Production Of Single Cell Protein Biology Essay

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ABSTRACT

The possibility of using Koji making fermentor for, Arachniotus citrinus and Candida utilis, single cell protein (SCP) production was investigated. The MBP was produced from deoiled rice bran in 300 Kg Koji making fermentor after optimize fermentation conditions in 250 ml flasks by solid state fermentation. The A. citrinus supported maximum values of substrate to water ratio (1:2), 0.05% MgSO4.7H2O, 0.075% CaCl2. 2H2O, 0.25% KH2PO4, C:N (12:1), 1ml molasses (10% solution), 0.6 ml yeast sludge, and 2 ml corn steep liquor while 2ml molasses (10% solution) and 0.25g urea for C. utilis for maximum crude protein productivity. The SCP in the 300 Kg Koji making fermentor contained crude protein, true protein, protein gain, ether extract, ash, crude fiber, and RNA content of 30.13 %, 23.74 %, 2.97 %, 14.71 %, 6.77 %, 3.383% respectively. The dried SCP showed a gross energy value of 3675 Kcal/kg and contained increase the levels of all essential and non-essential amino acids. The results suggest that A. citrinus and C. utilis cultures can be used for the production of SCP without extensive modification in Koji making fermentor on large scale solid state fermentation.

Keywords: Solid state fermentation; Rice bran; single cell protein; Arachniotus citrinus; Candida utilis; Koji making fermentor

1. Introduction

A growing alarm for the severe food scarcity for the world's increasing population has led to the utilization of non-conventional food sources as potential alternatives. Developing countries like Pakistan urgently need to increase livestock and poultry production to enhance meat, milk and egg supplies to meet protein requirement of increasing population. In Pakistan, poultry industry has played a main responsibility in providing animal protein (in the form of eggs and meat) to common man. But feed industry is facing massive shortage of both plant and animal based feed ingredients. These are the main constraints in the development of poultry industry. (Rajoka et al., 2006)

One possible alternative is to ferment cheap non-conventional agro-industrial by-products to produce single cell protein (SCP). These residues through fermentation will reduce the pollution as well as provide a potential source of carbon and energy for production of SCP which is an economical, quite comparable to animal protein and potential supplemental protein source. The SCP can replace costly conventional protein sources like soybean meal and fishmeal for feeding poultry, livestock and humans (Singh et al., 1991; Pacheco et al., 1997, Anupama and P. Ravindera., 2000).

Solid state fermentation (SSF) refers to the cultivation of microorganisms (mainly fungi) on a solid medium, with a moisture content that ensures growth and metabolism of microorganisms [5]. (Del Bianchi et al., 2001). In SSF, the solid material acts as physical support and source of different nutrients. SSF systems offer several economical and practical advantages such as: higher product concentration, improved product recovery, very simple cultivation equipment, reduced waste water output, lower capital investment and lower plant operation costs (Muniswaran et al., 1994). In SSF of agro-industrial byproducts can be increase their nutritional chemical composition, example, by increasing protein content [6,7]. (Rudravaram et al., 2006; Ravinder et al., 2003), improve the phenolic content and antioxidant potential of fermented foods by using different microorganisms. (Lin et al., 2006; Zhang et al., 2008, Lee et al., 2008, Randhir et al., 2004, Lateef et al., 2008; Bhanja et al. (2008).

The yeast Candida utilis has been frequently used in SCP production because of its ease of isolation, can grew very well at room temperature, ability to utilize a variety of carbon sources such as rice polishings (Rajoka et al., 2006), potato starch waste waters (Gélinas and Barrette, 2007), salad oil manufacturing wastewater (Zheng et al., 2005) and molasses (Nigam and Vogel, 1991), to support high protein yield, its minimal energy requirements and. It has been used for production of several industrial products both for human and animal consumption (Zayed and Mostafa, 1992; Pacheco et al., 1997; Kondo et al., 1997; Otero et al., 1998, Adoki, 2002). It has also been used as a host to produce several chemicals, such as glutathione (Liang et al., 2008), monellin (Kondo et al., 1997) and ethyl acetate (Christen et al., 1999).

Mycelia tips of fungi easily penetrate in hard substrate and produce much higher amount of the SCP as compared to submerged fermentation.6 A novel native fungal strain, Arachniotus citrinus is a white rot mesophillic fungus and has been used for the SCP on small scale by using different agro industrial wastes.7 [Shaukat et al., 2006] Previous studies of Arachniotus citrinus also proved that it has effective cellulases, glucoamylase producer in waste bread medium. strong resistance profile of from A. citrinus against proteases was observed. Jabbar et al., 2004; However, there is no literature reported to optimize the culture conditions for A. citrinus and C. utilis in rice bran on large scale by using Koji making fermentor for its reutilization in poultry rations and its biological evaluation in chickens. The main goal is to develop an optimal process on large scale SCP for rice bran with high protein content for poultry and livestock feed industry.

MATERIALS AND METHODS

2.1. Rice bran

Rice bran was obtained from National Feed Industries, Lahore, Pakistan. It was then sealed in polyethylene bags and stored at 4°C for further use.

2.2. Determination of different components in rice bran

The proximate analysis of rice bran was conducted to estimate its nutritive composition by following the methods of the Association of Official Analytical Chemists (AOAC,1994). Triplicate samples of rice bran were analyzed for moisture, crude protein, crude fat, crude fiber, total ash, nitrogen free extract and cellulose contents.

