World Journal of Microbiology & Biotechnology 20: 297–301, 2004. 2004 Kluwer Academic Publishers. Printed in the Netherlands.
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Production of single cell protein from rice polishings using Candida utilis M. Ibrahim Rajoka1,*, M.A. Tariq Kiani2,3, Sohail Khan2,3, M.S. Awan1,3 and Abu-Saeed Hashmi2,3 1 National Institute for Biotechnology and Genetic Engineering, P.O. Box 577, Faisalabad, Pakistan 2 Department of Animal Nutrition, University of Agriculture, Faisalabad, Pakistan 3 Department of Food Technology, University of Agriculture, Faisalabad, Pakistan *Author for correspondence: Tel.: þ92-041-651475/550815, Fax: þ92-041-851571/651472, E-mails: mirajoka@ nibge.org,
[email protected] Received 15 July 2003; accepted 25 November 2003
Keywords: Amino acids, Candida utilis, rice, single cell protein, yeast
Summary Microbial protein was produced from defatted rice polishings using Candida utilis in shake-flasks and a 14-l fermentor to optimize fermentation conditions before producing biomass in a 50-l fermentor. The organism supported maximum values of 0.224 h)1, 0.94, 1.35, 1.75, 2.12 g l)1 h)1, 0.62 g cells g)1 substrate utilized and 0.38 g g)1 for specific growth rate, true protein productivity, crude protein productivity, cell mass productivity, substrate consumption rate, cell yield, crude protein yield, respectively in 50-l fermentor studies using optimized cultural conditions. Maximum values compared favourably or were superior to published data in literature. The biomass protein in the 50-l fermentor contained 22.3, 27.8, 19.2, 9.5, 38.12, 8.5 and 0.27% true protein, crude protein, crude fibre, ash, carbon, cellulose and RNA content, respectively. The dried biomass showed a gross metabolizable energy value of 2678 kcal kg)1 and contained all essential and non-essential amino acids. Yeast biomass as animal feed may replace expensive feed ingredients currently being used in poultry feed and may improve the economics of feed produced in countries like Pakistan.
Nomenclature )1
cell mass (g l ) substrate (g l)1) rate of cell mass formation (g cells l)1 h)1) rate of substrate consumption (g l)1 h)1) cell yield coefficient (g cells g)1 substrate utilized) qS specific rate of substrate consumption (g g)1 cells h)1) l specific growth rate (h)1) QCP rate of crude protein formation (g l)1 h)1) YCP/X specific yield of crude protein (g crude protein g)1 cells) Protein concentrate dry whole fermented rice polishings containing cell mass DO dissolved oxygen (%) v/v/m air flow rate (volume of air per volume of fermentation medium per min) qCP specific rate of crude protein productivity (g g)1 cells h)1) X S QX QS YX/S
Introduction A growing concern for the acute food shortages for the world’s expanding population has led to the exploitation
of non-conventional food sources as potential alternatives. Among these, the single cell organisms probably present the best chances for the development of unique independence of agricultural (crop) based food supply. The protein obtained from the micro-organisms is cheap and competes well with other sources of protein and may provide good nutritive value depending, however, upon the amino acid composition. But balancing through high quality protein results in a higher cost of feeding. Hence, it is imperative to produce the biomass protein which is economical and quite comparable to animal protein (Singh et al. 1991). The poultry industry has played a major role in providing animal protein (in the form of eggs and meat) in Pakistan. The feed industry is facing immense shortage of quality feed ingredients both of vegetable and animal origin. One possible alternative is to ferment non-conventional agro-industrial wastes/residues which accumulate up to 50 million ton in this country (Anonymous 1988) including rice polishings (1.7 million ton annually) to produce single cell protein. It has a good potential as a supplemental protein source for feeding poultry, livestock and even humans (Pacheco et al. 1997). Candida utilis has been used frequently for production of single cell protein as it has the ability to consume
298 several types of substrates including wheat bran and produce several industrial products both for human and animal consumption (Christen et al. 1993; Kondo et al. 1997; Pacheco et al. 1997; Haddadin et al. 1998; Otero et al. 1998). Variables which are of great relevance to the economic evaluation of such biotechnological processes are cell mass and product formation kinetic parameters for upscaling to large scale bioreactors (Tobajas & Garcia-Calvo 1999). Effect of including rice polishings in the diet on rumen fermentation in vitro has been studied (Cardenas Garcia et al. 1992) but no information is available on its use for production of single cell protein. In this work, it was planned to develop and optimize a fermentation process for the production of biomass protein by culturing Candida utilis on defatted rice polishings consisting of 13.9, 11.82, 57.37, 40.35 and 9.7% crude protein, crude fibre, nitrogen free extract, carbon and cellulose, respectively and evaluate it for crude protein, RNA, metabolizable energy and amino acid content so as to find out its (single cell protein product) suitability for possible replacement/fortification of standard poultry rations.
