Potentials of Chemical and Biological Hydrolysis of Agricultural Wastes in the Production of Ethanol, Single Cell Protein (SCP) and Vinegar

The potentials of Chemical (acid) and Biological (Aspergillus niger and Rhizopus oryzae) hydrolysis on some agricultural wastes, namely: peels from ripe plantain (RPP), unripe plantain (UPP), red cocoyam (RCP), white cocoyam (WCP), yam (YP), cassava (CP) and garri processing chaff (GPC) into beneficial by products (Ethanol, Single Cell Protein and Vinegar) were investigated using standard methods. Glucose yield ranged from 4.68-17.24mg/g, 2.41-13.84mg/g and 1.28-5.22mg/g for A. niger R. oryzae and Acid hydrolysis of the various wastes. Inoculating the hydrolysates with Saccharomyces cerevisiae yielded significant (p < 0.05) amount of ethanol after 120hrs, with significant (p < 0.05) reduction in glucose contents. Ethanol contents ranged from 3.82-6.89%, 3.74-6.44% and 3.28-6.25%, while the amount of single cell protein (SCP) obtained from the biomass after ethanol fermentation ranged from 9.10-15.80%, 7.50-13.11% and 6.00-10.20% for A. niger, R. oryzae and Acid hydrolysates respectively. The vinegar obtained after seven days fermentation of the alcoholic product had acetic acid concentrations ranging from 0.14-1.10%, 0.13-0.99% and 0.11-0.95% for A. niger, R. oryzae and acid treatments respectively. This study has shown usefulness of microbial methods of hydrolysis over the chemical treatment method, for improved yield of fermentation by products.


Introduction
Efforts made for the expansion of food and energy resources through the conversion of glucose and other complex carbohydrates in wastes into ethanol, vinegar and single cell protein have heightened the interests of researchers in the exploitation of several sources of hydrolysis [1]. Hydrolysis is an essential step in the conversion of waste to food and energy since it is through this process that complex carbohydrates in agricultural waste are degraded into their constituent sugars thus becoming readily available for fermentation by microorganisms responsible for the production of ethanol vinegar and single cell protein (SCP). Carbohydrate based agricultural products are important staple foods in the diet of people in most developing countries of the tropics. They consist of renewable resource of great variety of biotechnological potential. However, the generation of waste materials (peels) resulting from their processing pose pollution problems [2]. Notwithstanding this, in many cases, these peels might have potentials for conversion into useful products of higher value after biological treatment [3,4]. The fermentable sugar produced from the hydrolysis of cellulosic fraction of the waste feed stock can serve as carbon source for microbes, especially Saccharomyces cerevisiae which is capable of fermenting sugar to ethanol. Ethanol production by fermentation faces competition with ethanol production from petroleum -based products as feed stocks and in this regard, the use of renewable materials would be more economical, since they are cheaper and easily available [5]. Ethanol serves as substrate for Acetobacter aceti for vinegar (acetic acid) production and presently, almost all acetic acid is produced synthetically [6] with a major disadvantage of the need of high cost catalyst and the dependence on non-renewable sources of raw materials such as crude oil. Hence acetic acid production by biological means become more attractive. The food and agricultural organization (FAO) of the United Nations established that vinegar is allowed for human consumption and that it must be produced from raw materials of agricultural origin by fermentation; by first converting glucose into ethanol followed by oxidation of the ethanol to acetic acid the main product of vinegar [7]. Another advantageous approach brought about by fermentation, is the increased accessibility of protein of agricultural waste products in single-cell proteins (SCPs). Single cell proteins (SCP) are the dried cells of microorganism or microbial biomass obtained after fermentation which serve as protein supplement in human foods or animal feeds. With increase in population and worldwide protein shortage, the use of microbial protein as food or feed is more highlighted and such protein from microorganisms is said to be cheap and competes well with other sources of protein with good nutritive value [2,8,9]. Base on this foregoing, to exploit the potentials of non-conventional food sources such as peels and chaff which are by products of the stable foods in the developing countries as potential alternatives for novel foods using chemical and biological methods of hydrolysis as a primary step. Therefore, this study was carried out to evaluate the potentials of peels from ripe and unripe plantain, red and white cocoyam, yam, cassava, and garri processing chaff to produce ethanol, acetic acid, and single cell proteins.

