EXTRACTION OF BIOETHANOL FROM PRETREATED SAWDUST

In this study, optimization of brix conversion of wood shavings by Saccharomyces cerevisiae was investigated. Wood shavings were collected from timber shade at Naze, Owerri Imo state. Chemical pretreatment and hydrolysis was evaluated. Brewer’s yeast ( Saccharomyces cerevisiae ) was used as the isolate for fermentation. A five factor box behnken design was used. The effect of incubation temperature(30-40oC), inoculum size(0.85),incubation time (72-120hours), wood concentration( 10-20g), and NaOH concentration (3.5-4.5%) were studied using response surface methodology (RSM). Brix concentration was used as a response in the resulting experimental design. The statistical analysis of the constructed model developed by RSM suggested that linear effects of NaOH concentration and wood concentration were both significant (p<0.05) for biomass

Cellulose hydrolysis 15g of wood shavings (substrate A) was measured into 3 conical flasks each containing 2%, 3% and 4% sulphuric acid. Also, 15g of wood shavings (substrate B) was measured into 3 conical flasks each containing 2%, 3%, and 4% sodium hydroxide. All six (6) conical flasks were heated at 1000C for 30-40minutes after pretreatment to remove impurities. They were allowed to cool and filtered afterwards using the whatman filter paper.

Determination of Brix level.
The Brix level were determined for all six flask using a refractometer.

Multiplication/standardization of the isolate (yeast)
10% glucose was prepared and the yeast was inoculated into it and incubated aerobically for 48hours to multiply the yeast. After incubation, the isolate were made in triplicates using a turbidity scale of 4, 5 and 6 respectively and the optical densities were standardized using the Mc farlands standard table with the aid of a spectrophotometer at 600nm.
After hydrolysis, the pH was reduced to a proper level of within 6.5-7.0 with distilled water using a pH conductivity meter. Then the products from the pH normalization were inoculated with yeast for fermentation to proceed.

Balancing optical densities.
Using McFarland's standard, the optical densities of the 46 extracts were balanced respectively.

Fermentation of hydrolyzed sawdust.
Fifteen mililitre of the yeast were inoculated into the 250ml conical flask for the whole forty six (46) extracts and incubated at a temperature of 30oC, 35oC, and 40oC respectively for 72hours, 90hours, 120hours…..

Statistical Analysis
Results obtained from this work was subjected to statistical Analysis at P<0.05 using the MINITAB 17 Software to obtain response surface plots, Statistical Significance of Factors as well as interval plots of differences and deviations across the Mean.

Results And Discussion:-Preliminary assessment of hydrolytic property of Acid and Alkaline on Saw dust
Brix levels of hydrolyzed wood, demonstrated by the amount of brix produced in the wood extract was measured and tabulated in Table 3.1. The results show that higher brix was recovered from the alkaline treatment than the Acid treatment.

Main effects plot for biomass converted.
The single effects of Wood concentration, sodium hydroxide concentration (NAOH), and inoculum size (OD), incubation time (hrs.), and incubation temperature on the conversion of biomass converted for the production of Bioethanol is shown in Figure 1. The production of bioethanol, characterized by biomass conversion to alcohol, was observed to increase with increase in wood concentration until a concentration of 15g/100ml was reached. Above this concentration, there was a considerable decrease in biomass converted. The biomass converted as a result of changes in the wood concentration had significant effects at p< 0.05.
On the other hand, the effect of NaOH concentration on the biomass converted was also studied. The results showed that there was a positive correlation between increase in the concentration of NaOH and Biomass converted until a concentration>4%. Afterwards biomass converted remained fairly constant with increase in NaOH concentration.
Similarly, the effect of the increase in NaOH in relation to Biomass converted was significant at P < 0.05. The effect of inoculum size and incubation time on the Biomass converted was also studied. Despite the fact that these two factors had no significant effect at p<0.05, results showed that an increase in each of both factors resulted to a slight increase in the biomass converted. Similar result was also recorded for the incubation temperature. A slight decrease in biomass converted was recorded as a result of a slight increase in incubation temperature for up to 380C which was followed by an increase till the end of the experiment. Also, this effect was not significant value at P < 0.05.    Res. 8(06), 23-33 29 Fig. 4a shows that at constant holding values of wood concentration, inoculum size and incubation time, the surface plot shows that the increase in biomass converted was not directly proportional to increase in NaOH concentration as seen in the figure. an increase in biomass converted showed a corresponding increase in incubation time. However, this result is not significant at P< 0.05.   Optimization of factors for the conversion of Biomass to bioethanol. Fig. 5a shows the optimization plots for the optimum production of Bioethanol as a function of Brix conversion. The result showed that the optimum conditions for the production of Bioethanol are wood concentration of 15.66g, NaOH concentration of 4.47%, Inoculum size with an Optical density of 0.85, Incubation time 72.0 hours and Incubation temperature 40.0oC. At these conditions, the isolate used was able to convert 60.97% of the Brix in the wood extract to ethanol. Experimentally, at the optimum conditions, a yield of 49.12% was obtained and the efficiency of the fermentation was calculated as

Discussion:-
During pretreatment process, the biomass is usually depolymerized, washed and the pH adjusted to 7.0. During this procedure, hydrolyzed sugars are lost and this work aims to utilize the already hydrolyzed sugars that are present, following a chemical pretreatment. This aspect of biomass conversion is poorly explored. The pretreatment is regarded as efficient when the formation of sugars in further enzymatic step of biomass processing is enhanced as well as the degradation of carbohydrates and the formation of inhibitory compounds are limited to minimum (Choi et al., 2018;Łukajtis et al., 2018;Kumari et al., 2018).
The result obtained from this work showed that the optimum conditions for the production of Bioethanol are wood concentration of 15.66g, NaOH concentration of 4.47%, Inoculum size with an Optical density of 0.85, Incubation time 72.0 hours and Incubation temperature 40.0oC. Ethanol was calculated from the Brix obtained before pretreatment which gave alcohol yield of 1.96%, 2.82% and 3.87%. After pre-treatment, ethanol was obtained at an optimum time of 72hours and a yield of 1.68-2.25% ethanol was produced. The estimated yield is higher when compared to other literatures nevertheless, the result may vary due to the different substrates others used.

Conclusion:-
Much energy is trapped in lignocellulosic systems and the need to depolymerize this natural polymer is unending. Truly, bioethanol has promising results and can be used to substitute fossil fuels in the nearest future. In order to encourage zero waste in the production of bioethanol from wood shavings, this work was done using what is regarded as product of chemical pretreatment and used for the production of bioethanol. If energy is lost in this 31 process, there is a need to harness this wood extract in the bioethanol production process as a solution for adjusting pH of the reaction flask rather than discarding it as effluent.
Also, the need for optimization cannot be overemphasized. the optimum conditions for the production of Bioethanol are wood concentration of 15.66g, NaOH concentration of 4.47%, Inoculum size with an Optical density of 0.85, Incubation time 72.0 hours and Incubation temperature 40.0oC. At these conditions, the isolate used was able to convert 60.97% of the Brix in the wood extract to ethanol. Alcohol yield was obtained which gave 1.68-2.25%respectively. Despite the fact that the theoretical yield was not equal to the actual yield, the production of ethanol through the optimized process is vital for production purposes.