The biochemistry of lipid accumulation in Mucor circinelloides and Mortierella alpina

1. The profile and biochemistry of growth and lipid accumulation in M. circinelloides and Mt. alpina were investigated. 2. A nitrogen-limited condition was vital in triggering lipid accumulation in both fungi, which was in agreement with previous work reported in oleaginous yeasts (Botham and Ratledge, 1979; Boulton and Ratledge, 1984). Good growth and lipid production by M. circinelloides were obtained only when it was grown in fermenters. Growth in stirred bottles (whirlipots) did not result in high lipid yields. This was caused by the anaerobic nature of cultivation in the whirlipots which affected the utilization of ammonium by the cells. As a result the cultures became carbon-limited instead of nitrogen-limited. Conversely, in a fermenter culture which had an efficient aeration, the culture reached a nitrogen-limited condition at an early stage of the incubation which led to a higher lipid production of the cells. 3. The lipid production in both fungi increased in parallel with the increase in the C:N ratio of the medium but the fatty acid compositions were not affected. 4. Ten enzymes potentially linked to the regulation of lipid biosynthesis (fatty acid synthase, acetyl-CoA carboxylase, ATP:Citrate lyase, AMP:deaminase, carnitine acetyl transferase, malic enzyme, glucose-6- phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, NADP:isocitrate dehydrogenase and NAD:isocitrate dehydrogenase) were detected in both fungi. In both fungi, the profile of all enzymes stated above was similar with the activities increased coincidentally with the depletion of ammonia in the medium. 5. The only differences in the enzymatic profiles of the two fungi was the early depletion of ME activity in M. circinelloides where it disappeared after approximately 40 h of incubation, coincident with the cessation of lipid accumulation, although other key enzymes of lipid biosynthesis (FAS, ACC and ACL) together with the activities of other NADPHgenerating enzymes were still active and the glucose was still present. Conversely, ME in Mt. alpina culture was present until the late stage of fermentation and the cell lipid continued to increase until the end of the fermentation. This suggests ME is a major provider of NADPH for lipid biosynthesis which was in agreement with previous observations in Aspergillus nidu/ans (Wynn and Ratledge, 1997) and M. circinelloides (Wynn et al., 1997). 6. The depletion of ME activity in M. circinelloides after approximately 40 h of incubation was as a result of the cessation of the protein from being synthesized, triggered by the depletion of ammonium in the culture. This - was evident as malic enzyme activity returned after its initial depletion when ammonium tartrate was added into the culture. Also, the restitution of malic enzyme activity was prevented when cycloheximide, a protein synthesis inhibitor, was added simultanously with the addition of ammonium tartrate. 7. The NAD:isocitrate dehydrogenase from both fungi showed an increased affinity for its substrate, isocitrate, in, the presence of AMP. However, the enzyme did not show an absolute requirement for AMP for its activity as it could still be activated in the absence of AMP at a saturating concentration of isocitrate. 8. ME was purified some 20-fold purification from both fungi. Both showed a similar Km values for NADP (approximately 0.04 mM) but a slightly higher Km value for malate was obtained in Mt. alpina compared to M. circinelloides (1 mM and 0.4 mM, respectively). 9. ME from both fungi showed various degrees of inhibition by tartronic acid, oxaloacetate, palmitoyl-CoA and oleoyl-CoA. At 10 mM, tartronic acid caused approximately 40 % inhibition in the activity of ME from both fungi while OAA inhibited ME from M. circinelloides more strongly (70 %) than that from Mt. alpina ( 45 % ). At a final concentration of 1 mM, palmitoyl-CoA and oleoyl-CoA caused a 100 % inhibition on ME from M. circinelloides and approximately 90 % on ME from Mt. alpina. 10. FAS purified from both fungi showed a similar Km values for malonyl-CoA and acetyl-CoA (approximately 0.013 and 0.016 mM, - respectively) while a higher Km value for NAOPH was observed in Mt. alpina compared to M. circinelloides (0.038 and 0.01 mM, respectively). 11. Despite a range of experiment using different approaches, no direct evidence of a physical association between FAS and ME was obtained when experiments were performed to observe the formation of complexes between the two enzymes in vitro

