1. Introduction

Cocoa (Theobroma cacao L) is a small cauliflorous and semi-deciduous tree believed to have originated from the Amazon valley of South America, from where it was introduced to other parts of the world ^[1] It is a beverage crop mostly grown for the utilization of its seed in chocolate production and the consumption of its pulp/juice. Today, the tree has found a wide range of importance in the food and beverage, agricultural and pharmaceutical industries ^[1].

The cocoa tree serves as shade and is ideally suited for long term land rehabilitation and conservation. The beans contain 50% fat and serve as a valuable source of vegetable fat. It is also used in producing cocoa butter. The residual powder is used in cakes, biscuits, drinking chocolate and has also been fermented to produce ethanol. The bean shell or testa is used as mulch and manure. It is also used as a feedstock ingredient for livestock and poultry. It provides fat and is also a source of vitamin D. It is also used as fuel. The cocoa leaves are regarded as medicinal and are used as a traditional medicinal plant. It has also been associated with mulch and manure ^[2].The pod husk has also been dried and incorporated as feedstock for poultry and can be fed fresh or dried. It is used as manure and also serves as a breeding ground for Midges which pollinates cocoa, enhancing efficacy and ultimate yield. It contains potassium oxide that can be used in soap making. It is a rich source of cellulose (about 45 – 50%) which can be acidically or enzymatically hydrolyzed to glucose and used by sugar-fermenting organisms to produce alcohol and increase in biomass ^[3].The pulp is directly consumed as food and can also be fermented to produce quality wine, jam, liquor, vinegar etc ^[4]. Gums and pigments of various importance have also been found to contain in the cocoa pod.

Fermentation is a process of anaerobic or partial oxidation of carbohydrates ^[5]. ^[6] Regard it as an energy-yielding process in which an organic molecule is oxidized without an exogenous electron acceptor. A carbohydrate intermediate, usually act as the electron donor. To industrial microbiologists, according to ^[5], it is also the growth of microorganisms in very large quantities for the production of industrially important products. Such products may include alcohol, organic acids, amino acids, nucleic acids etc referred to as primary metabolites. The fermentation microorganisms utilize the sugar in the fermentation medium as their carbon source, increase in number and produce these useful end products. These primary metabolites are produced in the log or exponential of the growth curve. Other products as antibiotics, mycotoxins, plants and animal hormones and other medicinal have also been produced by process of fermentation. These secondary metabolites are produced at the idiophase of the growth curve. Microbial cells accumulated during fermentation as biomass may serve to produce useful enzymes, single cell proteins or other products. The microbial cells used as single cell proteins may be directly consumed by humans or fed to animals as livestock feed where they provide rich source of protein to the animals directly and indirectly to humans on consumption of these animals ^[7] and as biomass they can be used as biofuel.

Ethanol is usually produced by yeasts and zymomonas in a process known as alcoholic fermentation ^[6]. Common among the yeasts involved in alcoholic fermentation is Saccharomyces cerevisae and is found to be present in local palm wine ^[8]. They are responsible for conversion of sucrose in palm wine to ethanol ^[9].

Ethanol, also known as ethyl alcohol, pure alcohol or grain alcohol is a volatile flammable liquid known and used by humans since prehistory as the intoxicating ingredient of alcoholic beverage ^[10]. It provides little energy on consumption and has been used as a psychoactive drug: one of the oldest recreational drugs. It is also used as fuel, can serve as spirits, scents, flavouring and colouring agent. In the medical laboratory, it is used as a antiseptic and also serve as an anti-poison ^[10].

The microbial production of ethanol from cellulosic materials has become an important source of valuable fuel particularly in regions of the world that have abundant supply of plant residues ^[11] and provides little environmental hazard than synthetic production. Agricultural wastes like coffee husk, cassava, yam tubers, pineapple peels, corn and carob cobs, sugar cane molasses, bagasses, rice straw, saw mills residue, peanut shells, cocoa pods etc, have been found to be rich in cellulose and can be utilized in production of ethanol ^[12]. This does not only result to conversion of waste to useful product, but also incorporates environmental cleanup and maximization of microbial proficiencies. ^[13] Refer to this approach as utilization of food wastes for sustainable development.

The aim of this work is to produce alcohol and biomass from waste (cocoa pods) using palm wine yeasts.

2. Methodology

Ripe cocoa pods and fresh palm wine were obtained from local farmers and a palm wine tapper in Umuariaga village in Ikwuano L.G.A of Abia State. The chemicals and reagents were purchased from Ariaria International Market, Aba while all other equipments used were gotten from the laboratories of Microbiology, and Food Science Technology of Michael Okpara University of Agriculture, Umudike.

The ripe cocoa pods whose seeds have been removed were cut into smaller pieces, dried and were finely blended to powder using an Electric Blender and the ash content determined according to ^[2].