2.3. Organism

Arachniotus citrinus and Candida utilis (a gift from the National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan) were obtained. Both microorganisms were maintained on potato dextrose agar (PDA) slants at 4°C and regular shifting on the PDA slant at the interval of 15 days to keep them viable. Both Arachniotus citrinus and Candida utilis were used to prepare seed culture by transferring a loopful of cells to 200 ml seed culture medium in a 1000 ml Erlenmeyer flask. The medium for Arachniotus citrinus was containing (g/L) rice bran, 20; CaCl2. 2H2O, 0.025; MgSO4.7H2O, 0.025; KH2PO4, 2; Urea 18.9 and grown at 35°C with pH 4 while the medium for C. utilis was containing (g L-1) KH2PO4,5.0; (NH4)2SO4, 5.0; CaCl2, 0.13; MgSO4, 0.5; yeast extract, 0.5 and grown at 35°C with pH 6 on an orbital shaker (150 rpm for 24 h). 13,14

2.6.1. Effect of moisture content on Arachniotus citrinus SCP production

Factors such as moisture content, ionic concentrations of MgSO4.7H2O, CaCl2 .2H2O, KH2PO4 , carbon to nitrogen ration(C:N), molasses (10% solution), yeast sludge, and corn steep liquor for Arachniotus citrinus and molasses (10% solution) and urea for C. utilis affecting the SCP production were standardized by adopting the search technique by varying one factor at a time. The optimized parameter of one experiment was followed for succeeding experiments.

In the first experiment, the effect of moisture content (ranging from substrate to water ratios of 1:2, 1:1, 1.5:1, and 2:1) on fungal SCP production, 5 g of rice bran was steamed, inoculated and incubated for 3 days at 35 °C for the optimization of water content. All media were adjusted to pH 4.0 with 1 M NaOH or 1 M HCl. A portion of SCP was used for the estimation of crude protein and true protein by following the methods of the Association of Official Analytical Chemists (AOAC, 1994). The protein gain in the fermented rice bran was calculated according to Equation 1, while, the correction factors of 5.7 for rice bran and of 6.25 for fermented biomass calculations.

Protein Gain %=[(NF-NF0) X6.25] X100

(NF0X5.7)

Where NF = nitrogen content in fermented rice bran on as such basis, NF0= nitrogen content in unfermented bran.

The moisture content favoring maximum fungal SCP production was followed for subsequent experiments.

2.6.2. Effect of ionic concentration on fungal SCP

To find out the influence of different ionic concentrations of MgSO4.7H2O, CaCl2 .2H2O and KH2PO4 on A. citrinus SCP production, SSF was carried out for 3 days at 35°C with pH 4 at ionic concentrations of MgSO4.7H2O (0, 0.025, 0.05, 0.075 and 0.1%), CaCl2 .2H2O (0, 0.025, 0.05, 0.075 and 0.1%) and KH2PO4 (0, 0.05, 0.1, 0.15, 0.20 and 0.25 %). The ionic concentrations giving high amount of SCP were taken as an optimum and applied for subsequent evaluation. All other chemicals were of analytical grade.

2.6.3. Effect of molasses

The effect of different levels of 10% molasses (0, 1, 2, 3, 4, 5, and 6 ml) on fungal SCP was also evaluated by conducting experiments for incubation period of 3 days at 35°C with pH 4. The other parameters were kept at their optimum levels.

2.6.5. Effect of yeast sludge

Experiments were conducted to find out the effect of various concentrations of yeast sludge on SCP production of A. citrinus by conducting SSF on sterilized 5g rice bran in 250 ml Erlenmeyer flask for 3 days at 35°C with pH 4. Optimum levels of all the other derived parameters were used. The yeast sludge giving maximum SCP production was determined as an optimum level of yeast sludge.

2.6.. Effect of corn steep liquor

The effect of various concentrations of corn steep liquor (0, 0.5, 1, 1.5, 2.0, and 2.5 ml) on fungal SCP was also evaluated by conducting experiments at 35°C with pH 4 for incubation period of 3 days. The other parameters were kept at their optimum levels. Corn steep liquor was obtained(a gift) from the Rafhan Maize Products (Pvt) Ltd, Faisalabad.

2.5. Effect of various concentrations of molasses and urea on Candida utilis SCP production

To demonstrate the influence of various concentrations of 10% molasses (0, 1, 2, 3, and 4 ml), and urea (0, 0.1, 0.15, 0.2, 0.25, and 0.3g) on yeast SCP production, experiments were conducted on 5g sterilized rice bran with C. utilis for 3 days at 35°C with the pH of 6.0. The media were adjusted to pH 6.0 with 1 M NaOH or 1 M HCl.

Large scale single cell protein production by solid state fermentation

The optimum conditions determined for SCP production by SSF of A. citrinus and C. utilis (in 250ml Erlenmeyer flask) were extended to ferment 300 kg rice bran in a Koji making fermentor (Fujiwara Techno- Art Co. Ltd, Japan) for the production of SCP(Fig 1). A simple SSF process was followed. Major components of the rotary bed koji maker are a round bed with a perforated bottom plate for up-flow aeration; a set of adjustable speed mixer for plowing up rice bran during SSF; a set of screw for sterilized substrate feed-in and SCP discharge, an air sterilizer and a humidifier. Temperature and humidity sensors are inserted for monitoring and control the temperature and humidity, respectively. pH was monitored frequently by using pH meter. There was some modification (the Koji bed was covered with cheesecloth) for large scale SCP production of A. citrinus and C. utilis in a more hygienic and controllable conditions with mechanized koji making facilities. The SCP product obtained on large scale was analyzed after drying at 70 °C in a hot air oven (AOAC Methods, 1994) and RNA content was analyzed as described previously (Pacheco et al., 1997; Rajoka et al., 2006).

2.6.2. Gross energy

It was determined by Parr oxygen method using Parr oxygen bomb calorimeter. The calorific value was calculated from the heat generated by the combustion of known weight of the sample in the presence of 20 atmospheric pressure of oxygen reaction.

Results and Discussion

Rice bran is a by-product of the rice milling industry and used in animal feed, in fertilizer and by the cosmetics industry. It has a high nutritive value and serves as a valuable feed for cattle, poultry, and pigs. The protein content (10-15 %) of rice bran supply almost the same amount of protein as wheat and oats and even its protein is of considerably better quality than maize. The chemical composition of rice bran used in this experiment for SCP protein contain moisture content 2.50%, crude protein 13.50%, crude fat 3.01%, crude fiber 11.82%, ash content 11.40%, carbon content 40.35% and cellulose 9.70%. Because of the high nutrient contents, it was selected as a potential alternative substrate for the production of SCP by using A. citrinus and C. utilis. The primary objectives of this study were to evaluate the potential of A. citrinus for SSF by using rice bran and production of fungal and yeast C. utilis biomass protein on large scale. Maximum microbial biomass protein from A. citrinus was obtained at optimal temperature 35 °C, pH 4 and incubation time 3 d while for C. utilis optimal temperature 35 °C, pH 6 and incubation time 3 d were selected (data not shown).

