SBR), under anoxic conditions. Acetate was fed as pulse during anoxic phase, sustained with external nitrate feeding. SBR operation involved three runs at steady state with COD/N ratios of 3.84, 2.93 and 1.54 gCOD/gN, where external nitrate concentrations gradually increased from 50 mg N/l to 114 mg N/l and 226 mg N/l, in 1st, 2nd and 3rd runs, respectively. In 1st run, acetate was fully converted into PHB with the storage yield value of 0.57-0.59 gCOD/gCOD, calculated both in terms of PHB formation and NO(X) utilization, confirming storage was the sole substrate utilization mechanism. In the following runs, PHB formation was reduced and the storage yield based on PHB dropped down to 0.40 and 0.33 gCOD/gCOD with increasing influent nitrate concentrations, indicating that higher portions of acetate were diverted to simultaneous direct growth. The observations suggested that nitrite accumulation detected at low COD/N ratios was responsible for inhibition of PHB storage.     

Production and characterization of poly-β-hydroxybutyrate (PHB) polymer from Aulosira fertilissima

Biopolymers such as polyhydroxyalkanoates (PHAs) are a class of secondary metabolites with promising importance in the field of environmental, agricultural, and biomedical sciences. To date, high-cost commercial production of PHAs is being carried out with heterotrophic bacterial species. In this study, a photoautotrophic N₂-fixing cyanobacterium, Aulosira fertilissima, has been identified as a potential source for the production of poly-β-hydroxybutyrate (PHB). An accumulation up to 66% dry cell weight (dcw) was recorded when the cyanobacterium was cultured in acetate (0.3%) + citrate (0.3%)-supplemented medium against 6% control. Aulosira culture supplemented with 0.5% citrate under P deficiency followed by 5?days of dark incubation also depicted a PHB accumulation of 51% (dcw). PHB content of A. fertilissima reached up to 77% (dcw) under P deficiency with 0.5% acetate supplementation. Optimization of process parameters by response surface methodology resulted into polymer accumulation up to 85% (dcw) at 0.26% citrate, 0.28% acetate, and 5.58?mg?L^?1 K₂HPO₄ for an incubation period of 5?days. In the A. fertilissima cultures pre-grown in fructose (1.0%)-supplemented BG 11 medium, when subjected to the optimized condition, the PHB pool boosted up to 1.59?g?L^?1, a value ～50-fold higher than the control. A. fertilissima is the first cyanobacterium where PHB accumulation reached up to 85% (dcw) by manipulating the nutrient status of the culture medium. The polymer extracted from A. fertilissima exhibited comparable material properties with the commercial polymer. As compared with heterotrophic bacteria, carbon requirement in A. fertilissima for PHB production is lower by one order magnitude; thus, low-cost PHB production can be envisaged.     

Biosynthesis of poly-3-hydroxybutyrate by Sphaerotilus natans

A sheathed bacterium Sphaerotilus natans could not survive at 4°C for 2 months, and mutants that exhibited different colony phenotypes were obtained only by repeating the short period of storage at 4 °C. The ability of these mutants and the parent strain to produce poly-3-hydroxybutyrate (PHB) was compared in batch cultures. The parent strain accumulated 30% (w/w) PHB, while one of the mutants defective in sheath formation, designated as T2, accumulated over 50% PHB. Because T2 did not require strict air or nitrogen limitation for polymer accumulation, its production was growth-associated, allowing one-stage fermentation. In a pH-controlled fermentation using a jar fermentor, 10 g/l glucose was converted into 2.0 g/l PHB in 24 h.     