Materials and methods Organism Candida utilis PPY 12 (a gift from Plant Pathology Culture Collection, University of Agriculture, Faisalabad) was maintained on rice polishings-agar slants. The inoculum was prepared by transferring a loopful of cells to 50 ml seed culture medium containing (g l)1) KH2PO4 5.0, (NH4)2SO4 5.0, CaCl2 0.13, MgSO4, 0.5, yeast extract 0.5 (pH 6.0 ± 0.1) (Tobajas & GarciaCalvo 1999) and grown at 30 C on an orbital shaker (150 rev min)1 for 3 days). Concentration of the organism was adjusted to contain 2.24 g dry cells l)1 of the fermentation medium in 1-l Erlenmeyer flasks, 14 l New Brunswick Microferm or 50-l fermentor. The ability of the organism to produce cell mass and protein from rice polishings as a sole carbon and energy source was examined in optimized salt medium containing (g l)1): KH2PO4 0.25, CaCl2, 0.025, KCl 0.5, MgSO4, 0.025, yeast extract 0.3, urea 4.22 [in place of (NH4)2SO4 5.0], and defatted rice polishings 9.0. All media were adjusted to pH 6.0 with 1 M NaOH or 1 M HCl. Cultivation Fermentation was carried out in 14-l fermentor (New Brunswick, USA) using 9 l of the optimized salt medium and in 50-l fermentor using 30 l medium using optimized cultural conditions. The medium was steam sterilized in situ in the case of fermentors. The medium was inoculated with a vegetative inoculum of 24-h-old culture. Standard operating conditions for the fermentor run were, temperature, 35 C; agitation, 400 rev min)1; controlled pH (pH 6.0), temperature (35 C), antifoam
M.I. Rajoka et al. and air flow rate, 2 v/v/m. The DO was controlled at 50% saturation through out the production period. Production process was carried out under four different percentages of oxygen i.e. 30, 40, 50 and 60% during the run. Analytical methods Flasks/samples in triplicate were taken at different times to determine concentration of cell, solid substrate, crude protein, true protein, crude fibre, nitrogen free extract and RNA (Pacheco et al. 1997; Paul et al. 2002). Culture samples (100 ml) were centrifuged (7000g at 10 C for 10 min) to remove substrate. The substrate was washed twice with saline and dried to estimate unutilized solid material. The culture broth (100 ml portion) was also centrifuged (10,000g, 10 min). The cell mass was washed twice with saline, suspended in 10 ml distilled water and dried at 70 C. The remaining 100 ml sample, containing cell mass and unutilized substrate was dried (called dried biomass). The dried biomass was routinely analysed for crude protein, true protein, and RNA (Pacheco et al. 1997; Paul et al. 2002). Micro-Kjeldahl’s nitrogen of dry biomass was multiplied with 6.25 to calculate crude protein. For determination of true protein, 0.5 g homogenized and dry biomass was treated with 20 ml (5% v/v) trichloroacetic acid for 5 min with shaking and then placed at 90 C (in an oven) for 15 min and shaken occasionally. It was filtered while hot and the residue was rinsed with hot water thrice and dried to a constant weight. Its nitrogen content was determined with micro-Kjeldahl’ method and true protein was calculated as above. Total RNA content was determined using orcinol–HCl reagent as described previously (Pacheco et al. 1997; Paul et al. 2002). The calorific value of dry biomass was determined using the Parr method (Hill & Anderson 1958) using a Parr oxygen bomb calorimeter. The calorific value was calculated by the amount of heat generated by the combustion of a known weight of the sample in the presence of 20 atmospheric pressure of oxygen. Moisture, dry matter, ash, ether extract, crude fibre, nitrogen free extract, carbon and cellulose were analysed according to AOAC (1984) methods. Amino acid analysis and chemical score The amino acid profile of biomass produced was determined by automatic amino acid analyser (Evans Electro-selenium Limited, UK). Chemical score of the biomass product was calculated following method of FAO/WHO (1957). Determination of kinetic parameters All kinetic parameters were determined as described earlier (Pirt 1975; Zayed & Mostafa 1992). Volumetric rate of crude and true protein production (QP) was determined from a plot between proteins (g l)1) and time of fermentation, process product yield (YP/S) was