3.1. Effect of moisture content

Solid-state fermentation is a well adopted method for cultivating fungi on agro-industrial wastes. It offers benefits for production of numerous enzymes and various chemicals. Solid-state fermentation has lower energy requirements, smaller effluent volumes, higher productivity, simple and easy operation of solid state fermentors. SSF is significantly affected by different factors such as selection of a proper strain, substrate and other processing parameters for fermentation such as moisture content, temperature, pH, incubation period, ionic concentrations of different anions and cations, different sources of carbon and nitrogen etc. In this study, different levels of moisture content were used to determine the optimum level of water to obtain maximum yield of fungal A. citrinus SCP.

The results of our study indicated that the maximum level of SCP production (in terms of crude protein %) was observed at substrate to water ratio of 1:2 by using fungal A. citrinus in SSF. A significant decrease (p< 0.05) in SCP production was observed at 1:1(2.24%), 1.5:1(16.39%) and 2:1(23.57%) as compared to 1:1. A similar trend was observed in true protein% and protein gain%. The maximum level of true protein% was observed at 1:2 while it was decreased at 1:1(1.15%), 1.5:1(15.78%) and 2:1(23.19%) as compared to 1:1. The highest protein gain % was observed at 1:2 while it was decreased at 1:1(5.08%), 1.5:1(37.27%) and 2:1(53.66%) as compared to 1:1.

It was already reported that at 6% moisture (w/v) corn stover had increased the microbial biomass protein production by sequential culture fermentation with Arachniotus sp., at pH 4, 35 °C for 72 h and then followed by C. utilis fermentation at pH 6, 35 °C for 72 h (Ahmad et al., 2010). Zambare., (2010) found that Aspergillus oryzae had increased the glucoamylase enzyme production at 100% (v/w) initial moisture by using different agro-industrial wastes of SSF. Sharma and Satyanarayana., 2012 found the highest production of a pectinase enzyme of Bacillus pumilus dcsr1 at moisture ratio of 1 : 2.5 by using different agro-residues in SSF.

Generally low moisture content has been reported for maximum fungal growth, more utilization of substrate and significant advantage is lowering the risk of bacterial species contamination. This variation in moisture content might be due to differences in fermenting fungal specie, and substrate. The reduction in SCP production at 1:1 of moisture content might be due to non-availability of nutrients due to lower moisture content and of lower water activity that affected the microbial activities because of limitation in the nutrient solubilization, lower degree of substrate swelling and decrease in diffusion of gas to the cell during fermentation (Nagadi and Correria, 1992; Ellaiah et al., 2004). Even higher concentrations of moisture also affected the microbial enzymes metabolic activities as a result of substrate stickiness, less porous nature of substrate and very limited oxygen transfer during the process of SSF in fermentor (Kumar et al., 2003; Pandey et al., 2000).

Effect of different ionic concentrations of MgSO4.7H2O, CaCl2 .2H2O, and KH2PO4 on SCP production

All the required metallic elements Mg, Ca and K can be supplied in the form of the cations of inorganic salts and they are normally required in relatively large amounts. Significant variation (p< 0.05) of SCP production was observed at different concentrations of MgSO4.7H2O. Maximum production was observed at 0.05%MgSO4.7H2O. Beyond 0.05%MgSO4.7H2O, the production of SCP was significantly lower. Concentrations above 0.05%MgSO4.7H2O also reduce the biomass production indicating the optimum level of MgSO4.7H2O for SCP production for A. citrinus was 0.05%.

A significant difference (p< 0.05) in SCP production (on crude protein% basis) was observed at control 0.0% (1.43%), 0.025% (0.24%) 0.075% (4.46%) and 0.10% (5.28%) as compared to 0.05% MgSO4.7H2O. When we compared supplementation of different levels of MgSO4.7H2O for SCP production, we found that there were increased in production of SCP at 0.025% (1.20%) and 0.05% (1.45%) while there were decreased at 0.075% (3.07%) and 0.10% (3.91%) as compared to control 0.0% MgSO4.7H2O.

The maximum levels of true protein% were observed at 0.025% and 0.05%. The average value of TP% was 17.08 ± 0.06. When we compared with the highest value of TP% with different levels of MgSO4.7H2O, it was found that the TP% was lower at 0.0% (1.87%) while it was decreased at 0.05% (0.23%), 0.075% (4.85%) and 0.1% (9.76%) of MgSO4.7H2O. When we compared the effect of different levels of MgSO4.7H2O on true protein%, we found that there were increased in TP% at 0.025% (1.37%) and 0.05% (1.66%) while there were decreased at 0.075% (3.03%) and 0.10% (8.04%) as compared to control 0.0% MgSO4.7H2O.

The highest protein gain% was observed at 0.05% MgSO4.7H2O 88.46 ± 0.11 while it was lower at 0.0% (3.91%), 0.025% (0.55%) and decreased at 0.075% (10.0%) and 0.10% (21.02%) as compared to 0.05% MgSO4.7H2O. When we compared the influence of different inclusion levels of MgSO4.7H2O on protein gain%, we found that there were increased in PG% at 0.025% (2.69%) and 0.05% (3.26%) while there were decreased at 0.075% (7.06%) and 0.10% (18.44%) as compared to control 0.0% MgSO4.7H2O.

Production of A. citrinus biomass protein was greatly influenced by different levels of CaCl2.2H2O. A significant increase in SCP production was observed in SSF by increasing the initial levels of CaCl2.2H2O from 0.025% to 0.075%. Maximum production of SCP was observed at 0.075% CaCl2.2H2O (24.66% ± 0.00). However, at 0.10% CaCl2.2H2O SCP production was decreased significantly (Table. 3).

