Lignocellulosic raw materials minimize the potential conflict between land use for food (and feed) production and energy feedstock production. The raw material is less expensive than conventional agricultural feedstock and can be produced with lower input of fertilizers, pesticides, and energy. Biofuels from lignocellulose generate low net greenhouse gas emissions, reducing environmental impacts, particularly climate change.

Brewer spent (BG) is a by-product of the brewing process, consisting of the solid residue remaining after mashing and lautering. It consists primarily of grain husks and other residual compounds not converted to fermentable sugars by the mashing process. BG is the most abundant brewing by-product, corresponding to around 85% of total by-products generated (Xiros et al., 2008) and is mainly used as low-value cattle food⁴. The chemical composition of BG varies according to barley variety, harvest time, malting and mashing conditions, and the quality and type of adjuncts added in the brewing process (Huige, 1995; Santos et al., 2003)^5,6. BG according to Xiros et al., 2008, contains mainly hemicellulose in the form of arabinoxylans from the barley grain and cellulose. BG has the potential to serve as a low-cost feedstock for the production of ethanol since hemicellulose and cellulose content corresponds to 52% w/w of dry BG. Other substances such as proteins, lignin and fat are also present in BG in significant quantities. Corn fiber (CF), a waste from maize pap is a heterogeneous complex of carbohydrate polymers and lignin. It is primarily composed of the outer kernel covering or seed pericarp, along with 10–25% adherent starch. Carbohydrate analyses of corn fiber vary considerably according to the source of the material. Generally, corn fiber has been reported to include 30–50% arabinoxylan and 15–20% cellulose (Leathers, 1998; Gaspar et al., 2005)⁷. The other carbohydrate component in corn fiber is hemicellulose, a well-branched polymer of xylose substituted with arabinose, galactose, mannose and glucose (Sun et al., 2000)⁸. Some of the side chains may also contain acetyl groups of felurate (Carpita and Gibeaut, 1993)⁹.

One to two per cent of bread baked in large bakeries is unsuitable for sale as is does not satisfy the required specifications with the bread ending up as cattle feed or waste. The bread residues can be fermented to get the ethanol yield around 0.35 g/g substrate (Ebrahimi et al., 2007)¹⁰.Bread has always been a valuable item – either home baked or purchased from the baker – although much cheaper in comparison to other foodstuffs. Every year, in Austria, an estimated amount of 60,000 to 65,000 tons of bakery goods are thrown away – not including private waste! Empirical studies being carried out by the Institute of Waste Management, BOKU – University of Natural Resources and Applied Life Sciences in accordance with internal extrapolation, branch experts agree that this figure is probably reliable (Laura Thomas, (2009)¹¹. Cellulosic biomass including forestry residue, agricultural residues, pulp mill refuse, switch grass and lawn, garden wastes and municipal solid wastes (MSW), is a potential feedstocks for the synthesis of biofuels. Lignocellulosic biomass is a renewable resource and has great potentials for the production of fuel ethanol because it is less expensive than starch (e.g. corn) and sucrose (e.g. sugarcane) producing crops and available in large quantities. Agricultural lignocellulosic residues are abundant renewable resources for bioconversion to sugars, which can then be fermented to fuel ethanol. The most important benefit of fuel ethanol production from biomass is reduced CO₂ emissions, thus reducing the greenhouse effect. Conventional production of ethanol from cellulose via fermentation involves a complex process of pretreatment in attempt to recover a maximum amount of sugars from the hydrolysis of cellulose and hemicellulose, and to ferment them into ethanol. Pretreatment is required to alter the biomass macroscopic and microscopic size and structure as well as its submicroscopic structural and chemical composition and to facilitate rapid and efficient hydrolysis of carbohydrates to fermentable sugars. The pretreatment aims to increase pore size and reduce cellulose crystallinity. In acid-catalyzed pretreatment, the hemicellulose layer is hydrolyzed, whereas in alkali-catalyzed pretreatment, mainly, a part of the lignin is removed and hemicellulose has to be hydrolysed by the use of hemicellulases. Hence, pretreatment is necessary to expose the cellulose fibres to the enzymes or to at least make the cellulose more accessible to the enzymes. An efficient pretreatment can substantially reduce the enzyme requirements, which make up a large part of the production cost. Pretreatment techniques have generally been divided into three distinct categories, including physical, chemical, and biological pretreatment.