Exactly 200g of the dried cocoa powder was reflux in 500ml of 1M HCl and incubated at 75°C in a water bath for 4 hours. The solution was filtered and neutralized with 2% NaOH to separate their salt [24]. This solution is known as hydrolysate.

Exactly 2ml of the hydrolysate was titrated against 2% NaOH to its phenolphthalein end point, as described by ^[17]. This was used to determine the volume of NaOH that will neutralize the HCl.

The salt formed by the reaction of HCl and NaOH was separated by filtration. The salt was collected as the solid residue according to ^[14].

The presence of reducing sugar (glucose) in the hydrolysate was determined by method of Fehling’s solution described by ^[15].

A specific gravity (SG) method described by ^[16] and the Abbe refractometer by ^[15] were used to determine the amount of soluble sugar in the solution.

A specific gravity bottle was weighed empty to obtain W₁. The bottle was then filled with water and weighed W₂. The bottle was emptied, dried and filled with the sample solution and weighed W₃. The specific gravity was obtained by:

Using a refractometer, the sample placed in contact with the prism and the SG and percentage examined through the telescope.

Exactly 65g of sugar was used to increase the concentration of fermentable sugar from 1.0625 SG (15°Brix) to 1.140SG (24°Brix) ^[16]. The sugar was weighed, dissolved in 100ml of distilled water and added to the hydrolysate. The specific gravity was then re-determined.

A freshly tapped palm wine was collected into a IL sterile container and allowed to stand for 16 – 18 hrs for the yeast(s) to use the wine sugar, grow and settle out of solution, according to ^[17]. The supernatant was discarded and the sediment used to inoculate a sterile Sabouraud Dextrose Agar (SDA) prepared according to the manufacturer’s instruction and incubated at 28°C for 48hrs ^[18]. Pure culture of the yeast isolate was obtained after sub-culturing and propagated (stepped up) for fermentation. 15ml of sterile broth was prepared, inoculated with 2 loopful of the yeast isolate and incubated for 48hrs at 30°C.

Exactly 15ml of the cultured broth was added to 15ml of the sterile hydrolysate and incubated for 48hrs at 30°C temperature. Exactly 5ml of the yeast hydrolyte was used to inoculate 50ml of sterile hydrolysate and incubated for 24hrs at 30°C. 50ml of the sensitized suspension was used to inoculate 500ml of the sterile hydrolysate and incubated at room temperature for 7 days. Temperature, pH change, sugar uptake, biomass formation and ethanol production was monitored daily throughout the period of fermentation.

The pH of the sample was determined using the pH meter (Hanna pH 211) and the temperature of the sample (medium) was determined using the Hanna pH meter (211) with temperature electrode according to ^[17].

Changes in fermentable sugar, was monitored by method of specific gravity. A fall in the specific gravity indicated sugar uptake and values in Brix were read from a table of ^[15].

Production of biomass was monitored by method of spectrophotometer as described by ^[16], at 600nm.

The alcoholic content of the sample was determined by multiplying the drop in the specific gravity during fermentation by 0.102 according to ^[19].

The ethanol produced was separated from the liquid medium by method of distillation described by ^[20].

3. Results

The physicochemical properties determined before fermentation are represented in Table 1.

Table 1. Physicochemical properties of the hydrolysate before fermentation

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The palm wine yeast grew as creamy-white slimy raised colonies on Sabouraud Dextrose Agar (SDA). The viability of the yeast during scale up was notified by the formation of Kraeusen and an increase in the amount of sediments in the medium. Presence of bubbles after 12hr of fermentation, palm wine flavor due to source of yeast and later, alcoholic flavor indicated that fermentation was taking place. The specific gravity, pH change, temperature and accumulation of biomass also revealed progress in fermentation.

The pH of the hydrolysate was 4.03 before fermentation and decreased to 3.91 after fermentation as shown in Figure 1, while the temperature of the hydrolysate was 26.5°C before fermentation and fluctuated between 28°C and 30°C during fermentation as shown in Figure 2.

Figure 1. Changes in pH during fermentation

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Figure 2. Changes in Temperature during fermentation

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Table 2. Changes in specific gravity and biomass formation (at OD of 600nm) during fermentation

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The change in specific gravity was used to monitor the up-take of fermentable sugar and decreased from 1.140 -1.003 with increase in fermentation days, as shown in Table 2. The corresponding Brix contents are presented in Figure 3.

Increase in biomass from 0.7 to 1.489 was observed within the fermentation days and is presented in Table 2.

Figure 3. Changes in Brix (⁰Bx) Content during fermentation

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10.3% ethanol was determined from the sample. 0.9472 of specific gravity was obtained from the distillate and extrapolated as between 50 and 60% ethanol. The alcohol meter also gave a 50% ethanol from the distillate.

