Abdul Rahim, Raha

Ariff, Arbakariya

Mohamad, Rosfarizan

Mohd Yusuf, Hirzun

Universiti Putra Malaysia Institutional Repository

UPM Research Report 1997-2000. Vol II.Section 2-Extended AbstractsImprovement of Baker's yeast production using fed-batch fermentationArbalcsrlya Bin Ariff, Ro5farlZJln Mohama", Raha Ab"ul Rahim anel Hirzun Mohel Yu50fInstitute of BioscienceFacultyof Food Science and BiotechnologyUniversiti Putra Malaysia43400 UPM, Serdang, SelangorMalaysiaTelephone Number of Corresponding Author: 03- fI65fi6427/lI946l1342E-mail o[CorrespondingAulhor:arharif@f.~h.upm.edu.myKey words: Sa ..,-haronrycescemisae, fed-batch fermentation, shear rate, stirred tank fermenterIntroductionYeast metabolism uses nutrients to synthesise new cell material as well as to generate energy for survival. Metabolically, yeastarc mostly facultative aerobes. capable of growing either in the absence of air (fermentative) or in its presence (oxidative).Therefore, in the absence of aeration, yeast has the ability to instantaneously change its respiratory metabolism from oxidativeto fermentative. This catabolic shift is referred to as the "Pasteur effect" or "Glucose effect".The growth characteristics of S. cerevisae are varied depending on the culture conditions and medium composition. Growth ofyeast is greatly influenced by the level of dissolved oxygen concentration in the culture and the different carbon sources used.The growth kinetics of yeast in adaptation to variation in sugar concentration has also been well documented. Growth kineticsinformation re required for the improvement of Baker's yeast fermentation for production of products in the preparation ofbread dough. Subsequently, studies in applications of genetic engineering techniques become very popular due to theincreasing demands of the industry to improve strains of yeast.Control strategies and suitable mode of fermenter operation in industrial aerobic fermentation have been developed tomaximize the growth of yeast and minimize the detrimental factors affecting the yeast's growth patterns. For example, fed-batch fermentation, the process whereby nutrients necessary for cell growth are fed into the culture during the fermentation,has been found to be particularly effective for processes in which effects such as substrate inhibition, catabolite repression, andproduct inhibition are important. In Baker's yeast fermentation, fed-batch culture is devised to prevent a detrimental effect toyeast growth because of high initial glucose concentration as normally employed in conventional batch fermentation. The rateof oxygen transfer from the dispersed air bubbles to the fed-batch culture of high cell density can become the rate limitingfactor which changes the growth characteristic of the yeast. This is. another problem associated with Baker's yeastfermentation. The main objective of the study was to investigate the effect of agitation speed; and hence volumetric oxygentransfer rate (Kja) on the performance of yeast cultivation. The feasibility of using fed-batch fermentation technique forimprovement of yeast cultivation was also investigatedMaterials and MethodsThe Baker's yeast, Saccharomyces cerevisae, obtained Global Yeast Industry, Turkey was used in this study. Lyophilizedculture was rehydrated in YM liquid medium and was incubated for 10 h at 30°C. Stock culture was maintained on a DifcoYM agar medium at 4°C and was transferred onto a new medium every month to maintain viability. Starter culture wasprepared by transferring two loops of the stock culture to a 500 mL Erlenmeyer flask containing 150 mL of an inoculummedium and incubated at 30°C for 18 h on a rotary shaker at 200 rpm.Batch fermentation studies of S. cerevisae in a 2 L stirred tank fermenter (B. Braun, Biotech., Germany) were undertaken togenerate kinetic growth data of the yeast to provide a background information for the design of fed-batch fermentation. Thevariables studied in batch fermentation include the use of different concentrations of carbon and nitrogen sources and the effectof agitation speed (ranging from 200 to 1200 rpm). The fermentation using a 2 L fermenter was started with the addition of150 mL of yeast starter culture to 1350 mL of the production medium at 30°C.The same fermenter was also used for fed-batch fermentation, but it was connected to the multifermenter control system(MFCS) for controlling the feeding rate of the substrate to culture and also to control some of the fermenter operatingvariables. Control of the peristaltic pump is facilitated using MFCS/win software, which is a fermenter supervisory control anddata acquisition system (SCADA) for simultaneous control of multiple fermenters. This 32-bit PC-based application, usingalgorithm or a profile inputs, was interfaced to the fermenter control unit for controlling the speed of the peristaltic pumpaccording to the given algorithm The exponential fed-batch fermentation was started after the glucose in the initial batch. 600mL of culture, became depleted. Several fed-batch fermentations were carried out at different required specific growth rate (p)of the yeast. In all fermentations. the culture pH was controlled at 5.0, while the level of dissolved oxygen tension (DOT; wasmonitored. During the fermentation, samples were withdrawn at time intervals for analysis. Cell growth was determined as drycell weight using filtranon and o..en dry method. Viability of yeast cell was determined using methylene blue staining method.Glucose was determmec using glucose analyzer (), while gas chromatography was used for ethanol determination.Results and DiscussionIn batch fermentation of Baker's yeast, after a very short lag phase (about 3 min), the yeast grew exponentially withconcomitant increased in ethanol concentration in the culture. Due to diauxic growth, after glucose in the culture became306Agricultural Sciencesdepleted, the yeast started to consume the ethanol for second phase of growth. Oxygen requirement for growth using glucosewas significantly lower than growth using ethanol as carbon source. Initial concentration of glucose greatly influenced thegrowth characteristics of the yeast. The final cell concentration attained at the end of fermentation was increasedproportionally with initial glucose concentration up to 120 g/L, which gave more or less a constant cell yield of 0.13 g cell/gglucose. However, the specific growth rate of the yeast was reduced with increasing glucose concentration. The