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 Cu 2+ 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 30�C 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 Fe 3+ 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 Mg 2+, 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, Mg 2+ and Fe3+ have significant positive effect on the lipid accumulation of this fungus, whereas Mn2+ and Cu2+ exert negative effects in combination.

The influence of the presence of ammonium ions in growth culture on malic enzyme (ME) isoforms activity and lipogenesis in Cunninghamella bainieri 2A1 was investigated. The fungus was cultivated in a nitrogen-limiting medium for 120 h at 30°C under two conditions. One of the cultures was intermittently fed with ammonium tartrate to maintain the ammonium concentrations above 0.5 g/L. The second culture was performed without any feeding to allow N limitation, thus promoting lipid accumulation. Activity staining of ME isoforms was carried out for both cultures. The culture which was not intermittently fed with ammonium tartrate achieved a maximum lipid content of 35% (g/g biomass) at 48 h. This culture possessed five ME isoforms (A, B, C, D and E) with isoform E showing a parallel correlation to lipid accumulation profile. In contrast, intensity of bands representing isoform D decreased as lipid accumulated. No appreciable differences of all other isoforms were observed. However, the culture which was intermittently fed with ammonium tartrate, accumulated only up to 16% lipid (g/g biomass). All isoforms were present but with a more pronounced activity of isoform D and a lower activity of isoform E was observed. These findings support further evidence that isoform E is the key isoform for lipid synthesis in C. bainieri 2A1.

Inoculum for a local isolate of oleaginus fungi, Cunninghamella bainieri 2A1 was developed by establishing the inoculum types, age and size for growth, lipid and gamma linolenic acid (GLA) production. Cultivation was carried out in 500 mL shake flask containing 200 mL of nitrogen limiting medium at 30°C and 250 rpm agitation. Direct inoculation of spores into the production cultures gave higher productivity of lipid production 0.71 (g/L/day) than using 24 and 48 vegetative cells cultures which gave only 0.51 and 0.45 (g/L/day), respectively. Besides, production of GLA (5.3 × 10 -2 g/g lipid less biomass) in the cultures which directly inoculated with spores (1 × 10 5 spores/mL) was 23% higher than those produced in the cultures started with vegetative cells. Spore concentration of 1 × 10 3 spores/mL produced pelletal growth in size of 1.04 mm which contain 40% (g/g biomass) lipid and 8.34 × 10 -2 (g/g lipid less biomass).

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 CLA 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 x 10-2(g/g lipid less biomass), respectively.

Strategic feeding of ammonium and metal ions (Mg2+, Mn 2+, Fe3+, Cu2+, Ca2+, Co 2+, and Zn2+) for enhanced GLA-rich lipid accumulation in C. bainieri 2A1 was established. When cultivated in nitrogen-limited medium, the fungus produced up to 30% lipid (g/g biomass) with 12.9% (g/g lipid) GLA. However, the accumulation of lipid stopped at 48 hours of cultivation although glucose was abundant. This event occurred in parallel to the diminishing activity of malic enzyme (ME), fatty acid synthase (FAS), and ATP citrate lyase (ACL) as well as the depletion of metal ions in the medium. Reinstatement of the enzymes activities was achieved by feeding of ammonium tartrate, but no increment in the lipid content was observed. However, increment in lipid content from 32% to 50% (g/g biomass) with 13.2% GLA was achieved when simultaneous feeding of ammonium, glucose, and metal ions was carried out. This showed that the cessation of lipid accumulation was caused by diminishing activities of the enzymes as well as depletion of the metal ions in the medium. Therefore, strategic feeding of ammonium and metal ions successfully reinstated enzymes activities and enhanced GLA-rich lipid accumulation in C. bainieri 2A1. � 2014 Shuwahida Shuib et al.