Effects of Mg2+, Fe3+, Mn2+ and Cu2+ Ions on lipid accumulation by cunninghamella bainieri 2A1

Cunninghamella bainieri 2A1 is an oleaginous fungus whose lipid accumulation profile is significantly influenced by metal ion concentrations in growth medium. Mg2+, Fe3+, Mn2+ and Cu2+ were found to be the important elements affecting lipid accumulation in this fungus. This study employs a statistical method (Response Surface Methodology – RSM) to study the combined effects of Mg2+, Fe3+, Mn2+ and Cu2+ on lipid accumulation of C. baineri 2A1. Cultivation was carried out in 250 mL Erlenmeyer flasks containing 100 mL nitrogen limited medium at 30ºC and 250 rpm agitation for 120 h. A thirty-run central composite design experiment was employed to identify and optimize the significant factors. In addition to Mg2+ and Fe3+ which were shown to have significant effects on lipid accumulation, the interactions between Mg2+ and Cu2+, as well as the effect of Cu2+ in quadratic terms were also found to have significant effect on the process (p<0.05). The highest amount of lipid obtained in this study was 39% g/g biomass with optimal levels of Mg2+, Fe3+ and Cu2+ at 5.00, 0.017 and 0.0005 g/L, respectively, while Mn2+ was omitted. A 32% increment in lipid yield was recorded, where the lipid content increased to 38%, compared to initial yield of 29% g/g biomass prior to optimization. In conclusion, Mg2+ and Fe3+ have significant positive effect on the lipid accumulation of this fungus, whereas Mn2+ and Cu2+ exert negative effects in combination

Optimisation of ammonium tartrate and glucose concentration for gamma linolenic acid production by Cunninghamella sp. 2A1.

The effects of ammonium tartrate and glucose concentration on biomass, lipid and GLA accumulation in Cunninghamella sp. 2A1 were investigated using Response Surface Methodology (RSM). Cultivation was carried out in 250 mL shake flask containing 100 mL of nitrogen limiting medium (with various combinations of concentration of ammonium tartrate (1-3 g/L) and glucose (30-60 g/L) at 30°C and 250 rpm agitation for 120 h. The concentration of both compounds significantly affected the biomass, lipid and GLA yield (p<0.05), with the production of each of them being represented by quadratic models. Higher concentration of ammonium tartrate and glucose (2.99 and 59.33 g/L, respectively) was required for enhanced biomass production whereas low nitrogen content with excess glucose was otherwise favoured for lipid and GLA production. Ammonium tartrate and glucose concentration at 1 and 43 g/L, respectively were estimated by the model and proven to give the highest lipid production and GLA yield of 31.06 % (g/g biomass) and 4.15 ×10-2 (g/g lipid less biomass), respectively

Profil pengekspresan gen mengekod delta 6 dan delta 12-asid lemak desaturase Cunninghamella bainieri semasa biosintesis asid gamma-linolenik

Delta 6-asid lemak desaturase dan delta 12-asid lemak desaturase merupakan enzim yang diperlukan bagi langkah desaturasi semasa proses biosintesis asid gamma-linolenik (GLA) oleh kulat oleaginus. Objektif kajian ini ialah untuk menganalisis profil pengekspresan gen mengekod enzim delta 6-asid lemak desaturase (des6) dan delta 12-asid lemak desaturase (des12) kulat oleaginus Cunninghamella bainieri semasa penghasilan GLA. Jujukan gen separa bersaiz 1372 pb bagi des6 dan 1008 pb bagi des12 telah dipencil daripada C. bainieri. Analisis pengekspresan gen menggunakan kaedah tindak balas berantai polimerase kuantitatif masa sebenar (RT-qPCR) menunjukkan perubahan kadar pengekspresan des6 adalah lebih tinggi berbanding kadar pengekspresan des12 semasa penghasilan GLA. Pengekspresan des6 adalah tertinggi selepas 24 jam dikultur dalam medium penghasilan GLA. Namun, kadar pengekspresannya menurun hingga jam ke-96 pertumbuhan tetapi meningkat semula pada jam ke-120. Bagi des12, kadar pengekspresannya adalah lebih sekata dengan pengekspresan tertinggi dikesan pada jam ke-120. Analisis penghasilan GLA menunjukkan jumlah GLA dalam sel berkolerasi dengan kadar pengekspresan des6. Hasil kajian mencadangkan bahawa aras pengekspresan des6 adalah penting dalam menentukan aras GLA dalam C. bainieri