amount ofethanol accumulated in the culture was also increased proportionally with increasing glucose concentration. In term of overallproductivity, the highest (0.35 g/Lh) was obtained in fermentation using 80-120 g/L glucose.Nitrogen source used in the medium formulation also greatly influenced growth characteristics of the yeast. Growth wasgreatly inhibited in fermentation using inorganic nitrogen source (NH4CI and NH42S04) as the sole nitrogen source. Growthwas enhanced when organic nitrogen source (peptone and yeast) was used as nitrogen source. In this study, the optimizemedium formulation for batch cultivation of yeast was proposed.Different growth characteristics of yeast were also observed at different agitation speeds, which provided different volumetricoxygen transfer rate (Kla) to the culture. The Kla at 200 rpm was only 3.7 h-' and the Kja was increased to 82.46 h-I at 1000rpm. The final cell concentration attained during the fermentation was increased from 29.8 g/L at agitation speed of200 rpm to41.9 g/L at 1000 rpm. However, a slight decrease in cell viability was observed with increasing agitation speed, indicating thathigh shear rate caused damage to the cells.High density cell cultivation was achieved in exponential fed-batch fermentation process. During the fed-batch fermentation,the feed rate of the substrate is increased according to the exponential growth of the yeast at specific growth rate below themaximum. Among the specific growth rate (0.1 to 0.4 h-I) employed for the exponential fed-batch fermentation, the highestcell concentration (90.0 g/L) was obtained at specific growth rate of 0.1 h-I, which was associated with very small quantity ofethanol accumulated in the culture during the fermentation. The cell yield (0.5 g cell/g glucose) and overall productivity (6.2g/Lh) obtained in fed-batch fermentation was significantly higher than those obtained in batch fermentation, with more or lessabout the same cell viability was maintained.From this study, it was found that the fed-batch fermentation can be used to avoid engineering limitation and also useful toexert a metabolic control in Baker's yeast fermentation. The sugar limitation that can be maintained during exponential fed-batch fermentation can be utilized to avoid extensive overflow metabolism, which otherwise would result in too high ethanolproduction and an accompanying reduction of biomass yield. Even if the ethanol is subsequently consumed, the total biomassyield from glucose is reduced when the combustion to carbon dioxide passes via ethanol, as can be concluded from yieldcalculations. This decrease of the yield has been observed in batch fermentation where growth of yeast was in anaerobic mode,i.e. part of glucose supplied to eulture was used for ethanol production. The overflow metabolism of sugar to ethanol is due toa restriction in the carbon metabolism, somewhere after the pyruvate. When subjected to sugar concentrations above a certaincritical value, the glycolysis rate exceeds a critical value and the cells can not fully oxidize all the available sugar to CO2,Some of the sugar uptake is converted to ethanol, even under aerobic conditions. This critical value was determined to be I IQmg/L glucose during Baker's yeast fermentation with feed of molasses. For the cultivation using a defined glucose medium,the critical value of 40 and 50 mg/L have been reported for fed-batch and continuous fermentation, respectively. A sugarconcentration above the critical value, ethanol is formed. On the other hand, at concentration below the critical value, ifethanol is present, it will be consumed concomitantly with sugar.In our study, fed-batch fermentations of Baker's have also been carried out in 100 L fermenter. When cultivation areperformed in large scale fermenters, concentration gradient of substrate will arise due to the combined effect of limited mixingand mass transfer, and microbial activities. Limitation in mixing is increased with increasing fermenter size and become morepronounced when the fed-batch fermentation is used, since the rate limiting substrate is fed as a concentrated solution, oftenfrom only one point of the fermenter.ConclusionsFed-batch fermentation with suitable control strategies has been established for high density cell cultivation of Baker's yeast.Using optimize medium, glucose as a carbon source and yeast extract as a nitrogen source, a final cell concentration of 90.0g/L was achieved in fed-batch fermentation. This gave a yield and overall productivity of 0.5 gig and 6.2 gIL.h. This techniqueof fermentation greatly improved the performance on Baker's yeast fermentation as compared to conventional batch process.Benefits from the studySeveral locally isolated yeasts have been isolated and characterized. Some of which have potentials to be used for commercialapplications. Standard operating procedure for industrial production of Baker's yeast, using efficient fed-batch fermentationtechnique, has been established. Important parameters for the scaling-up of the process have been identified. proposed anddescribed. The same technology could also be used for production of various products from yeast (eg. amino acids,gluthathione, single cell protein and ~-glucosidasc). This is a strategic and important future technology for the country and theprocess is environmental friendly. Small scale production for laboratory use is being carried out at UPMPatent{s), if applicable:NilStage of Commercialization, if applicable:Nil307UPM Research Report 1997-2000, Valli, Section 2-Extended AbstractsProject Publications in Refereed Journals:NilProject Publications in Conference Proceedings:NilGraduate Research--- ._-------------------------_._-----_._---- ----------------Name of Graduate Research Topic Field of Expertise Degree Awarded Graduation YearTan Li Lung Characterization oflocally isolatedyeast strain towardsindustrialapplicationFennentationTechnologyMSc 2003---------_. ----------Ahmad AriIT Development ofFed-batchfermentation forefficient Baker'syeast productionFermentationTechnologyMSc Expected to begraduated 2004IRPA Project number: 03-02-04-0135UPM Research Cluster:BAB308

Improvement of Baker's yeast production using fed-batch fermentation

http://psasir.upm.edu.my/42177/1/improvement%20of%20baker%27s%20yeast.pdf

Improvement of Baker's yeast production using fed-batch fermentation

Abstract

Similar works

Full text

Available Versions

Universiti Putra Malaysia Institutional Repository