Significant variation (p< 0.05) in SCP production (on crude protein% basis) was observed at control 0.0% (1.01%), 0.025% (0.64%) 0.05% (0.28%) and 0.1% (1.45%) as compared to 0.075% CaCl2.2H2O. When we compared supplementation of different levels of CaCl2.2H2O for SCP production, we found that there were increased in production of SCP at 0.025% (0.36%), 0.05% (0.73%) and 0.075% (1.02%) while there was decreased at 0.10% (0.45%) as compared to control 0.0% CaCl2.2H2O.

The maximum levels of true protein% were observed at 0.05% and 0.075% (average value 17.26 ± 0.01). When we compared the highest value of TP% with other levels of CaCl2.2H2O, it was found that the TP% was lower at 0.0% (1.01%), 0.025% (0.64%) and 0.05% (0.28%) while it was decreased at 0.01% (1.45%) of CaCl2.2H2O. However, when we compared the effect of different levels of CaCl2.2H2O on true protein%, we found that there were increased in TP% at 0.025% (0.35%), 0.05% (0.93%) and 0.075% (1.22%) while there was decreased at 0.10% (0.05%) as compared to control 0.0% CaCl2.2H2O.

The highest protein gain% was observed at 0.075% (90.69% ± 0.05) CaCl2.2H2O while it was lower at 0.0% (2.28%), 0.025% (1.43%) and 0.05% (0.61%) and decreased at 0.10% (3.21%) as compared to 0.075% CaCl2.2H2O. When we compared the influence of different inclusion levels of CaCl2.2H2O on protein gain%, we found that there were increased in PG% at 0.025% (0.86%), 0.05% (1.70%) and 0.075% (2.33%) while there was decreased at 0.10% (0.95%) as compared to control 0.0% CaCl2.2H2O.

The maximum level of fungal SCP production was observed at 0.25% KH2PO4 level. A significant increase (p< 0.05) was observed in SCP production from 0.05-0.25% KH2PO4 after SSF of rice bran with A. citrinus.

Significant variations (p< 0.05) in the SCP production was observed at 0.0% (15.29%), 0.05% (14.30%), 0.10% (11.55%), 0.15% (7.35%) and 0.20% (2.68%) as compared to maximum increase production of SSP at 0.25% KH2PO4. When we compared supplementation of different levels of KH2PO4 for SCP production, we found that there were increased in production of SCP at 0.05% (1.17%), 0.10% (4.42%), 0.15% (9.37%), 0.20% (14.89%) and 0.25% (18.06%) as compared to control 0.0% KH2PO4.

A similar trend was observed in true protein% and protein gain%. The maximum level of true protein% was observed at 0.25% KH2PO4 (20.39% ± 0.02). The true protein% of different levels of KH2PO4 were observed at 0.0% (15.49%), 0.05% (14.46%), 0.10% (11.47%), 0.15% (7.55%) and 0.20% (2.79%) as compared to 0.25%. However, when we compared the effect of different levels of KH2PO4 on true protein%, we found that there were increased in TP% at 0.05% (1.21%), 0.10% (4.75%), 0.15% (9.40%), 0.20% (15.03%) and 0.25% (18.34%) as compared to control 0.0% KH2PO4.

The highest protein gain% was observed at 0.25% (126.69% ± 0.17). The protein gain% of different levels of KH2PO4 were observed at 0.0% (28.57%), 0.05% (26.71%), 0.10% (21.58%), 0.15% (13.75%) and 0.20% (5.00%) as compared to 0.25% KH2PO4. When we compared the influence of different inclusion levels of KH2PO4 on protein gain%, we found that there were increased in PG% at 0.05% (2.60%), 0.10% (9.79%), 0.15% (20.74%), 0.20% (32.99%) and 0.25% (40.00%) as compared to control 0.0% KH2PO4.

These finding agree with the studies of Baig et al (2002); Xu and Yun (2003); Xiao et al (2004), Athar et al., 2009 and Ahmad et al., 2010. At these concentrations of 0.05% MgSO4.7H2O, 0.075% CaCl2.2H2O, and 0.25%KH2PO4 maximum SCP was produced.

It has been reported that mineral ions play a pivotal role in fungal growth and in their secondary metabolite formations. Chardonnet et al.(1999) found that external Ca2+ can play an indirect role in fungal growth by altering internal Ca2+, which controls the cytoplasmic Ca2+ gradient, and the activity of fungal enzymes involved in cell wall expansion. The direct effect of Ca2+ on the fungal cell wall may also be a significant factor in cell membrane permeability interactions. In contrast, Papagianni (2004) found that increased concentrations of Ca2+ inhibit the synthesis of fungal biopolymers might be due to effect on enzymes such as b-glucan synthesis. For higher CaCl2.2H2O concentrations, the calcium ion content of the cell wall increased, resulting in reduced protein and neutral sugar contents. Mg2+ is also an essential metal ion to all fungi. It act as a cofactor in enzymatic reactions, stabilizes the plasma membrane, and its uptake is ATP dependent. Potassium ion is very important for the regulation of osmotic strength and intracellular pH while phosphorus plays an important role in all phases of cellular metabolism (Conn and strumpf,1976; Verchtert, 1990).

PO4-3 (phosphate), in the form of K- salt, was added because K+ is required for the absorption of phosphate. On the other hand, when Na2HPO4 and (NH4)2HPO4 were added to bacterial, yeast and fungal cultures, poor growth rates and higher resting oxygen consumption were observed as compared to K fed microbes(Conway and Moore,1954). This could be probably due to the death of fermenting microorganisms caused by reverse osmosis in the presence of higher concentrations of ions. A combination of Ca2+ Mg2+ and K+ ions gave rise to enhanced mycelia growth of A. citrinus in SSF of rice bran.