Fermentation is an ATP-generating process in which organic compounds act as both donors and acceptors of electrons; it could either be aerobic or anaerobic, by which oxidation of a substrate occurs, with an inorganic substance acting as the final electron acceptor (Verhagen, 1981; Lange et al., 2000). Two groups of microorganisms - enteric bacteria and some yeasts - are able to ferment pentoses, but with low ethanol yields. Furthermore, in the case of xylose fermenting yeasts (Pachysolen tannophilus, Candida shehatae, and Pichia stipitis), large-scale utilization is hampered by their sensitivity to high concentrations of ethanol (≥40 g/l), the requirement for carefully monitored microaerophilic conditions, high sensitivity to inhibitors, and the inability to ferment xylose at low pH. Some filamentous fungi have been shown to ferment most of the sugars found in pretreated biomass hydrolysates, such as glucose, mannose, galactose, xylose, and arabinose. Some fungi, such as Monilia, Fusarium, Rhizopus, Aspergillus, Neocallimastix, and Trichoderma, have been reported to possess the ability to convert cellulose to ethanol.

Recent genetic improvements focused on the transformation of Saccharomyces cerevisiae and Zymomonas mobilis could result in good fermentative performances on pentoses (Hahn-Hagerdal et al., 2006)³. Three approaches have been attempted to enable the xylose to be utilized: (i) to clone genes from pentose utilizing species into S. cerevisiae; (ii) to co-culture two different strains of genetically modified Zymomonas mobilis; and (iii) to clone pentose-utilizing genes into ethanol-resistant strains of E. coli. The demand for bioethanol is expected to increase dramatically until 2020. In 1999 the US signed an executive order specifying a tripling in the production of biobased products and bioenergy by the year 2010. As a consequence, US oil imports will be reduced by nearly 4 billion barrels over that time. Efforts to decrease greenhouse gas (GHG) emissions are expected to spur the production of renewable energy sources by 6% within the European Union by 2010 (Zaldivar et al., 2001)². In France, the approval of a clean air act could increase ethanol production to 500 million liters. Similar projects in Spain, Sweden and the Netherlands are expected to increase the utilization of ethanol to account for 15 % of transportation fuels by 2010 (Mansson and Foo, 1998)¹². The EU market for fuel ethanol will grow considerably in the coming years, as a result of the EU policy to substitute 8% of fossil transport fuels by renewable biofuels by the year 2020. The aim of this work is hydrolysis and bioconversion of lignocellulosic byproducts from Breweries, fermented corn pomace (‘Ogi’) and bakery (bread) waste to ethanol. Economically acceptable production of ethanol from lignocellulosic material could be also solution to dilemma: food, feed or biofuel, which has appeared in last years as a result of growing demand for biethanol on world’s market. Industrial production of fuel ethanol is predominantly from agricultural crops, which also serve as food or animal feed. In order to meet the increasing demand for alternative biofuels, biomass sources other than those used as food need to be explored. We have identified spent grains from breweries, ‘Ogi waste’ and waste bread as a potential biomass source for bioethanol.

MATERIALS AND METHODS:

Materials include fermenter, a fractional distillation column, and an attrition mill, among others.

Feedstocks:

Raw materials used include spoilt Bread (2–3 weeks old), obtained from a bakery and stored below 5 °C. Corn fibre was obtained from maize slurry made according to the method of Akingbala et al., (1981), and Brewer’s spent grain was obtained from International Brewery, Ilesha, Nigeria.

Chemicals and reagents

Chemicals and reagents were of analytical grade and products of SIGMA chemical Company Limited, USA and British Drug Houses (BDH) Limited, Poole, England. Dried bakers yeast (Saccharomyces cerevisiae) is the product of GYMA ZI. Le Terradou 84200 CARPENTRAS, TEWEX 431184, France.

Methods

The wastes materials collected were sundried and milled using an attrition mill, the milled raw materials were passed through 1.7mm screen according to Badal et al., 2006, prior to pretreatment, and 500 gm powder of each raw material was used as carbon source.

Pretreatment

A two-stage process which combines the Dilute Acid Prehydrolysis (DAPH-100-121) and alkaline delignification using NaOH as described by Dehnavi (2009). In this step, dry materials were submitted to a reaction with dilute sulfuric acid which consisted in the use of 1.25% (w/v) H₂SO₄ solution in a 1:8 g: g solid: liquid ratio.