Optimization of polyhydroxybutyrate production from a wild type and two mutant strains of Rhodobacter sphaeroides using statistical method

Interaction studies using central composite design (CCD) gave the optimum concentrations of acetate at 4 g l(-1) and (NH4)2SO4 at 0.01 g l(-1) with an optimum temperature of 35 degrees C. Rhodobacter sphaeroides N20 gave the highest PHB (7.8 g l(-1)) and biomass (DCW) (8.2 g l(-1)) values compared to the wild type strain and the mutant strain U7. The CCD results predicted that the optimum medium for the mutant strain N20 consisted of 3.90 g l(-1) acetate, 0.01 g l(-1) (NH4)2SO4 at 33.5 degrees C (R2=0.985). Validation of this model by culturing the mutant strain in this optimum medium exhibited similar values of PHB (7.76 g l(-1)), biomass (8.32 g l(-1)) and the PHB content in the cell 93.2% of DCW. Similar amounts of PHB were also obtained in batch fermentations using a 5-l bioreactor. The effect of pH and aeration rate was also studied and the optimum values were found to be pH 7.0 with an aeration rate of 1.0 vvm. Under these optimal conditions, strain N20 produced the highest amount of PHB production (8.76 g l(-1)), PHB content (95.4% of DCW) as well as the product yield (Yp/x) (0.72). These results are the highest values ever obtained from photosynthetic bacteria reported so far.     

Degradation of polyesters by a novel marine Nocardiopsis aegyptia sp. nov.: application of Plackett-Burman experimental design for the improvement of PHB depolymerase activity

This is the first report on the degradation of poly(3-hydroxybutyrate) (PHB), and its copolymers poly(3-hydroxyvalerate) P(3HB-co-10-20% HV) by Nocardiopsis aegyptia, a new species isolated from marine seashore sediments. The strain excreted an extracellular PHB depolymerase and grew efficiently on PHB or its copolymers as the sole carbon sources. The degradation activity was detectable by the formation of a transparent clearing zone around the colony on an agar Petri plate after 25 days, or a clearing depth under the colony in test tubes within 3 weeks. The previous techniques proved that the bacterium was able to assimilate the monomeric components of the shorter alkyl groups of the polymers. Nocardiopsis aegyptia hydrolyzed copolymers 10-20% PHBV more rapidly than the homopolymer PHB. The bacterial degradation of the naturally occurring sheets of poly(3-hydroxybutyrate), and its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) was observed by scanning electron microscopy (SEM). The samples were degraded at the surface and proceeded to the inner part of the materials. Clear morphological alterations of the polymers were noticed, indicating the degradative capability of the bacterium. Plackett-Burman statistical experimental design has been employed to optimize culture conditions for maximal enzyme activity. The main factors that had significant positive effects on PHB depolymerase activity of Nocardiopsis aegyptia were sodium gluconate, volume of medium/flask and age of inoculum. On the other hand, MgSO4.7H2O, KH2PO4, K2HPO4 and NH4NO3 exhibited negative effects. Under optimized culture conditions, the highest activity (0.664 U/mg protein) was achieved in a medium predicted to be near optimum containing (in g/L): PHB, 0.5; C6H11O7Na, 7.5; MgSO4.7H2O, 0.35; K2HPO4, 0.35; NH4NO3, 0.5; KH2PO4, 0.35; malt extract, 0.5 and prepared with 50% seawater. The medium was inoculated with 1% (v/v) spore suspension of 7 days old culture. Complete clarity of the medium was achieved after 3 days at 30 degrees C.     

Comparison of recombinant Escherichia coli strains for synthesis and accumulation of poly-(3-hydroxybutyric acid) and morphological changes

A stable high-copy-number plasmid pSYL105 containing the Alcaligenes eutrophus polyhydroxyalkanoic acid (PHA) biosynthesis genes was constructed. This plasmid was transferred to seven Escherichia coli strains (K12, B, W, XL1-Blue, JM109, DH5alpha, and HB101), which were subsequently compared for their ability to synthesize and accumulate ploy- (3-hydroxybutyric acid) (PHB). Growth of recombinant cells and PHB synthesis were investigated in detail in Luria-Bertani (LB) medium containing 20 g/L glucose. Cell growth, the rate of PHB synthesis, the extent of PHB accumulation, the amount of glucose utilized, and the amount of acetate formed varied from one strain to another. XL1-Blue (pSYL105) and B (pSYL105) synthesized PHB at the fastest rate, which was ca. 0.2 g PHB/g true cell mass-h, and produced PHB up to 6-7 g/L. The yields of cell mass, true cell mass, and PHB varied considerably among the strains. The PHB yield of XL1-Blue (pSYL105) in LB plus 20 g/L glucose was as high as 0.369 g PHB/g glucose. Strains W (pSYL105) and K12 (pSYL105) accumulated the least amount of PHB with the lowest PHB yield at the lowest synthesis rate. JM109 (pSYL105) accumulated PHB to the highest extent (85.6%) with relatively low true cell mass (0.77 g/L). Considerable filamentation of cells accumulating PHB was observed for all strains except for K12 and W, which seemed to be due either to the overexpression of the foreign PHA biosynthesis enzymes or to the accumulation of PHB. (c) 1994 John Wiley & Sons, Inc.     