4. Discussion

A constant weight obtained after drying the cocoa both under the sun and in the oven at 70°C suggests both methods are good in removing the moisture content of the cocoa pod, though blasting in hot air oven will be more efficient in large scale business and during wet seasons. ^[21] Observed that insufficient drying of the cocoa pods would encourage fungal growth, causing the pods to lose some of its sugars and might also interfere with the ash content. The 3% ash determined from the dried cocoa sample can be used in soap making. Concentrated Hydrochloric (HCl) acids are capable of swelling and dissolving cellulose as also observed by ^[22]. Hydrolysis of dried cocoa powder with 1M HCl at 75°C gave a sugar yield of 15⁰Brix. This result though different from the report of ^[22], the sugar content is greater than the fermentable sugar concentration used in beer production (<12°Brix). Treatment of cellulose materials with acids disrupts the hydrogen bonding between cellulose chains releasing glucose. 430ml of NaOH was able to neutralize 250ml of HCl acid and precipitate out 0.8g of its salt. Separation of this salt by method of filtration took care of the disadvantage described by ^[23]. 65g of sugar added to the hydrolysate increased the concentration of fermentable sugar from 15°Brix to 240 Brix (Table 1). Palm wine yeasts were capable of growing in the medium and ferment the sugar to ethanol and CO₂. The glucose in the medium is catabolized via the Embdem Meyerhof-Parnas (EMP) pathway or glycolytic pathways to pyruvate as indicated by ^[19].

The pyruvate is then decarboxylated by pyruvate decarboxylase with the formation of aldehyde and CO₂. The enzyme uses a co-factor thiamine pyrophosphate (TPP) for activity. The acetaldehyde, thus act as an electron acceptor and is used to oxidise NADH with the formation of ethanol. The fermentation was observed to be taking place by formation of bubbles after 12 hrs of set up indicating the release of CO₂, palm wine flavour, then an alcoholic flavor. These observations were also reported by ^[17] on fermentation of Sorghum using S. cerevisiae for burukutu production. When the specific gravity was determined, it was found to be decreasing and this is a direct indication of the metabolic activities of the yeast carrying out fermentation (Table 2). The release of CO₂ caused the pH of the medium to decrease from 4.03 – 3.91 (Figure 1). Though the upper range (4.03) was below that reported by ^[19], the values were within the range of pH changes during beer and wine production as reported by ^[20]. The sweet wine flavor produced by the yeasts during the fermentation suggests that the cocoa pod could be fermented to produce sweet wine. Monitoring the temperature, the values revealed fluctuations in the temperature of the medium between 28°C and 30°C (Figure 2). A fall in the temperature was observed on the 6th day and this could be attributed to environmental factors that may affect fermentation. However, the temperature range is important in regulating the enzymatic activity of the yeast because too low the temperature slows down their metabolic activities while too high the temperature denatures the enzymes. Many reports reveal optimal temperature for growth of yeast as 30°C. The continual increase in cell biomass from 0.7 – 1.489 (Table 3) is in agreement with the definition of fermentation by ^[5]. The then fall on the biomass at 7th day, also depicts its correspondence to the microbial growth curve.

These microorganisms, when recovered, could serve as fuel, animal feeds, single cell proteins or source of enzymes. Due to high solubility of these cells in liquid, immobilization could serve a better means of recovering them for effective use. End of fermentation was observed by no other changes in the fermenting sugar indicative of the specific gravity (1.003) for both the 7th and 8th day and a decrease in the O.D of the spectrophotometric reading. These observations suggested maximum ethanol production and correspond with observation of ^[23].

Following the method described by ^[19], 10.3% ethanol was produced by the end of fermentation. Though the ethanol could be said to be smaller compared to the report of ^[22], the result was in agreement with the result of ^[23]. The difference in the result with ^[22] may be due to the variety of cocoa pod used, medium of isolation of the yeast, or use of controlled- fermentation. The general shortcomings of batch fermentation described by ^[23], may also affect yield, suggesting continuous fermentation, increase of the yeast population by recycling and the removal of ethanol during the fermentation as a better method. An advancement in separating ethanol from the fermentation liquid (by partial distillation), recovered about 50% ethanol by volume which was reported by ^[17] on cassava waste. Further distillation of the sample under controlled conditions could produce a higher percentage of ethanol.

5. Conclusion

This study was undertaken to investigate the production of ethanol and biomass from cocoa pod using palm wine yeast, as a means of converting our waste to useful products. The results obtained revealed that wastes can be utilized in the production of value added products like ethanol. Acids can be used to digest cellulose releasing glucose for other purposes. Also palm wine yeasts can be effectively used in converting simple sugars to ethanol and CO₂ with subsequent production of microbial cell (biomass) for other uses. It can also as serve a good substrate for wine production. Cocoa pod, a waste material can therefore be utilized for production of ethanol, biomass and other value added products.

Acknowledgement

We wish to acknowledge the contribution and support of all management staff and nurses associated with the maternity section of the Federal Medical Centre Umuahia, Abia State, Nigeria.

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