One step Dilute Acid Prehydrolysis (DAPH-100) was performed in water bath at 100 ◦C for 1 hour.

One step Dilute Acid Prehydrolysis (DAPH-121) was performed in autoclave at 121 ^oC for 17 minutes. The solids were treated with 5% (w/v) sodium hydroxide solution in a solid: liquid ratio of 1:20 g: g, 120 ◦C for 90 min. After that the residual solid material (cellulose pulp) separated by filtration was washed with water to remove the residual alkali, and was dried at 50±5 ^oC for 24 hours.

Preparation of yeast culture A modified method of Kirimi et al., (2006) was used¹³. The inoculums were prepared using media containing (g/l): glucose, 100; yeast extract, 10; (NH₄)₂SO₄, 15; K₂HPO₄, 7; MgSO₄·7H₂O, 1.5; CaCl₂·2H₂O, 2 and 0.05M buffer citrate at pH 5.5±0.1. Volumes of 100 ml media were autoclaved at a pressure of 103.4KNm^-2 temperature 121^oC for 15 min to allow for sterilization of the flask content without degrading the essential nutrients for yeast growth. The sterilized content was cool and inoculated in 500 ml cotton-plugged Erlenmeyer flasks, and then incubated for 30 h at 35±0.5 ^oC and shaked at 150 rpm. Urea 5g, KOH, phosphate 5g, Yeast 20g were added to 100ml of H₂O using magnetic stirrer to stir. Adaptation was allowed for 4 hours. It was later poured into the fermentor which contains total sample.

Fermentation

A modified method of Akin-Osanaiye et al., (2008) was used. 400g portion of the pretreated sample was weighed into conical flask and 400 cm³ distilled water was added¹⁴. This was pasturized by boiling in a water bath for 15 min. It was allowed to cool and the inoculums added. The fermentation was then monitored from day 1 (24h) to day 7. The pH of the sample were adjusted with 0.5M NaOH, between 4.2-5.0. At the end of the fermentation, the fermented sample was poured into a cheese cloth to drain out the fermented broth. This procedure of carried out in triplicates.

Analytical methods:

(i) Determination of total carbohydrate content: The carbohydrate content of untreated and pretreated raw materials for fermentation were measured by phenol sulphuric acid method with glucose as standard.

(ii) Determination of crude fibre: The crude fibre content was determined as described by Pearson (1976). 3g of sample of sample was weighed into a Soxhlet apparatus and extracted with petroleum ether. The extracted sample was air dried and transferred to a dry 100ml conical flask and added 200ml of 0.128M H₂SO₄. The mixture was later boiled for exactly 30 minutes with the constant rotation of the flask every few minutes so as to mix the contents and remove particles from the sides. After boiling for 30minutes the mixture was allowed to cool for a minute and gently poured into an already prepared Buchner funnel. The suction was adjusted so that the filtration of the bulk of 200ml was completed within 10 minutes. The insoluble matter was washed with boiling water until the washing were free from acid, then washed back into the original flask by means of a wash bottle containing 200ml of 0.313M NaOH solution measured at ordinary temperature and brought to boiling point. The mixture was further boiled for another 30minutes and allowed to stand for 1 minute and then filtered immediately through a Whatman filter paper. The insoluble matter was later transferred to a weighed ashless filter paper and dried at 100^oC to a constant weight. The filter paper and its content were incinerated to an ash at a dull red heat. The weight of the ash was subtracted from the increase of the weight on the filter paper due to the insoluble material and the difference reported as fibre content.

(iii) Determination of Ash content: The ash content was determined as described by AOAC (1990).

(iv) Glucose standard: As the fermentation commenced, 10g of glucose was placed in a 100ml volumetric flask and was dissolved with distilled water. The solution was made up to 100ml to give a concentration of 100mg/ml of glucose solution. Serial dilutions of the solution were made to obtain glucose solution of various concentrations. 1ml of each of the solution was placed in a test tube and two drops of alkaline 3, 5-dinitrosalicylic acid (DNS) were added and shaken. The test tube was then placed in boiling water for 5 minutes. The solution was allowed to cool to room temperature and the extinction was measured at 540nm. The glucose concentration (mg/ml) of the various glucose solutions versus their extinction values at 540nm were used to plot a glucose standard curve.