Optimization of medium for the production of β-cyclodextrin glucanotransferase using Central Composite Design (CCD)

Production of cyclodextrin glucanotransferase (CGTase) from Bacillus G 1, a new bacterial isolate was optimized in shake-flask cultures using statistical design approach. An efficient fermentation medium producing CGTase with high activity (54.9U/ml) was determined: A 2(5) Central Composite Design (CCD; half fraction) has been chosen to elucidate the combined effect of five process variables; tapioca starch, peptone, yeast extract, magnesium sulphate and potassium phosphate concentration. Concentrations of 4% tapioca starch, 2% peptone, 0.04% magnesium sulphate and 1 % sodium carbonate have been shown to be optimum for the production of CGTase

Effect of aeration and dilution on continuous bioethanol production in a packed-bed bioreactor by saccharomyces cerevisiae

Stability of cell growth was achieved continuously at a steady state in a packed-bed bioreactor. The highest productivity of ethanol was achieved (0.02 g/L/h) when 0.003 vvm was employed. The productivity of bioethanol increases when dilution rate increases. The highest production of 0.037 g/L/h was recorded when the dilution rate (D) was at 0.05 per hour. The production of bioethanol was sucessfully maintained in a non 100% anaerobic condition. The best aeration for the continous production of bioethanol in a condition of steady state growth was at an aeration rate of 0.003 vvm

Production of docosahexaenoic acid, DHA using different modes of cultivation by Aurantiochytrium sp. SW1

Thraustochytrids, such as members of the genus Aurantiochytrium, are rich in docosahexaenoic acid (DHA, C22:6n-3) and represent a promising source of omega-3 fatty acids which plays a vital role in the enhancement of human health, particularly for neurological and visual functions. Different modes of cultivation (batch, fed-batch and repeated-batch) by Aurantiochytrium sp. SW1 were studied for effective docosahexaenoic acid (DHA) production. In this study, three different modes of fermentation were carried out in 1 L shake flasks with a working volume of 500 mL, incubated at 30 ºC and 200 rpm. Batch cultivation significantly exceeds the rest of cultivation modes, achieving maximal lipid and DHA concentrations of 11.22 g/L and 5.87 g/L, respectively, and DHA productivity of 0.061 g/L/h. Lipid and DHA concentration of the repeated-batch process decreased through the cycles for all three different types of replacement ratio (80, 90 and 95%). The average decrease percentage of DHA concentration for cycle one and cycle two were 21.76 and 32.52%, respectively. However, the fatty acid composition of lipids obtained in the cycles remained consistent with 16:0 and DHA being the most abundant fatty acids indicating that this mode of fermentation is highly useable for industrial applications

