Cryptococcus curvatus Essays

Cryptococcus curvatus Essays Cryptococcus curvatus Essay Cryptococcus curvatus Essay Introduction An buttery barm, Cryptococcus curvatus, once named as Apiotrichum curvatum and Candida curvata D, was found to be accumulated more than 60 % [ 1,2,4 ] of it dry weight in the signifier of intracellular lipoid when grown under extra C beginning with restricting growing of N. This accrued lipoid is formed as triglyceride in which the fatso acerb composing is similar to the works seed oil in specific for the chocolate butter equivalents ( CBE ) production. Cocoa butter is widely used in cocoa fabrication which obtained from the seeds of chocolate tree ( theobroma chocolate tree ) . This fat chiefly consist of its chief triacrylglycerol 1,3-disaturated-2-unsatuated triacrylglycerols with high sum of stearic acid ( 32-37 % , C 18:0 ) , palmitic acid ( 23-30 % , C16:0 ) and oleic acid ( 30-37 % , C18:1 ) . The monetary value of chocolate butter is really fluctuating and even exceeds $ 8000/ton in early 1980s. The high monetary value made this yeast oil becomes an attractive market. It was found that the fatty acid profile of Cryptococcus curvatus, it has low per centum of stearic acid C18:0 ( 12-15 % ) , and high per centum of oleic acid C18:1 ( 44-49 % ) . The undertaking of production of executable yeast oil as CBE is to increase the stearic acid content and guarantee the right distribution of fatty acerb profile of the ensuing triacylglycerol that warrant this yeast oil run into the rigorous demand of CBE in cocoa. In effort to successfully commercialize barm oils, the production has to accomplish feasibleness and profitableness. These factors in individual cell oil ( SCO ) production chiefly depend on whether the procedure can bring forth high valued lipoids and fatty acid with specific physical, chemical and nutritionary belongingss. [ 1-2 ] Meanwhile, the procedure cost in agitation is besides of import that determined by substrate and fermentor cost. Therefore, the lipid production rate and output should be every bit high as possible with the usage of the inexpensive C beginning like whey permeate in order to accomplish economic sciences executable. [ 1 ] Biochemistry Lipid accretion in Cryptococcus curvatus begins when foods ( normally nitrogen ) is exhausted with extra C beginning ( normally in the glucose signifier ) in the medium. Excess glucose is assimilated continuously and converted to triacylglycerols. Under the limited N supply, the cells can no longer proliferate and unable to turn and split. Lipid may roll up in the bing cells continuously, cells rich in lipoid. ( a ) ( B ) Fig3. ( a ) Cryptococcus curvatus, lipid droplet lend 70 % of its cell weight ( B ) Extracted oil from Cryptococcus curvatus in negatron micrograph with boundary beds within the oil droplets ( triacylglycerol ) . The first biological reaction after nitrogen exhaustion is the AMP deaminase activation: AMP a† Ã¢â‚¬â„¢ IMP + NH4+ The rapid lessening in AMP concentration accompany with the alteration in O2 ingestion and CO2 end product of the cells. This will decelerate down or even discontinue the activities of isocitrate ( NAD+ -dependent ) dehydrogenase ( ICDH ) within chondriosome. The enzymatic activity is greatly influenced by AMP concentration. However, this activity is independent on the non-oleaginous barm. Nitrogen exhaustion consequence in the addition of AMP deaminase and lessening of AMP concentration with effect of rapid bead in ICDH activity. Isocitrate is no longer metabolized efficaciously and so equilibrate with citrate though aconitase. Isocitrate i?i?i?z Aconitate i?i?i?z Citate Citrate is transported outside the chondriosome and cleaved by an enzyme Adenosine triphosphate: citrate lyase ( ACL ) in the cytol, which is absent in the non-oleaginous barm. Citrate + ATP + CoA a† Ã¢â‚¬â„¢ Acetyl-CoA + ADP + Pi + oxalacetate The citrate cleaving enzyme, ACL is really important in fatty acerb synthesis. If cell absent from ACL like non-oleaginous micro-organisms, it is unable to roll up lipoid. Fig. Pathway of lipid accumlation in Cryptococcus curvatus The oxalacetate formed in above reaction converted into malate instantly by the enzyme, malate dehydrogenase. Malate + NADP+ a† Ã¢â‚¬â„¢ pryuvate + NADPH + CO2 Fatty acerb synthesis requires the uninterrupted supply of both acetyl-CoA and NADPH ( cut downing power ) . Acetyl-CoA are used to make the C edifice unit which the turning long fatty acyl concatenation is derived from acetyl-CoA. NADPH are used to cut down the turning long fatty acyl concatenation. Fatty acerb synthesis in Cryptococcus curvatus follows fatty acerb synthase ( FAS ) path. Elongase act on palmitic acid ( 16:0 ) to bring forth stear ( 18:0 ) . I-9 desaturase act as enzyme to catalyse the dual bond interpolation between C9 and C10 in fatty acid concatenation. Stearic acid ( 18:0 ) is converted to oleic acid ( C18:1 n-9 ) . Meanwhile, I-12 desaturase infix a dual bond between C12 and C13 in mono-unsaturated fatty acid concatenation. It facilitate the transition of oleic acid ( C18:1 n-9 ) to linoleic acid ( 18:2 n-6 ) . Production Agitation is utilized in the growing of micro-organisms and its biogenesis. Fermentater provides an effectual reactor to accomplish optimum conditions of micro-organisms by seting different parametric quantities. Within fermenter, there are few dimensions to optimise lipid production of Cryptococcus curvatus which are C: N ratio of substrate, civilization manners, the O demand in the civilization medium. Substrate used Many researches with Cryptococcus curvatus chiefly concentrate on using whey permeate in agitation of milk sugar. An attractive feature of this barm is it s able to turn on assortment of substrates. This implicated that many nutrient processing wastes apart from whey permeate can be used as substrates for lipid production. Other than assorted sugars as a C beginning, glycerin is besides an interested feedstock for lipoids production when biodiesel is produced with big commercial graduated table. [ 4 ] Influences on C to N ( C: N ) ratio There are two stage growing rhythm of Cryptococcus curvatus. The first phase is the growing stage where the cells grow up to the high cell denseness with low lipid accretion. The 2nd stage is the nitrogen exhaustion stage which the residuary C beginnings in medium are converted to the signifier of lipid, ensuing in the lipid accretion. When cells grow in the balanced civilization medium, with uninterrupted supply of N, lipid production by Cryptococcus curvatus will be less effectual. Nitrogen facilitate the synthesis of nucleic acid and protein for cell growing and cell division. Oxygen demand The capableness of O transportation is the most of import consideration in aerophilic agitation. The fermenter should be ensured to reassign equal O with high rate into the civilization medium so that non to restrict the growing Cryptococcus curvatus. However, the rate should non be excessively high that cause inordinate energy ingestion. In batch agitation of Cryptococcus curvatus turning on whey permeate, the extremum of the O uptake rate ( OUR ) is 13 mmoles/L/h under N limited status. This is a turning point from the growing stage of Cryptococcus curvatus to lipid accretion stage. The OUR is so bit by bit decreased at N exhaustion which is low oxygen demand with non inordinate cell denseness in fermenter. Therefore, a simple concept fermenter like airlift or bubble column constellation could be utilized. Comparing to the industrial stirred reactor, airlift or bubble column can be erected and constructed on site easy. Fig.1 shows the airlift fermenter design of using pealing sparger. Ring sparger is used to pump air into bill of exchange tubing. The stock is circulated upward in bill of exchange tubing vertically and downward in outer ringed part. The circulation provides good blending features in agitation. Airlift bubble column was used in industrial procedure. It was found that the lipid production rate ( LPR ) in airlift fermenter of 0.5m3 pilot works and 8.2m3 mill was 0.12 g/L/h and 0.06g/L/h severally. Both LPR was much lower comparing to the moved armored combat vehicle reactor on research lab graduated table. Using stirred armored combat vehicle reactor in a fed batch procedure has proven efficient in the growing and lipid production of Cryptococcus curvatus [ Meesters, 1995 ] . However, it is hard to scale up moved armored combat vehicle reactor taking to alter blending features and lessening O transportation rate in larger armored combat vehicle. The optimum status achieved in Meesters, 1995 is hard to execute in big graduated table, ensuing in the slower growing and production rate. Furthermore, when the agitation procedure require high O transportation rates, stirred armored combat vehicle fermenters is non sufficient. Oxygen transportation, good commixture and stirring may go restricting factors of production rate in syrupy systems like high cell denseness agitations. New type of reactor, Surer reactor is used to scale up the agitation. It consists of an external pumped cringle and 2 inactive sociables. Surer reactor mixes the medium exhaustively with no dead country by its forced flow feature. Air bubbles are reduced to really little bubbles by inactive sociables, ensuing in high O transportation rate. Installing more inactive sociables can accomplish much high O transportation rate. Besides, the commixture features will non be affected by the reactor s breadth. Scale up of Cryptococcus curvatus cultivation in Surer reactor achieve LPR 0.42 g/l/h which is much higher than scale up procedure of airlift fermenter 0.06 g/l/h mentioned by [ Davies 1992 ] [ 7 ] Culture manners There are four different civilization manners in cultivation of Cryptococcus curvatus: batch agitation ; fed batch agitation, uninterrupted agitation ; partial recycling agitation. Assorted civilization manners performed otherwise in the lipid production rate. Ykema [ 1 ] found that lipid production rates are faster in uninterrupted civilizations than in batch civilizations. However, highest lipid production rate rates will be achieved in