Sample Preparation
These seven agricultural wastes samples mentioned above were washed and oven-dried along with the garri processing chaff (GPC) at 60 0 C for 24 hours, then pulverized, sieved to 60µm particle size and packaged for further analysis.

Hydrolysis of Complex Carbohydrate Acid Hydrolysis
Processed and stored samples of the Agricultural wastes were separately hydrolyzed following the method of Zainab et al., (2011) as previously reported by Ogunka-Nnoka et al., (2011). Briefly, samples (20g) of each peel were slurried in 120ml of 0.25M H 2 SO 4 and extracted in water bath at 70-80 0 C for 90mins. Samples were cooled, neutralized then filtered through No. 1 Watman filter paper. Glucose content of filtrate was determined following the dinitrosalicylic acid method as described by Amadi and Ifeanacho, [5].

Source of Culture
Pure cultures of Aspergillus niger and Rhizopus oryaze were obtained from the Microbiology Department of the University of Port Harcourt, Rivers State, Nigeria. The Microbial cultures were maintained on potato dextrose Agar (PDA) slant.

Hydrolysis
The method of Abouzied and Reddy (1986) was used. Briefly, 7.5g of each pregelatinized sample waste was suspended in a 250ml conical flask containing 200ml of mineralized media and autoclaved at 121 0 C for 30mins. The samples were cooled and a 5% (v/v) inoculation of A. niger or R .oryzae was used. Inoculation lasted for 72hrs and 10ml aliquot was centrifuged for 20min at 5,000xG. The supernatant was used for reducing sugar analysis following the Dinitrosalicylic acid method as described by Amadi and Ifeanacho, [5].

Determination of Ethanol, Single Cell Protein, and Acetic Acid
The hydrolysates from the Acid, A. niger and R. oryzae were inoculated each with 5% (v/v) S. cerevisiae under sterile condition. The fermentation lasted for 120hr under room temperature at pH 5.0. Aliquot of 20ml each of the sample waste was collected and centrifuged for 20min at 5,000xG and the supernatant was used for ethanol estimation using Gas Chromatography (HP6890 powered with chemostation Rev. A09.01 1206 Software). The single cell protein content was estimated using the biomass residue obtained after ethanol fermentation [10]. While the acetic acid (vinegar) was estimated using 33ml of the centrifuged alcoholic products inoculated with Acetobacter aceti inoculum [11].

Statistical Analysis
All data were analyzed using the analysis of variance. When analysis of variance revealed a significant effect, means were separated using Duncan's New Multiple Range Test as explained by [12].
Potentials of Chemical and Biological Hydrolysis of Agricultural Wastes in the Production of Ethanol, Single Cell Protein (SCP) and Vinegar Figures 1-7 shows the effect of various hydrolysis on glucose yield and also the residual glucose content after ethanol fermentation.  Glucose yield ranged from 4.68-17.24mg/g, 2.41-13.84mg/g and 1.28-5.22mg/g for A. niger, R. oryzae and acid hydrolysis respectively. There was significant (p < 0.05) reduction in the glucose content after fermentation of all the hydrolysates. Ripe plantain peel, had the highest glucose content irrespective of the treatment used; while, low glucose yield was observed in the cocoyam variety (using microbial hydrolysis) and garri processing chaff (using acid hydrolysis). The effect of different hydrolysates on glucose yield showed that these organic wastes had appreciable glucose yield, with RPP ranging highest. The high content of glucose obtained in RPP from the various hydrolysates could be deduced from the fact that during ripening, starch molecules are hydrolyzed to sugar [7]. From the result, it suggests that the cocoyam peels were more resistant to microbial hydrolysis. The result of Figure 4 corroborates the study of Omemu et al., [13] on cocoyam tuber. They stated that the reason for the variation could be as a result of some phytoconstituents present in the cocoyam cultivars that resist microbial attack. While for samples that had better glucose yield with microbial hydrolysis, it was suggested that it could be due to the fact that the complex carbohydrate was pregelatinized prior to hydrolysis [14,15].  Table 1 shows the data on the ethanol yield from the various hydrolysate. Ethanol yield ranged from 3.28-6.25%, 3.82-6.89%, and 3.74-6.44% for Acid hydrolysate, A. niger, and R. oryzae hydrolysates respectively. Results obtained based on the different treatments showed that microbial hydrolysates had significantly (p < 0.05) higher values for the fermentation/products compared to acid hydrolysate. However, apart from the results shown for white cocoyam peels, the ethanol yields from both of the microbial hydrolysates of all the other substrates were comparable. Based on the sample performance, GPC had the highest ethanol yield and RPP had the lowest from the three hydrolysates. According to Omemu et al. [13], Akpan et al. [16], and Adesanya et al. [17], ethanol yield is expected to be directly proportional to glucose yield, however, a contrary result was obtained in this study for GPC and RPP. Ripe plantain peel had the highest glucose yield (Figure 1) with the lowest ethanol yield (Table 1) while the reverse was the case for GPC. This observation could have resulted from the presence of other products of hydrolysis; for instance, other sugars like sucrose and xylose which are not metabolized by the yeast (S. cerevisiae) may have been produced [18]. S. cerevisiae is specific for glucose, mannose and galactose, while other strains of yeast ferment sucrose, maltose and raffinose. Again, there might be an inhibitory effect of fermentation by-products like furfural, which has been reported to inhibit the activities of some glycolytic enzymes particularly dehydrogenases present in S. cerevisiae [19,20]. In addition, the yeast might have utilized much of the nutrient for growth rather than for ethanol production [21]. On the other hand, GPC with moderate glucose yield had the highest ethanol yield and since the starch content depends largely on the processing method, more starch component with amylose moiety which can easily be hydrolysed to glucose, might have been deposited in the chaff, [3,18]. The results obtained in this study, followed closely the observations made by Akpan et al., [16] that reported maximum ethanol content of 8.2% in a mixture of groundnut shell and corncobs using acid hydrolysates, and 34 Potentials of Chemical and Biological Hydrolysis of Agricultural Wastes in the Production of Ethanol, Single Cell Protein (SCP) and Vinegar