Biochemical Characterization for Lipid Synthesis in Aspergillus niger

A niger, a fungus which doesn't have high ability to production lipid, this fungus has been select to investigate the non oleaginicity. In this search, there are explorations about: i) growth profile ii) enzymes profile iii) isoforms. Growth profile shows that this fungus doesn't have ability to accumulate lipid more than 6% while bio mass are around 10g/l in spite of the presence of glucose in the media till the end of cultivation time and excision of nitrogen within 24 hrs. In enzyme study, we investigate all lipogenic enzymes Malic enzyme (ME), Fatty acid synthase (FAS), ATP: Citrate lays (ACL), NAD+ isocitrate dehydrogenase (NAD+ICDH), Glucose-6-phosphate (G6PD), and 6-phosphogluconate dehydrogenase (6PGD), all these enzymes show, activities till the end of cultivation time including ACL which is regarded the key enzyme to differentiate between the two species oleaginous and non oleaginous. So, there is no main reason to non oleaginicity for this fungus. A further experiment has been done using Polyacrylamide gel electrophoresis to identify ME isoforms. The result of Polyacrylamide gel electrophoresis shows multi isoforms (A, B, C, D & E), with low intensity of isoform E, the isoforms that may involve in lipid synthesis. We have now studied the biochemistry of A.niger grown under conditions designed to promote lipid accumulation and can now advance a coherent hypothesis to explain why A niger could not accumulate lipid more than 6%. So the absence of isoforme E is the main reason for non oleaginicity in A niger

Enhanced Butanol Production by Clostridium acetobutylicum

The production of biobutanol was studied by the cultivation of Clostridium acetobutylicum NCIMB 13557 in P2 medium including date fruit as the sole substrate. The effect of P2 medium and the effect of different concentrations of date fruit ranging from 10 to 100 g/L on biobutanol production were investigated. Anaerobic batch culture was carried out at 35°C incubation temperature and pH 7.0 ± 0.2 for 72 h. Experimental results showed that the lowest yield of biobutanol and acetone-butanol-ethanol (ABE) was 0.32 and 0.35 gram per gram of carbohydrate consumed (g/g), respectively, when an initial date fruit concentration of 10 g/L was utilized. At this fruit date concentration a biobutanol production value of 1.56 g/L was obtained. On the other hand, the maximum yield of biobutanol (0.48 g/g) and ABE (0.63 g/g) was produced at 50 g/L date fruit concentration with a biobutanol production value as high as 11 g/L. However, when a higher initial date fruit concentration was used, biobutanol and ABE production decreased to reach the yield of 0.22 g/g and 0.35 g/g, respectively, where 100 g/L date fruit was used. Similar results also revealed that 10.03 g/L biobutanol was produced using 100 g/L date fruit

Kinetics of surfactin production by Bacillus subtilis in a 5 L stirred-tank bioreactor

A kinetic model of bacterial growth and metabolite production can adequately explain the trends and interaction of important parameters in a fermentation process. Production of surfactin by two bacterial strains, namely, Bacillus subtilis MSH1 and Bacillus subtilis ATCC 21322, in a 5 L bioreactor was investigated using Cooper’s media with 4% (v/v) glucose. The present kinetic study was carried out in order to determine the correlation between microbial cell growth, surfactin production and glucose consumption. Batch fermentation was performed by cultivation of each selected strain in a bioreactor at 30°C for 55 h. The experimental results showed production of surfactin in the culture medium after 5 and 10 h of incubation for B. subtilis ATCC 21332 and B. subtilis MSH1, respectively, at which the bacterial cells were at an early stage of the log phase. The maximum concentration of surfactin (Pmax) achieved by B. subtilis MSH1 and B. subtilis ATCC 21332 was 226.17 and 447.26 mg/L, respectively. The kinetic study of bacterial cell growth of both strains indicated that B. subtilis MSH1 had a specific growth rate (μmax) of 0.224 h-1 and attained a maximum biomass concentration (Xmax) as high as 2.90 g/L after 28 h of fermentation, while B. subtilis ATCC 21332, with μmax of 0.087 h-1, attained an Xmax of 2.62 g/L after 45 h of incubation. B. subtilis MSH1 showed higher growth kinetics, thus exhibited higher values of μmax and Xmax compared with B. subtilis ATCC 21332 under identical fermentation conditions. The Pmax achieved by B. subtilis ATCC 21332 was 447.26 mg/L, two times higher than that achieved by B. subtilis MSH1 (226.17 mg/L). The results obtained provide kinetics information including values of Pmax, μmax and Xmax for better understanding of interactions of bacterial cell growth and glucose consumption towards surfactin production by a commercial strain of B. subtilis ATCC 21332 and a local isolate of B. subtilis MSH1