Effect of carbon: nitrogen ratio on SCP production by fermentation with A. citrinus

The Carbon to Nitrogen (C/N) ratio is important in a biological process. Microorganisms require a proper nitrogen supplement for metabolism during fermentation. It is a major nutrient for fungal growth. High concentrations of nitrogen have increasing the fungal growth and biomass yield. It is necessary to maintain proper composition of the growth media for efficient fermentation process so that the C:N ratio remains within desired range. Microorganisms generally utilize carbon 25-30 times faster than nitrogen during anaerobic digestion.

The C: N ratio of 12:1 produced maximum SCP production (29.91 ± 0.02) by fermentation with A. citrinus (Table. 5).

A significant variation (p< 0.05) in the CP% production was observed. We found that there were decrease in the production of CP% when we supplied different C:N ratios of 15:1 (14.91%), 17:1 (0.70%), 19:1 (8.72%), 21:1 (10.76%) and 23:1 (15.84%) as compared to C: N of 12:1.

The maximum level of TP% was observed at C: N of 12:1 (29.94% ± 0.04). However, when we compared the effect of different ratios of C:N on TP%, we found that there were decreased in TP% at 15:1 (40.40%), 17:1 (28.72%), 19:1 (36.13%), 21:1 (37.54%) and 23:1 (41.11%) as compared to C: N of 12:1.

The highest PG% was observed at C: N of 12:1 (133.33% ± 0.22). When we compared the influence of different ratios of C:N on PG%, we found that there were decreased in PG% at 15:1 (27.16%), 17:1 (1.29%), 19:1 (15.89%), 21:1 (19.63%) and 23:1 (28.86%) as compared to control C: N of 12:1.

in agreement with Kiani (1989), Gutierrez et al (2004) and Zheng et al (2005) Rajoka et al., 2004, Athar et al., 2009; Ali et al., 2010 . This could be due to the fact that when C:N was 12:1, maximum production of biomass protein was produced. If the ratio was increased above this level, excess urea was produced which was responsible for the increase in pH and ultimately reduced the production of single cell protein.

Replacement of one nitrogen source for another in the medium causes a change in protein synthesis as well as product formation. To explore the influence of nitrogen sources on production of crude protein and RNA, were compared to, urea, and corn steep liquor (which are cheap nitrogen sources) when added to rice polishings medium. The results (Table 1) show that these nitrogen compounds influenced the production of protein productivity and RNA content to varying degrees. Generally, the results confirmed that corn steep liquor, a low-cost by-product of the starch industry, supported the maximum kinetic parameters of crude protein compared to those of other nitrogen compounds. The organism produced lower SCP from sodium nitrate and ammonium nitrate and was attributed to low nitrate reductase activity in the organism.

However, the maximum EPS production was achieved when yeast extract was employed as nitro-gen source An appropriate amount of C: N ratio is the key to get maximum yield of SCP.19,20 Urea is a low cost fertilizer and supported maximum SCP production which was in agreement with previous studies.21,22

Effect of supplementation with molasses (10% solution) on SCP production by fermentation with A. citrinus

Fermentation was carried out at different concentration of cane molasses (10% solution) to standardize the optimum level of molasses. High levels of SCP formed at 1ml of molasses (10% solution). Further addition of molasses results in decreased SCP production (table. 5). Significant variations (p< 0.05) in the SCP production were observed at 0.0 ml (3.28%), 2 ml (2.78%), 3 ml (7.99%), 4 ml (19.34%), 5 ml (34.20%) and 6 ml (53.90%) as compared to the highest production of SCP at 1 ml of molasses (10% solution). However, when we compared supplementation of different levels of molasses (10% solution) for SCP production, we found that there were increased in production of SCP at 1 ml (3.39%) and 2 ml (0.59%) levels. However, further addition of molasses (10% solution) at 3 ml (4.87%), 4 ml (16.60%), 5 ml (31.97%) and 6 ml (52.33%) decreased the SCP production when we compared these levels with control 0.0 ml molasses (10% solution).

A similar trend was observed in true protein% and protein gain%. The maximum level of true protein% was observed at 1 ml of 10% molasses (22.26% ± 0.15). The true protein% of different levels of 10% molasses were observed at 0.0 ml (3.68%), 2 ml (3.14%), 3 ml (8.04%), 4 ml (19.72%), 5 ml (34.54%) and 6 ml (54.08%) as compared to the highest production of SCP at 1 ml of molasses (10% solution). However, when we compared supplementation of different levels of molasses (10% solution) for SCP production, we found that there were increased in production of SCP at 1 ml (3.82%) and 2 ml (0.55%) levels. However, further addition of molasses (10% solution) at 3 ml (4.80%), 4 ml (16.65%), 5 ml (32.04%) and 6 ml (52.33%) decreased the SCP production when we compared these levels with control 0.0 ml molasses (10% solution).

The highest protein gain% was observed at 1 ml of 10% molasses (147.32% ± 0.86). The PG% of different levels of 10% molasses were observed at 0.0 ml (5.73%), 2 ml (4.87%), 3 ml (13.94%), 4 ml (33.77%), 5 ml (59.66%) and 6 ml (94.01%) as compared to the highest production of SCP at 1 ml of molasses (10% solution). However, when we compared supplementation of different levels of molasses (10% solution) for SCP production, we found that there were increased in production of SCP at 1 ml (6.08%) and 2 ml (0.93%) levels. However, further addition of molasses (10% solution) at 3 ml (8.71%), 4 ml (29.74%), 5 ml (57.2%) and 6 ml (93.65%) decreased the SCP production when we compared these levels with control 0.0 ml molasses (10% solution).

Flasks experiments using molasses and sucrose for enzyme production showed a pH increase during the fermentation. High pH affects the enzyme stability. Consumption of sucrose or glucose as carbon source is not cost-effective in the production of microbial biomass protein. Low cost substrates such as cane molasses can be used for the production of microbial biomass protein for animal feed supplements.23,24 In addition, molasses is widely available from the sugar industry and consist of water, sucrose (47-50%, w/w) which is the disaccharide most easily exploited by yeast cells. It also contain 0.5-1% nitrogen, proteins, vitamins, amino acids, organic acids and heavy metals.25 Therefore, it is a very attractive carbon source for SCP production economically. In this study, molasses were added to the fermentation medium to enhance the SCP production. Among different concentrations of molasses, 1 and 2 ml molasses gave higher SCP production by fermentation with Arachniotus sp. and C. utilis, respectively (Fig. 6 and Fig. 7). The results of our experiment were agreed with the previous studies.17, 26 The present results showed the potential of Arachniotus sp. and C. utilis to grow on cheap substrates like rice bran along with molasses for SCP production.