(v) Determination of reducing sugar in the fermenting sample: 2ml of the fermenting sample was placed in a test-tube and 1gm of activated charcoal was added. The mixture was shaken thoroughly. The mixture was filtered with filter paper until a colourless filtrate was obtained. 1ml of filtrate was placed in attest-tube and two drops of alkaline DNSA were added and the tube was placed in a boiling for 5minutes. The mixture was allowed to cool and the extinction was measured at 540nm. This measurement was taken as the absorbance value at zero hour. 24 hour of the commencement of the fermentation, 2ml of the fermenting sample was withdrawn and decolourized as earlier described this procedure was repeated at 48,72, 96, 120, 144 and 168 hours respectivel.

(vi) Analysis of the Percentage Alcohol Produced: The alcohol content was determined as described by AOAC (1990).

RESULTS AND DISCUSSION:

The production of ethanol from food and industrial waste by the efficient lignocellulolytic enzyme system of Saccharomyces cerevisiae is essential for the bioconversion of Corn fibre, Brewer’s spent grain and spoilt bread to useful and economic important product (Ethanol) (Murai et al., 1998)¹⁵. The optimization of the bioconversion of these wastes took into consideration the pH, temperature and considering the waste as the carbon growth substrate and other important factors.

Fig. 1 shows the concentration of the reducing sugar and total carbohydrate of Waste bread, Corn fibre and Brewer’s waste (before and after treatment). The result showed that the BW (bread waste) has the highest value of total carbohydrate (0.317mg/ml) with a corresponding reducing sugar (0.107mg/ml). The table also revealed that all other waste that was pretreated lost some of their total carbohydrate and gained more of the reducing sugar.

Figure 1: The Reducing Sugar and Total Carbohydrate of the Samples

KEY

CP: Corn Fibre; CPP: Pretreated Corn fibre, BSGA: Acid treated brewer’s waste, BSGB: Base treated brewer’s waste, BSGU: Untreated brewer’s waste, BW: Bread waste

Fig. 2: The percentage yield of Ethanol and Proof Spirit of the Distillate

KEY

CP: Corn Fibre, CPP: Pretreated Corn fibre, BSGA: Acid treated brewer’s waste, BSGB: Base treated brewer’s waste, BSGU: Untreated brewer’s waste, BW: Bread waste

The initial total carbohydrate values of unpretreated Corn fibre (CP) and Brewers spent (BSGU) was 0.20 and 0.215mg/ml respectively and reducing sugar 0.03 and 0.032mg/ml respectively. And for the pretreated sample samples of brewers spent; acid pretreated (BSGA) and Alkaline pretreated (BSGB) has values 0.14 and 0.033 for total carbohydrate respectively and 0.084 and 0.051mg.ml respectively for the reducing sugar. This result indicated that pretreatment with either acid (H₂SO₄) or alkaline (NaOH) generally reduce the availability of total carbohydrate and reducing sugar for fermentation (Kumar et al., 2009)¹⁶. The levels of reducing sugar in the sample determine to some extent the percentage of ethanol that will be produced from the fermenting medium. Fig. 2 shows the percentage yield of ethanol and proof spirit. There was indication of high percentage of proof spirit in the BW (bread waste) with value 4.12%, with corresponding 1.8% in ethanol yield. The results for Acid treated brewers spent (BSGA) for ethanol and proof spirit were 1.16 and 0.53% respectively, while the value of ethanol for Alkaline treated brewers spent brewers waste (BSGB ) and Corn fibre were not significant. This indicated that using some wastes containing between 0. 1- 0.5% reducing sugar a maximum ethanol yield of about 4.5% and a minimum yield of 0.5% can be achieved. The specific gravity of the fermented waste bread (0.99). Corn fibre (1.0) and brewer’s waste pretreated with acid, BSGA, (0.99), brewer’s waste pretreated with alkaline, BSGB, (1.0) were shown in figure 3. The specific gravity is defined as the ratio of the mass of that product to the mass of an equal volume of water at 4°C. It can also be used to determine the ethanol content in the fermented sample using hygrometer (Akpan et al., 2005)¹⁷. Table 1. Shows the crude fibre of the Waste bread, Corn fibre and brewer’s waste (before and after treatment). the crude fibre of the untreated brewers spent (BSGU); 34.23±0.16% was higher than the rest of the fermented waste, this was followed by acid treated brewer’s spent grain (BSGA); 31.43±0.33%, alkaline treated brewer’s spent grain (BSGB); 29.41±0.78%, treated corn fibre (CPP); (0.05±0.01) and untreated corn fibre (CP); 7.25±0.14 and bread waste (BW); 0.05±0.01. After pretreatment the level of the crude fibre was found to have significantly reduced for all the studied samples. The crude fibre quantity will affect the production of bioethanol from the samples. As expected, bread waste with the least amount of crude fibre produces the highest quantity of bioethanol. The acid treated brewer’s spent grain though with higher concentration of crude fibre than corn fibre- treated and untreated, produces more bioethanol. The only explanation that could be adduced to this is that the higher concentration of total carbohydrate and reducing sugar in the acid treated brewer’s spent grain are responsible for the more bioethanol production as explained in figures 1 and 2.