Poly-3-hydroxybutyrate production byAzotobacter chroococcum

Thirty-seven soil isolates and mutants ofAzotobacter chroococcum tested for poly-3-hydroxybutyrate (PHB) production using Sudan black B staining method were found to be positive. One mutant showed a higher number of PHB-producing cells and maximum number of granules per cell. Using 2% glucose and 15 mmol/L ammonium acetate, PHB production was found to be maximum at 36 and 48 h of growth under submerged cultivation and under stationary cultivation, respectively. PHB production was found to be higher on sucrose and commercial sugar (as carbon sources) as compared to glucose and mannitol. As commercial sugar is cheaper than sucrose it was selected as carbon source for PHB production, that being found to be maximum at 1% concentration. Inorganic nitrogen sources seemed to have no stimulatory effect on the production of PHB. However, ammonium acetate (15 mmol/L) was found to be best for PHB production. Peptone (0.2 %) gave a better yield of PHB under both growth conditions. Using all optimized conditions, PHB production was studied in ten selected strains. Two of them were found to be best PHB producers under both growth conditions, one producing 621 and 740 μg/g dry mass under submerged cultivation and under stationary cultivation, respectively, while the second one produced 589 and 733 μg/g.     

Simultaneous nitrification and denitrification using stored substrate (PHB) as the electron donor in an SBR

The potential for PHB (poly-beta-hydroxybutyrate) to serve as the electron donor for effective simultaneous nitrification and denitrification (SND) was investigated in a 2-L sequencing batch reactor (SBR) using a mixed culture and acetate as the organic substrate. During the feast period (i.e., acetate present), heterotrophic respiration activity was high and nitrification was prevented due to the inability of nitrifying bacteria to compete with heterotrophs for oxygen. Once acetate was depleted the oxidation rate of PHB was up to 6 times slower than that of soluble acetate and nitrification could proceed due to the decreased competition for oxygen. The slow nature of PHB degradation meant that it was an effective substrate for SND, as it was oxidised at a similar rate to ammonium and was therefore available for SND throughout the entire aerobic period. The percentage of nitrogen removed via SND increased at lower DO concentrations during the famine period, with up to 78% SND achieved at a DO concentration of 0.5 mg L(-1). However, the increased percentage of SND at a low DO concentration was compromised by a 2-times slower rate of nitrogen removal. A moderate DO concentration of 1 mg L(-1) was optimal for both SND efficiency (61%) and rate (4.4 mmol N x Cmol x(-1) x h(-1)). Electron flux analysis showed that the period of highest SND activity occurred during the first hour of the aerobic famine period, when the specific oxygen uptake rate (SOUR) was highest. It is postulated that a high SOUR due to NH(4) (+) and PHB oxidation decreases oxygen penetration into the floc, creating larger zones for anoxic denitrification. The accumulation of nitrate towards the end of the SND period showed that SND was finally limited by the rate of denitrification. As PHB degradation was found to follow first-order kinetics (df(PHB)/dt = -0.19 x f(PHB)), higher PHB concentrations would be expected to drive SND faster by increasing the availability rate of reducing power and reducing penetration of oxygen into the floc, due to the corresponding increased SOUR. Process control techniques to accumulate higher internal PHB concentrations to improve PHB-driven SND are discussed.     