Results and Discussion
Akin-osanaiye et al. [8], reported 3.83 to 4.18% in peels of pawpaw. The result for the single cell protein contents of the agricultural wastes using different hydrolyzed methods, are presented in Table 2. Single cell protein, content ranged from 6.00-10.20% 9.10-15.80%, and 7.50-13.11% for acid, A. A. niger and R. oryzae catalyzed hydrolysis respectively. For these fermented samples, garri processing chaff had the highest SCP yield and WCP had the least judging from the various hydrolysates. Also, A. niger hydrolysates produced the highest amount of SCP in all the samples compared to other hydrolysates. Single cell protein is the most readily measured biomass component [22]. Ajao et al., [2] reported low levels of SCP for cocoyam peel inoculated with Penicillium expansum. Also, Akinyele and Agbro, [23] reported SCP level of 8.5 and 11.4% for ripe and unripe plantain peels respectively when subjected to solid state fermentation using A. niger. This result obtained, shows the possibility and effectiveness of the enrichment of agrowastes through the fermentation by S. cerevisiae and is thus suggestive of the applicability and usefulness in the poultry feed manufacturing industries among others. As shown in Table 3, the amount of vinegar produced ranged from 0.11-0.95%, 0.14-1.10%, and 0.13-1.00% for A. niger, R. oryzae and Acid hydrolysates respectively. The production of acetic acid from these wastes was studied using Acetobacter aceti. Following the above trend, A. niger hydrolysate had significantly higher yield over R. oryzae and acid hydrolysates for YP and CP samples studied. GPC had the highest yield (1.10%), while RPP had the least (0.11%). The study by Parrondo et al., [11] revealed that fermentation of whey supplemented with lactose, using Acetobacter aceti for a longer period produced vinegar having concentration of acetic acid between 5-6% (v/v) with efficiency of biotransformation of 84%. Other researchers reported acetic acid yield of 0.80 to 0.90g acetic acid/glucose using Clostridium sp. [8] [24]. The fermentation process used in this study and the substrate employed satisfied FAO requirements in order to serve for food [11]. In this study, agricultural based domestically generated wastes were successfully converted into useful products of economic value using chemical and microbial approach.

Conclusions
From the result of this study, it was deducible that Aspergillus niger produced the best glucose yield and fermentation by products in majority of the agricultural wastes evaluated, and in general, the hydrolytic potentials of the chemical method was less effective than the applied microbiological sources.