Effect of Yeast Sludge

Significant variation (p< 0.05) of SCP production was observed at different yeast sludge levels. Maximum production was observed at 0.6 ml (Table. 2). Beyond 0.6 ml, the production was significantly reduced. Yeast sludge above 0.6 ml also reduces the SCP production indicating the optimum level of yeast sludge for biomass production for A. citrinus was 0.6 ml. Significant variations (p< 0.05) in the SCP production were observed at 0.0 ml (5.22%), 0.2 ml (3.55%), 0.4 ml (1.79%), 0.8 ml (8.09%), 1.0 ml (15.32%) and 1.2 ml (22.04%) as compared to the highest production of SCP at 0.6 ml of YS.

However, when we compared supplementation of different levels of YS for SCP production, we found that there were increased in production of SCP at 0.2 ml (1.76%), 0.4 ml (3.62%) and 0.6 ml (5.51%) levels. However, further addition of YS at 0.8 ml (3.02%), 1 ml (10.65%), and 1.2 ml (17.74%) decreased the SCP production when we compared these levels with control 0.0 ml SY.

A similar trend was observed in true protein% and protein gain%. The maximum level of true protein% was observed at 0.6 ml of SY (23.47% ± 0.05). The true protein% of different levels of YS was observed at 0.0 ml (5.28%), 0.2 ml (3.66%), 0.4 ml (1.74%), 0.8 ml (8.09%), 1.0 ml (15.33%) and 1.2 ml (22.19%) as compared to the highest production of SCP at 0.6 ml of YS. However, when we compared supplementation of different levels of YS for TP%, we found that there were increased in TP% at 0.2 ml (1.70%), 0.4 ml (3.73%) and 0.6 ml (5.57%) levels. However, further addition of YS at 0.8 ml (2.96%), 1 ml (10.61%), and 1.2 ml (17.85%) decreased the TP% when we compared these levels with control 0.0 ml SY.

The highest protein gain% was observed at 0.6 ml of SY (162.27% ± 0.40). The PG% of different levels of SY was observed at 0.0 ml (8.78%), 0.2 ml (5.95%), 0.4 ml (3.00%), 0.8 ml (13.59%), 1.0 ml (25.68%) and 1.2 ml (36.94%) as compared to the highest PG% at 0.6 ml of YS. However, when we compared supplementation of different levels of YS for PG%, we found that there were increased in PG% at 0.2 ml (3.10%), 0.4 ml (6.33%) and 0.6 ml (9.63%) levels. However, further addition of YS at 0.8 ml (5.26%), 1 ml (18.52%), and 1.2 ml (30.86%) decreased the PG% when we compared these levels with control 0.0 ml SY.

YS is a brown colored semi solid material, which is a by-product of brewing industry. It contains of moisture content 73.8% ± 1.93% , crude protein 29.57± 0.74 and true protein 20.78± 0.24 %, crude fat 1.2± 0.03, ash content 30.0± 0.17, RNA content 1.82 ±0.12 ( on dry matter basis). It is considered a waste material and not utilized properly. Y.S produced by brewing industry in Pakistan is estimated to be 16,000 tons/year. This sludge has a great potential in itself. It contains a substantial quantity of protein but limiting in lysine. Yeast cell mass also contains mannaoligosaccharides (4.6%) in its cell wall which work against aflatoxin B1 and pathogens.

Effect of supplementation of corn steep liquor on SCP production

Experiments were also carried out to study the effect of corn steep liquor(CSL) in the medium as a nitrogen source for SCP production. Corn steep liquor, one of a low cost byproduct of starchy industry and the least expensive nitrogen source.

The production of crude protein by A. citrinus fermentation at different CSL levels (0.0- 2.5 ml) was carried out in the 250 ml Erlenmeyer flasks (Table 3). Maximum production of crude protein (34.85% ± 0.04) was realized when the CSL level of fermentation was maintained at 2 ml. When it was increased above 2 ml, production of crude protein decreased. Significant variations (p< 0.05) in the CP% production were observed at 0.0 ml (4.76%), 0.5 ml (3.47%), 1.0 ml (2.15%), 1.5 ml (0.74%), and 2.5 ml (1.06%) as compared to the highest production of SCP at 2.0 ml of YS.

However, when we compared supplementation of different levels of CSL for CP% production, we found that there were increased in production of CP% at levels of 0.5 ml (1.35%), 1.0 ml (2.74%), 1.5 ml (4.21%), 2.0 ml (5.00%), and 2.5 ml (3.88%) as compared with control 0.0 ml CSL.

A similar trend was observed in TP% and PG%. The maximum level of TP% was observed at 2.0 ml of CSL (24.42% ± 0.04). The TP% of different levels of CSL was observed at 0.0 ml (4.75%), 0.5 ml (3.52%), 1.0 ml (2.21%), 1.5 ml (0.69%), and 2.5 ml (1.10%) as compared to the highest production of TP% at 2.0 ml of CSL. However, when we compared supplementation of different levels of CSL for PG%, we found that there were increased in PG% at 0.5 ml (1.28%), 1.0 ml (2.66%), 1.5 ml (4.25%), 2.0 ml (4.98%) and 2.5 ml (3.82%)as compared with control 0.0 ml CSL.