civilization manners that enable the cultivation at high cell densenesss like fed-batch procedure. It was found that the fed-batch agitation performed most effectual in general. The procedure is two phases experiment. During the first stage, there is no restriction on foods, this status facilitate biomass production. The 2nd stage performs in N restrictions which cause lipid accretion. The fatty acerb composing varied in this two stage. The comparative sum of stearic acid C18:0 is low in the first biomass production stage, while the sum of oleic acid C18:1 addition, linoleic acid lessening C18:2 in the 2nd lipid accretion stage. Partial recycling civilizations aloe uninterrupted operation and increased productiveness by extinguishing batch downtime. Though partial recycling cultivation can accomplish a hi ghest lipid production rate, it is merely suited for small-scale operation. [ 4 ] Fed-batch agitation performed most efficaciously see to the big lipid production rate and big graduated table pertinence. Therefore, high lipid production rate can be achieved by seting C/N ratio and choose the civilization modes that the barm cultivated at high cell denseness. [ 1 ] Fig. LPR in different cultivation manner In the 80-90s century, different attacks have been performed to optimise the oil production of barm with the fatty acerb composing comparable to cocoa butter. For this ground the procedure has to increase the sum of steric acid in barm lipoid. It was shown that the consequence of low Os uptake rate altered the fatty acid profile. The grade of impregnation in fatty acid profile was increased by take downing the O uptake rate. With the low O concentration, I-9 desaturase enzyme used to covert stearic acid to oleic acid, elongation enzyme used to change over palmitic to stearic acid, are affected. Hence, the sum of stearic increased. The other straggy used a mutation in which theI-9 desaturase for the transition of strearate to oleate ( see besides fig.1 ) was partly blocked so that stearate accumulated at the disbursal of oleate. This guaranting lipid so had the correct belongingss for its usage as a chocolate butter equivalent. [ 3 ] Familial attack can besides spread out the scope of oils formed in Cryptococcus curvatus. It requires the transmutation and look system for the barm consequence in modifying fatty acerb biogenesis in the barm. I-9 desaturase cistron will be cloned and so to be use as a selective marker for the I-9 desaturase mutation of Cryptococcus curvatus. [ 5,8 ] The barm, Cryptococcus curvatus are possible manufacturers of lipoids similar to vegetable oils and fats. Research on their applications as a dietetic addendum in nutrient or indispensable pharmacological constituents in medical specialty has non progressed to desirable bounds. Since barms are polyploidy in nature, similar to workss, they may be appropriately engeernied so as to synthesise fresh lipoids which may happen public-service corporation in bring forthing value added oils anf fat for usage in the nutrient and biomedical industries. Rerouting of the barm s metabolic tract for the synthesis of biochemically of import polyunsaturated fatty acids has non received much attending. [ 4 ] Future Development Mentions 1. Adrie Ykema, Elizabeth C. Verbree, Martin M. Kater and Henk Smit, Optimization of lipid production in the buttery barm Apiotrichum curvatum in wheypermeate, Applied and Microbiology Biotechnology ( 1988 ) 29:211-218 2. R. Julian Davies, Jane E. Holdsworth and Sarah L. Reader, The consequence of low Os uptake rate on the fatty acid profile of the buttery barm Apiotrichum curvatum, Applied and Microbiology Biotechnology ( 1990 ) 33: 569-573 3. Z. Jacob, Advances in applied microbiology Vol 39, 1993 p.185-208 4. Meesters PAEP, Huijberts GNM, Eggink G. High-cell-density cultivation of the lipid roll uping yeast Cryptococcus curvatus utilizing glycerin as a C beginning. Appl Microbiol Biotechnol 1996 ; 45:575-9. 5. PATRICIA A. E. P. MEESTERS AND GERRIT EGGINK Isolation and Characterization of a A-9 Fatty Acid Desaturase Gene from the Oleaginous Yeast Cryptococcus curvatus CBS 570, Agrotechnical Research Institute, VOL. 12: 723-730 ( 1996 ) 6. Mainul Hassan, Philippe J. Blanc, Louis-Marie Grangerf Alain Pareilleux A ; Gerard Goma, Influence of Nitrogen and Iron Limitations on Lipid Production by Cryptococcus curvatus Grown in Batch and Fed-batch Culture, Process Biochemistry Vol. 31, No. 4, pp. 355-361, 1996 7. Patrica A.E.P. Meesters, Hetty van der Wal, Ruud Weusthuis and Gerrit Eggink, Cultivation of the buttery barm Cryptococcus curvatus in a new reactor with improved commixture and mass transportation characteristic, Biotechnology Techniques, Vol 10 No.4 ( April 1996 ) p.277-282 8. Colin Ratege and James P. Wynn, Advances in applied microbiology, Vol 51, 2002 p.1-46 9. C. Ratledge, Fatty acid biogenesis in micro-organisms being used for Single Cell Oil production, Biochimie 86 ( 2004 ) 807-815 10. Colin Ratledge, Peter Dawson, James Rattray, Biotechnology for the oils and fats industry, American Oil Chemists Society, c1984