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Single cell protein production from rice polishings determined from dP/dS, specific product yield (YP/X, g g)1 cells) was determined using relationship dP/dX, and volumetric rate of substrate consumption was determined from a plot between solid substrate (g l)1) present in the fermentation medium and time of fermentation. Cell mass productivity expressed as g dry cells l)1 h)1 was determined from a plot of g dry cells l)1 and time of fermentation. Specific growth rate was determined from the relationship lt ¼ ln Xt/X while specific productivity was a multiple of l and YP/X.
Results and discussion Optimum conditions for biomass production Initially, studies were performed in shake flasks (in time course studies) to produce crude protein to optimize fermentation conditions. Among concentrations of rice polishings, 9% rice polishings supported significantly (P £ 0.05) higher protein productivity and other parameters except cell yield. Thus 90 g rice polishings l)1 in the medium was found to be adequate to support 0.88 g l)1 h)1, 0.36 g g)1, 0.84 g g)1 cells, 0.53 g g)1 and 0.29% crude protein productivity, crude protein yield, crude protein’s specific yield, cell mass yield and lower RNA accumulation in the cells, respectively. Gradual reduction in protein formation, cell mass synthesis and substrate consumption rates was observed when the rice polishings concentration was increased.
There was enhanced substrate metabolism by aerobic pathway, resulting in build up of high cell mass (maximum cell mass (X) is equal to 32.5 g l)1) (Figure 1). The values of above process variables were higher than those reported previously (Zayed & Mustafa 1992; Nigam 2000). Effect of different carbon:nitrogen ratios Carbon:nitrogen ratios in fermentation processes influence formation of protein concentrates. To explore the influence of this variable on production of crude protein and RNA, ratios were obtained by increasing the urea concentration in the medium. A ratio of 10:1 supported the maximum values of 0.97 g l)1 h)1, 0.35 g g)1, 0.85 g g)1 cells, 0.53 g g)1substrate and 0.29% crude protein productivity, crude protein yield, crude protein’s specific yield, cell mass yield and l RNA accumulation in the cells, respectively. All these kinetic parameters were higher than those obtained with other ratios. Generally, the results confirmed that urea, a low-cost fertilizer, supported maximum profiles of crude protein productivity compared to other ratios, and confirmed the findings of Hashmi (1986). Effect of aeration rate and agitation intensity (DO) in 14-l fermentor Various experiments were conducted in 14-l fermentor to establish fermentation conditions like dissolved
Figure 1. Kinetics of crude protein (CP), true protein (TP) and cell mass (X, g/l) production and solid substrate (S, g/l) present in shake flask (a) 14 l (b) and 50 l (c) fermentors, respectively during fermentation of rice polishings with Candida utilis. The pH of the medium was 6.0, inoculum size 10%, substrates 9% (w/v), C:N ratio 10:1 and fermentation temperature 35 C in each case. Optimum dissolved oxygen concentration was 50% and air flow rate 2.0 v/v/m in case of fermentors. Error bars show standard deviation among three observations. s ¼ Crude protein, n ¼ true protein, h ¼ cell mass and d ¼ solid substrate in the fermentation medium.