The highest PG% was observed at 2.0 ml of CSL (173.48% ± 0.40). The PG% of different levels of CSL was observed at 0.0 ml (7.77%), 0.5 ml (5.68%), 1.0 ml (3.53%), 1.5 ml (1.22%), and 2.5 ml (1.75%) as compared to the highest PG% at 2.0 ml of CSL. However, when we compared supplementation of different levels of CSL for PG%, we found that there were increased in PG% at 0.5 ml (2.26%), 1.0 ml (4.59%), 1.5 ml (7.10%), 2.0 ml (8.43%) and 2.5 ml (6.52%) as compared with control 0.0 ml CSL.

The corn steep liquor (CSL) used as nitrogen source in this study was a complex mixture of free amino acids, peptides, proteins, inorganic salts, organic acids and many other compounds. On analyzing the data referring to biomass production, it can be observed that the highest result (P< 0.05) was obtained with the medium containing glucose (8.18 g L1) when compared with medium with sucrose (5.94 g L1). Because of high biomass, the culture containing glucose showed the highest yield (0.82 g g1) and productivity (0.085 g L1h1) when compared to the data obtained with sucrose, 0.59 g g1 and 0.041 g L1h1,respectively. Among different concentration of yeast sludge 0.6 ml (Fig. 8) and CSL 2 (Fig. 9) ml gave higher SCP production by fermentation with Arachniotus sp. These findings agree with previous studies.14,20,26,27 At higher levels of CSL, Arachniotus sp. showed slow rate of fermentation due to change in its own structure. It might be that CSL have some additional growth factors (some amino acids, nitrogen free extract, minerals, certain vitamins etc.) due to which it gave better results as compared to other nitrogen sources.20 Our finding indicates that inorganic nitrogen source yielded lower mycelial growth and EPS production by Stemphylium sp., in comparison with organic nitrogen source. Xu and Yun11, as well as Fang and Zhong 5 , also reported that organic nitrogen sources enhanced EPS production and mycelial growth of different fungi. It should be mentioned here that some of the enhancements obtained from using the organic sources of nitrogen may indeed not only reflect the form that the nitrogen is in, but also the fact that other non-nitrogen components could play a role in the improvements. Addition of corn steep liquor upto 1.0% level in culture medium supplied additional nutrients and sugars as energy source. Further addition of corn steep liquor decrease the production of a-amylase which may be due to catabolic repression.

Addition of molasses (10% solution) for maximum production of SCP by C. utilis

The influence of various concentrations of cane molasses (10% solution) on yeast biomass protein production by using SSF with C. utilis on fungal SCP was quantified. Maximum production was observed when 2 ml was given (Table. 4). However significant variation (p< 0.05) was observed in CP% production at various levels of 10% solution of molasses. When molasses (10% solution) level lower or higher than the optimum was used for fermentation process yeast SCP production decreased. Significant variations (p< 0.05) in the CP% production were observed at 0.0 ml (0.37%), 1 ml (2.49%), 3 ml (13.56%) and 4 ml (26.41%) as compared to the highest production of SCP at 2 ml of molasses (10% solution). However, when we compared supplementation of different levels of molasses (10% solution) for yeast SCP production, we found that there were increased in production of SCP at 1 ml (0.37%) and 2 ml (2.49%) levels. However, further addition of molasses (10% solution) at 3 ml (13.56%) and 4 ml (26.41%) decreased the SCP production when we compared these levels with control 0.0 ml molasses (10% solution).

A similar trend was observed in TP% and PG%. The maximum level of TP% was observed at 2 ml of 10% molasses (35.73% ± 0.02). The TP% of different levels of 10% molasses was observed at 0.0 ml (0.36%), 1 ml (2.49%) 3 ml (13.50%) and 4 ml (26.26%) as compared to the highest production of SCP at 2 ml of molasses (10% solution).

However, when we compared supplementation of different levels of molasses (10% solution) for yeast SCP production after fermentation of rice bran with A. citrinus, we found that there were increased in production of SCP at 1 ml (0.36%) and 2 ml (2.49%) levels. However, further addition of molasses (10% solution) at 3 ml (13.50%) and 4 ml (26.26%) decreased the SCP production when we compared these levels with control 0.0 ml molasses (10% solution).

The highest PG% was observed at 2 ml of 10% molasses (173.56% ± 0.05). The PG% of different levels of 10% molasses was observed at 0.0 ml (0.60%), 1 ml (4.04%), 3 ml (22.16%), and 4 ml (43.10%) as compared to the highest production of SCP at 2 ml of molasses (10% solution). However, when we compared supplementation of different levels of molasses (10% solution) for SCP production, we found that there were increased in production of SCP at 1 ml (0.60%) and 2 ml (4.04%) levels. However, further addition of molasses (10% solution) at 3 ml (22.16%), and 4 ml (43.10%) decreased the SCP production when we compared these levels with control 0.0 ml molasses (10% solution).

C.utilis can utilize various carbon sources to synthesize its own cell mass. Utilization of sucrose or glucose as carbon source is not economical in production of SCP and a cheap carbohydrate source would be beneficial. Low cost substrates such as cane molasses can be used for the production of SCP for animal feed supplements (19). Molasses, cheap by-products, are widely available from the sugar industry and consist of water, sucrose (45-50 % w/w) which is the disaccharide most easily utilized by yeast cells, 0.5-1% of nitrogen source, protein, vitamins, amino acids, organic acids, and heavy metals(20). Hence it is very attractive carbon source for SCP production from C.utilis from economic point of view. One criterion that is crucial in the selection of a yeast strain for the SCP production is its ability to grow on cheap substrates. This criterion is satisfied with the results obtained with the current strain of C.utilis used in this study, which was found to grow well and produce SCP production on rice bran along with cane molasses.

Addition of urea for maximum production of SCP by C. utilis

With the addition of urea from 0.10 g, SCP production increased to maximum (38.46 %) at 0.25 g after fermentation of 3 days (Figure 10). Therefore, the urea level of 0.25g was considered as optimum for the growth of C. utilis.

The maximum level of yeast SCP production was observed at 0.25g urea level. A significant increase (p< 0.05) was observed in SCP production from 0.1-0.20g urea after SSF of rice bran with A. citrinus and C. utilis.