辅酶Q_(10)产生菌发酵条件的优化

对辅酶Q10生产菌株鞘氨醇单胞菌YZ0803的发酵条件进行优化,确定发酵时间为90 h,250 mL摇瓶装液量为30 mL。培养基组成(质量分数,下同):葡萄糖1.5%,淀粉2.5%,黄豆饼粉2.5%,(NH4)2SO40.5%,NaCl0.03%,K2HPO40.02%,MgSO40.005%。优化后的辅酶Q10产量达到192 mg/L,比采用基础培养基的产量(138mg/L)提高了39.13%。     

2,3-丁二醇生产菌株生产能力的比较和培养基优化

对5株克雷伯氏肺炎杆菌 (包括两株乳酸途径被敲除的工程菌株) 发酵生产2,3-丁二醇能力进行了比较,其中K. pneumonia HR521 LDH (乳酸合成途径中ldhA基因被敲除) 具有最佳的发酵性能。通过正交试验优化了其发酵培养基的主要组分,优化后的培养基组成为：葡萄糖 90 g/L,(NH4)2HPO4 3 g/L,玉米浆 (CLSP) 6 g/L,乙酸钠 5 g/L,KCl 0.4 g/L,MgSO4 0.1 g/L,FeSO4·7H2O 0.02 g/L,MnSO4 0.01 g/L。在优化后的发酵培养基中进行摇瓶发酵,24 h发酵乙偶姻和2,3-丁二醇的终浓度为37.46 g/L,比未优化前增加了10 g/L,2,3-丁二醇得率达到了理论得率的90.53％,生产强度1.56 g/(L·h),检测不到副产物乳酸的生成,利于后提取工艺的进行和工业生产的应用。     

Effects of alternative carbon sources on biological transformation of nitrophenols

The removal of nitrophenols under denitrifying conditions was studied in bench-scale upflow anaerobic sludge blanket (UASB) reactors (R1, R2, R3 and R4) using three different carbon sources. Initially acetate was used as carbon source (substrate) in all the four reactors followed by glucose and methanol. Reactor R1 was kept as control and R2, R3, R4 were fed with 30 mg/l concentration of 2-nitrophenol (2-NP), 4-nitrophenol (4-NP), and 2,4-dinitrophenol (2,4-DNP), respectively. Throughout the study the hydraulic retention time (HRT) and COD/NO₃ ^-–N ratio were kept as 24 h and 10, respectively. 2-Aminophenol (2-AP), 4-aminophenol (4-AP) and 2-amino,4-nitrophenol (2-A,4-NP) were found as the major intermediate metabolites of 2-NP, 4-NP and 2,4-DNP degradation, respectively. Methanol was found to be a better carbon source for 4-NP and 2,4-DNP degradation as compared to acetate and glucose, while 2-NP degradation was not influenced much by the change of substrate. Nitrate nitrogen removal was always more than 99%. COD removal efficiency of the nitrophenol fed reactors varied from 85.7% to 97.7%. The oxidation-reduction potential (ORP) inside the reactors dropped, up to –300 mv, with glucose as carbon source. As the reactors were switched over to methanol, ORP increased to –190 mv. The granular sludge developed inside the reactors was light brown in colour when acetate and glucose were used as substrate, which turned dark brown to black at the end of methanol run. Biomass yield in terms of volatile suspended solids was observed as 0.15, 0.089 and 0.14 g per gram of COD removal for acetate, glucose and methanol, respectively.     

Poly-beta-hydroxybutyrate production by Pseudomonas sp. RZS 1 under aerobic and semi-aerobic condition

Pseudomonas sp. RZS1 was isolated from distillery effluent and identified based on phenotypic characters and 16s rRNA sequencing. It accumulated optimum amount (703.79 microg/mg of biomass) of poly-beta-hydroxybutyrate (PHB) under aerobic process of fermentation and 75 microg/mg of biomass under the anaerobic process of fermentation. Aerobic fermentation yielded 9.3-fold more PHB than semi-aerobic fermentation. Acetone alcohol method proved to be the best suitable recovery method as it gave 703.79 microg PHB per mg of biomass with a percentage recovery yield of 70.37. It started to accumulate PHB at the end of lag phase (from 6 h of incubation). Optimum amount of PHB (20 microg/ml) was reported during early stationary phase (30 h of incubation). Extracted PHB showed two peaks, minor one at 248 nm and major one at 365 nm. IR spectra revealed the presence of functional groups characteristics of PHB.     