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oxygen tension (DOT), air flow rate in the growth medium for the maximum production of biomass protein from defatted rice polishings before upscaling to 50-l fermentor. The incubation temperature was kept at 35 C throughout the fermentation period of 3 days. Agitation speed of the stirring was kept at 400 rev min)1 and the aeration rate was maintained at 2.0 v/v/m to control dissolved oxygen (DO) at 50% during control operation. Sterilized silicone oil was used to control the foaming during fermentation. The effect of varying DO levels indicated that crude protein of 26 g l)1 (Figure 1) was realized in 72 h when the DO was maintained at 50% saturation. It was also observed that increasing the DO level above 50% saturation did not further enhance product formation, thereby indicating that maintaining DO level above a certain value was important for protein production rather than the saturation percentage of DO. The presence of antifoam agent also permitted increased aeration. Similarly 2.0 l l)1 min)1 level of aeration was optimized for crude protein production in stirred fermentor. Maximum values of 0.94 g l)1 h)1, 0.36 g g)1, 0.84 g g)1 cells, 0.54 g g)1 and 0.28% crude protein productivity, crude protein yield, crude protein’s specific yield, cell mass yield and RNA in the cells, respectively were noted. The values of all kinetic parameters are higher than those reported by Nigam (2000) and Paul et al. (2002). Further experiments were performed with Candida utilis in shake flasks, 14- and 50-l fermentors, respectively in optimized fermentation conditions. Figure 1 shows the kinetics of crude protein production, cell mass formation and solid substrate present in the medium in shake flasks (a), 14-l fermentor (b) and 50-l fermentor (c). All values of fermentation variables increased progressively from shake flask to 14- and 50-l fermentors (Table 1) and were significantly higher than those reported by Nigam & Vogel (1991), Ichii et al. (1993) and Lee & Kim (2000) and were attributed to the
Table 1. Kinetic parameters for crude protein, true protein and cell mass formation following growth of Candida utilis on rice polishings in 14- and 50-l fermentors under optimized fermentation conditions. Parameter
Shake flaska
14-l fermentorb 50-l fermentorb
lm(per h) QTP (g l)1 h)1) QCP (g l)1 h)1) QX (g l)1 h)1) QS (g l)1 h)1) YX/S (g g)1) YTP/S (g g)1) YCP/S (g g)1)
0.24 0.60 0.90 0.45 0.59 0.45 0.19 0.28
0.24 0.75 0.98 0.69 1.09 0.54 0.20 0.34
a
± ± ± ± ± ± ± ±
0.012 0.05 0.06 0.07 0.04 0.03 0.01 0.01
± ± ± ± ± ± ± ±
0.015 0.06 0.072 0.083 0.091 0.026 0.011 0.012
0.32 0.94 1.13 1.15 1.28 0.62 0.26 0.38
± ± ± ± ± ± ± ±
0.021 0.057 0.076 0.079 0.078 0.031 0.013 0.023
Culture was grown under optimized conditions: substrate concentration 9%, C:N ratio 10:1, fermentation temperature 35 C and shaking speed 150 rev min)1. b Culture was grown under optimized conditions: substrate concentration 9%, C:N ratio 10:1, fermentation temperature 35 C and DO level 50%. Each value is a mean of three independent readings. ± stands for standard deviation among three readings.