Significant variations (p< 0.05) in the CP% was observed at 0.0 (1.40%), 0.10g (3.55%), 0.15g (4.98%), 0.20g (7.79%) and 0.30g (1.62%) as compared to maximum increase of CP% at 0.25g urea. When we compared supplementation of different concentrations of urea for CP%, we found that there were increased in production of CP% at 0.10g (1.40%), 0.15g (3.55%), 0.20g (4.98%), 0.25% (7.79%) and 0.30% (1.62%) as compared to control 0.0g urea.

A similar trend was observed in TP% and PG%. The maximum level of TP% was observed at 0.25g urea (26.96% ± 0.03). The TP% of different levels of urea was observed at 0.0g (1.32%), 0.10g (3.48%), 0.15g (4.96%), 0.20g (7.84%) and 0.30g (1.52%) as compared to 0.25g urea. However, when we compared the effect of different levels of urea on TP%, we found that there were increased in TP% at 0.10g (1.32%), 0.15g (3.48%), 0.20g (4.96%), 0.25% (7.84%) and 0.30% (1.52%) as compared to control 0.0g urea.

The highest PG% was observed at 0.25g (202.80% ± 0.28). The PG% of different levels of urea was observed at 0.0g (2.25%), 0.10g (5.72%), 0.15g (8.02%), 0.20g (12.55%) and 0.30g (2.63%) as compared to 0.25g urea. However, when we compared the effect of different levels of urea on TP%, we found that there were increased in PG% at 0.10g (2.25%), 0.15g (5.72%), 0.20g (8.02%), 0.25% (12.55%) and 0.30% (2.63%) as compared to control 0.0g urea.

which was consistent with previous reports.6,17,19

Candida species are preferred for microbial biomass production since they have a better regulation of metabolic pathways which prevent waste of substrate. The predominantly aerobic metabolism of C.utilis and active participation of the pentose phosphate pathway for sugar metabolism predisposes this yeast to carbon balance in favors of SCP production as compared with other yeasts such as Saccharomyees cerevisae, which are glucose sensitive and largely fermentative (15,16).

The omission of nitrogen in the medium greatly affects fungal growth and metabolite production. Nitrogen source may be supplied to media in the form of ammonia, nitrate, or as organic compounds, such as amino acids or proteins. It has been reported that the optimal nitrogen source of

mycelial biomass. In comparison with organic nitrogen sources, inorganic nitrogen sources often yield relatively lower mycelial biomass and EPS production than organic sources in liquid cultures of higher fungi. It could be suggested that when the inclusion level of urea was above 0.25g, the pH was increased which reduced the production of single cell protein.

Thus, if there were a further nutrient increase in the culture medium, it would be possible to obtain higher mycelial concentrations. However, this would compromise the economic feasibility of the medium, since the cost of soy protein and yeast extract can be significant, when in higher concentrations in the broth.

Chemical composition of mixed SCP produced in 300 Kg Koji Making Fermentor

Arachniotus sp. was grown for 3 d in a 300 Kg fermentor followed by C. utilis fermentation of rice bran medium for an additional 3 d. The aeration was kept constant at 2.0 vvm and agitation was varied. The nutrient composition of the mixed SCP was crude protein (13.50%), true protein (23.74%), crude fiber (6.77%), ash (14.71%), ether extract (2.97%), nitrogen free extract (48.68%), caloric value (3675 Kcal/Kg) and RNA content (3.383). The crude protein% was increased from 13.50 to 30.13. The true protein content of 23.74% and gross energy of dried SCP 3675 Kcal/kg indicated that the SCP can serve as an energy source beside protein particularly when it fed to poultry. There were 125.92% increased in CP content, and increased in ash content 27. 28%, while, there were 3.65% decreased in crude fat content and in 43.40% in crude fiber content. A previous study reported 55.3% crude protein of C. utilis SCP.28

A similar results reported that the single cell protein product contained 30.4 % crude protein.29 while Kluyveromyces fragilis biomass grown on deproteinized whey supplemented with 0.8% diammonium hydrogen phosphate contained 37% crude protein. 30 Recently, the mixed microbial biomass protein produced in the 75-L fermentor contained true protein 16.41%, and crude protein 23.51% by sequential culture fermentation of Arachniotus sp. and C. utilis by using corn stover as a substrate.17 The SCP obtained in 300 Kg fermentor in this study by the sequential culture fermentation of Arachniotus sp. and C. utilis offers potential as an excellent protein and energy supplement for animal feed.

Both Cordycepsspecies investigated in this study utilized sucrose as the preferred carbon source. This may make it possible to use a cheaper alternative sources of sucrose, such as raw sugar or molasses, for mass production of mycelial biomass and EPS by large-scale submerged cultivations. As medium cost is one of the critical factors affecting the economy of the industrial fermentation process, further study using less expensive ingredients should be considered.

This indicated that the SCP can serve as an energy besides protein particularly when it may be used in poultry feed.

Hence in this study, it was found that rice bran , can molasses and CSL can be used to generate SCP without costly pretreatment. The demonstration of its successful production at laboratory as well as on large scale is an incentive for the production of SCP on commercial level using rice bran and other nutrients.

CONCLUSION

Rice bran, a byproduct of rice industry can be converted into single cell protein using predetermined conditions. The study findings suggested that single cell protein of Arachniotus sp and Candida utilis can be replaced upto 20% but its maximum suitable level in chicken feed was10% of the protein supply by soybean meal. This practice not only can provide a cheap source of protein for the feed of live stock and chickens but also provides an opportunity to utilize agriculture waste and recover resources. The authors found that protein content and biomass were not always proportional.

These results indicate that the substrate used was supplying the necessary nitrogen for the production of fungal protein.

There are many factors (i.e. temperature, humidity, pH, etc.) which can influence A. aleyrodis, and there must be interactions among these factors. Basic and essential information for the mass production of A. aleyrodisin culture allows for some insights into the physiology of this fungus. It is necessary to further research the other factors and optimize the fermentation conditions in a fermenter using complex media to achieve the demands of large-scale mycelia production and sporulation, which are an ongoing project in this laboratory.

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