Fig. 3: The Specific Gravity of the Distillate

KEY

CP─Corn Fibre, CPP─Pretreated corn fibre, BSGA─Acid treated brewer’s waste, BSGB─Base treated brewer’s waste, BSGU ─Untreated brewer’s waste, BW ─Bread waste

Table 1: The crude fiber content of Feedstock for Ethanol Production

SAMPLES	% FIBRE
Bread waste	0.05±0.01^f
Untreated corn fibre	7.25±0.14^e
Treated corn fibre	5.86±0.25^f
Untreated brewer’s spent grain	34.23±0.16^a
Acid treated brewer’s spent grain	31.43±0.33^b
Base treated brewer’s spent grain	29.41±0.78^c

Table 2: Ash of the distillate

SAMPLES	%ASH
Bread waste	2.08±1.96^a
Corn fibre	0.12±0.00^b
Acid treated brewer’s waste	0.27±0.00^b
Base treated brewer’s waste	0.21±0.00^b

Table 2. Shows the ash content of the distillate from the Waste bread, Corn fibre and Brewer’s waste. The result indicated that there was a significant higher level of ash in the ethanol distillate from the bread waste (2.08±1.96) than all other waste distillate for ethanol. The ash content, which is a measure of the mineral content of food (Nnamani et al., 2009), was found to be significantly high in the distillate of waste bread and least in distillate of corn fibre (0.12±0.00)¹⁸.

APPENDIX

Fig. 4: Graph of standard glucose concentration (mg/ml) vs. the extinction values at 540nm

Table 3. Shows the pH of the waste feedstock for the production of ethanol, from the result the pH of the feedstock generally decreased during the period of fermentation. This is an indication that the fermentation process becomes more acidic as a result of the production of other secondary metabolites and activities of other lactobacteria in the fermentation medium. In addition, the decrease in pH remain constant on the 5^th day for the bread waste, on the 4^th day for the acid treated brewer’s spent grain, and on the 3^rd day for both the base treated brewer’s spent grain and corn fibre. This also is in consonance with the ethanol production as bread waste with the longest fermentation as deduced from the time the pH become constant has highest ethanol production rate, followed by acid treated brewer’s spent grain, base treated brewer’s spent grain and corn fibre in that order^19,20.

Table 3: The pH of the Feedstock for Ethanol Production

Days	Bread waste	Brewer’s spent grain (acid treated)	Brewer’s spent grain (base treated)	Corn fibre
1	6.36	7.00	7.11	6.29
2	6.32	6.35	7.07	6.24
3	6.26	6.30	6.73	6.19
4	6.20	6.27	6.73	6.19
5	6.08	6.27	6.73	6.19
6	6.08	6.27	6.73	6.19
7	6.08	6.27	6.73	6.19

CONCLUSION:

The result of this study show that the rate of alcohol production through fermentation of industrial and food waste by baker’s yeast (Saccharomyces cerevisiae) increases with fermentation time. The finding of this work suggests that bioethanol can be produced from bread waste and brewer’s spent grain that has been pretreated with acid. In addition, the quantity of bioethanol production is directly proportion to the amount of total carbohydrate and reducing sugar in the samples and inversely proportion to the fibre content of the sample.

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Received on 31.12.2010

Modified on 22.01.2011

Accepted on 27.01.2011

Research J. Science and Tech. 3(2): March-April. 2011: 65-69