Optimization of polyhydroxybutyrate production by mixed cultures submitted to aerobic dynamic feeding conditions

Activated sludge submitted to aerobic dynamic feeding conditions showed a good and stable capacity to store polyhydroxybutyrate (PHB). The system, working for 2 years, selected a microbial population with a high PHB storage capacity. The influence of carbon and nitrogen concentrations on the PHB accumulation yield was studied in a range of 15-180 Cmmol/l for acetate and 0-2.8 Nmmol/l for ammonia. Low ammonia concentrations favored PHB accumulation. The maximum PHB content, 67.5%, was obtained for 180 Cmmol/l of acetate supplied in one pulse. However, such high substrate concentration proved to be inhibitory for the storage mechanism, causing a slowdown of the specific PHB storage rate. In order to avoid substrate inhibition, 180 Cmmol/l of acetate was supplied in different ways: continuously fed and in three pulses of 60 Cmmol/l each. In both cases the specific PHB storage rate increased and the PHB content obtained were 56.2% and 78.5%, respectively. The latter value of PHB content is similar to that obtained by pure cultures and was never reported for mixed cultures. Addition of acetate by pulses controlled by the oxygen concentration was kept for 16 days, the PHB content being always above 70% of cell dry weight.     

粗壮假丝酵母(Candida valida 20231)产脂肪酶发酵条件的研究

对粗壮假丝酵母(Candida valida 20231)产脂肪酶所需的培养基成分和发酵条件进行了优化。结果表明,粗壮假丝酵母在接种后3 h即进入对数期,18 h后转入稳定期,66 h后进入衰退期;发酵36 h达到最大酶活,至72 h仍然无明显下降趋势。最适合的培养基成分为:4.0%大豆粉,2.0%菜籽油,0.3%葡萄糖,0.3%NH_4NO_3,1.2%K_2HPO_4,0.42%NaH_2PO_4,0.4%MgSO_4·7H_2O。使用优化的培养基,最佳产酶的条件为:装液量80 mL(250 mL摇瓶),初始pH 6,接种量为2.0%,种子菌龄为12 h。在最佳产酶条件下,脂肪酶活力达到32.6 U/mL。     

Statistical medium optimization and production of a hyperthermostable lipase from Burkholderia cepacia in a bioreactor

AIM: Statistical medium optimization for maximum production of a hyperthermostable lipase from Burkholderia cepacia and its validation in a bioreactor. METHODS AND RESULTS: Burkholderia cepacia was grown in shake flasks containing 1% glucose, 0.1% KH2PO4, 0.5% NH4Cl, 0.24% (NH4)2HPO4, 0.01% MgSO4.7H2O and 1% emulsified palm oil, at 45 degrees C and pH 7.0, agitated at 250 rev min(-1) with 6-h-old inoculum (2% v/v) for 20 h. A fourfold enhancement in lipase production (50 U ml(-1)) and an approximately three fold increase in specific activity (160 U mg(-1)) by B. cepacia was obtained in a 14 litre bioreactor within 15 h after statistical optimization following shake flask culture. The statistical model was obtained using face centred central composite design (FCCCD) with five variables: glucose, palm oil, incubation time, inoculum density and agitation. The model suggested no interactive effect of the five factors, although incubation period, inoculum and carbon concentration were the important variables. CONCLUSIONS: The maximum lipase production was 50 U ml(-1), with specific activity 160 U mg(-1) protein, in a 14 litre bioreactor after 15 h in a medium obtained after statistical optimization in shake flasks. Further, the model predicted reduction in time for lipase production with reduction in total carbon supply. SIGNIFICANCE AND IMPACT OF THE STUDY: Statistical optimization allows quick optimization of a large number of variables. It also provides a deep insight into the regulatory role of various parameters involved in enzyme production.