Table 2. Nutrient composition (%) of defatted rice polishings and the Candida utilis single cell protein product. Nutrients
Rice polishings
SCP product
Moisture Dry matter Crude protein True protein Ether extract Ash Crude fibre Nitrogen free extract Carbon Cellulose RNA Calorific value (kcal kg)1)
2.50 97.50 13.90 0 3.01 3.01 11.82 57.37 40.35 9.76 0 0
0 100.00 32.75 23.62 4.68 12.95 11.50 0 38.12 10.50 0.275 2971.70
± 0.1 ± 3.5 ± 0.5 ± ± ± ± ± ±
0.2 0.1 0.6 2.0 1.5 0.5
± ± ± ± ± ±
4.5 1.45 1.1 0.15 0.52 0.51
± ± ± ±
1.65 0.52 0.01 75
Each value is a mean of three readings. ± stands for standard deviation among three independent analyses.
presence of high enzyme activities of the organism (Villas-Boas et al. 2002) and in the case of fermentors, due to higher aeration and mass transfer rates. Compositional analysis of single cell protein product obtained with C. utilis (Table 2) revealed that dried biomass was rich in crude protein, true protein, amino acids and low in RNA and was found superior as compared with those reported by Paul et al. (2002), Singh et al. (1991) and Nigam (2002). RNA content of dry fermented rice polishings with yeast cells was found to be 0.275% which is significantly lower than the values reported by Paul et al. (2002) and Singh et al. (1991) and is apparently toxicologically non-offensive as observed with higher values of RNA in fungi and yeasts (Nigam & Vogel 1991; Singh et al. 1991; Paul et al. 2002). The improvement of crude protein from 13.90 to 27.8% with 22.3% as true protein (Table 2) indicated a great deal of urea metabolism by the yeast. Of this nitrogen, most was utilized in the formation of true amino acids. The single cell protein product reported by Singh et al. (1991), contained 30.4% crude protein while Kluyveromyces fragilis biomass grown on deproteinized whey supplemented with 0.8% diammonium hydrogen phosphate contained 37% crude protein (Paul et al. 2002). The dried biomass showed a gross metabolizable energetic value of 2678 kcal kg)1. The calorific value clearly indicated that the biomass can serve as an energy source besides protein, and amino acids particularly when it may be fed to poultry. As evident from Table 3 and 4, the biomass protein contains 16 amino acids, like rice polishings. The chemical scores for seven selected essential amino acids indicate comparative values to FAO (1957) reference pattern in mg g)1 and confirmed the findings of Nigam (2000). The only limiting amino acid was found to be valine. Acknowledgements This work was supported by the Pakistan Atomic Energy Commission and University of Agriculture,
Single cell protein production from rice polishings Table 3. Amino acid profile (g 100 g)1) of defatted rice polishings and SCP protein product obtained through its conversion with C. utilis.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Amino acids
Rice polishings
SCP product
Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine
0.93 0.43 0.45 1.82 0.30 0.66 0.60 0.39 0.22 0.43 0.77 0.27 0.54 0.71 0.16 0.91
1.32 0.60 0.64 3.20 0.74 0.75 1.18 0.54 0.44 0.81 1.44 0.86 0.98 1.24 0.19 0.82
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.07 0.03 0.03 0.08 0.01 0.025 0.023 0.015 0.01 0.025 0.04 0.02 0.04 0.05 0.01 0.04
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.12 0.05 0.04 0.2 0.045 0.06 0.12 0.04 0.032 0.045 0.12 0.054 0.057 0.11 0.012 0.045
Test samples were hydrolysed with HCl and analysed using an automatic amino acid analyser (Evans Electroselenium Limited, UK) in triplicate. Each value is a mean of three independent readings. ± stands for standard deviation among three independent analyses.
Table 4. Chemical score of SCP product from defatted rice polishings with C. utilis using FAO (1957) amino acid pattern. Amino acid
Amino acid pattern (mg g)1)
SCP product amino acid (mg g)1)
Available amino acid (%)
Lysine Leucine Isolecucine Phenylalanine Methionine Threonine Valine
42 48 42 28 22 28 42
44.59 51.78 29.14 35.59 15.83 21.58 19.42
106.17 107.88 69.36 127.11 71.96 77.08 46.24
Each value in column 3 is a mean of three independent analyses. Standard deviation among replicates varied between 3.50 and 7.5% and have not been presented. Valine was found to be deficient according to FAO (1957) standard.
Faisalabad. The authorities of both organizations are appreciated for their support. These studies formed a part of thesis works of SK, and MATK. Technical staff of both Institutes are thanked for expert technical assistance.
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