Bioethanol production using vegetable peels medium and the effective role of cellulolytic bacterial (Bacillus subtilis) pre-treatment

Abbreviations

SC1 - yeast isolates from sugarcane juice; DJ1 - yeast isolates from date juice; pH - Negative logarithm of hydrogen ion concentration; °C - Degree Celsius; % - Percentage; CH₃CH₂OH - ethanol or ethyl alcohol; YEPD - Yeast Extract Peptone Dextrose; nm - Nanometer; gm - Gram; ml - Milliliter; rpm - Round per minute; v/v - volume per volume; w/v - weight per volume; spp. - Species; et al. - And others.

Introduction

With the aims of protecting the environment and reducing dependence on petroleum and nonrenewable energy sources, the development of renewable energy sources has become increasingly important. Ethanol can be produced chemically from petroleum, and from biomass or sugar substrates fermentation¹. Fermentation-derived ethanol (CH₃CH₂OH) or ethyl alcohol is commonly known as bioethanol. This organic chemical is a flammable, clear and colorless liquid which can be used as fuel. Other functions of ethanol include its use as a solvent, antifreeze and germicide².

Several processes of bioethanol production currently exist, such as microbiological production from fermentable organic substrates or carbohydrates by yeast. Fermentation of cellulosic biomass, molasses, vegetable peels or food wastes can be considered as an economical process of bioethanol production³. Bioethanol produced from cellulosic materials by direct conversion is utilized in countries such as Brazil, Canada and, USA⁴. The economical production of bioethanol requires an easily available supply of inexpensive raw materials. Organic food waste is one of the topmost suitable materials for that process. Solid food wastes from household, restaurants or food processing industries can be obtained as a substrate to be used as fermentation medium for bioethanol production. Food wastes can also be recycled as animal feed and fertilizer after specific treatment.

The foremost focus of this ethanol production technology is the optimized utilization of biomass resources and microbial action on fermentation. One promising technique is the fermentation of lignocellulosic biomass where hydrolysis by specific microbial cellulase enzymes is involved^5,6. Ethanol can be derived from the fermentation of sugar-containing materials. Different yeast varieties are reported for the fermentation of lignocellulosic substrates to produce ethanol^7,8.

The objective of the project is to establish a highly efficient microbial fermentation process by natural yeast isolates to produce bioethanol. Other objectives included the characterization of the microbial strain. It is to be mentioned that ethanol production rate from insoluble lignocellulosic biomass is currently not economical. Therefore, commonly available cellulosic kitchen wastes were used as raw material. Proper treatment of the substrate was done to optimize the fermentation condition which has resulted in a highly efficient and economical production rate. Potential wild-type yeast strains were isolated from date juice, sugarcane juice, grapes, and pineapples. Wild-type yeasts were identified by the biochemical and physiological characterization and taken under comparative studies and experiments to obtain a strain with high productivity. Cellulose degrading bacteria (Bacillus subtilis) was used for pre-treatment of the fermentation media. Cellulolytic microorganism debased celluloses present in the lignocellulosic fermentation media and the degraded materials were easier and more readily available to be fermented by yeast.

Methods

Identification

A compound microscope (Model-CX-21, Olympus, Japan) was used to observe the cell morphology and the presence of yeasts were confirmed which were isolated from sugarcane juice (Figure 1A) and date juice (Figure 1B) (named SC1 and DJ1 respectively). Identification of each isolate of yeast up to species level was carried by the methods demonstrated by Kreger-Van Rij (1984)⁹ based on the morphology, sporulation and fermentation characteristics, as well as the assimilation of nitrogen and a range of carbon sources. Yeast specific API® identification kit (bioMérieux, Marcy-l'Étoile, France) was also used by inoculating 48-hrs culture broths into the chambers according to the protocol.

Figure 1.

The cell morphology (unicellular ovoid shape, multipolar budding; white and creamy texture) of yeasts under the compound microscope (100X) from sugarcane juice (A) and date juice (B).

Results

Morphology was visually observed as white and creamy texture, ovoid shape, multipolar budding pattern, under microscope (Figure 1A and B). Sporulation was confirmed due to the presence of ascospore. Nitrate reduction was not exhibited in the nitrate assimilation test (Figure 2A). Carbohydrate assimilation tests were conducted using API® 20C test strips (bioMérieux, Marcy-l'Étoile, France) (Figure 2B). In all cases, positive results were obtained for glucose, galactose, maltose, starch, and fructose (Table 1). Therefore, carbohydrates and nitrate assimilation test results signified the strong probability of isolates being the species Saccharomyces cerevisiae. Yeast isolates from sugarcane juice (SJ1) had a good growth at 25°C, 30°C, and 37°C, but showed poor growth at 40°C and 44°C. Yeast isolates from date juice (DJ1) had a good growth at 30°C, and 37°C, moderate growth at 40°C, but propagated poorly at 25°C and 44°C. Yeast isolate SC1 and DJ1 showed a variable growth result at pH 2–10. Overall, pH 5 and 6 was optimum growth conditions where the isolate SC1 exhibited the highest growth at pH 6 and DJ1 had its best growth at pH 5. All isolates showed excellent growth at 5% and 10% ethanol concentrations throughout the entire 48-hour incubation period (Table 2).

Figure 2. Biochemical tests to identify yeast species.

(A) Negative nitrate reduction test indicated by the yellow color (positive test would result in red color change) (B) API 20 C X kit results for different carbohydrates fermentation using API kit after 48 hours. Color in the chamber indicates a positive result, negative results retain the yellow color of the broth. Active ingredients contained in each chamber from left to right: 0 – none, 1 – D-glucose, 2 – Glycerol, 3 – Calcium 2-keto-D-gluconate, 4 – L-arabinose, 5 – D-xylose, 6 – Adonitol, 7 – Xylitol, 8 – D-galactose, 9 – Inositol, 10 – D-sorbitol, 11 – Methyl α-D-glucopyranoside, 12 – N-acetylglucosamine, 13 – D-cellobiose, 14 – D-lactose, 15 – D-maltose, 16 – D-saccharose/Sucrose, 17 – D-trehalose, 18 – D-melezitose, 19 – D-raffinose.

With fermentation conditions of 30°C incubation temperature with a pH of 6, the highest rate of alcohol production from a cellulosic medium (a mixture of papaya and potato peels pretreated with Bacillus subtilis) was 14.17% v/v or 141.7 gm/L (w/v) by yeast isolate SC1 (Figure 3A; Dataset 1). On the other hand, under the same fermentation conditions, the highest rate of alcohol production using the same cellulosic medium not treated with cellulolytic bacteria was 6.21% v/v or 62.1 gm/L (w/v) by the isolate SC1 (Figure 3B; Dataset 2). The highest rate of alcohol production from the pretreated potato and papaya media was 12.24% v/v or 122.4 gm/L (w/v) by isolate DJ1 (Figure 3A; Dataset 1) under a 48-hour fermentation condition at 30°C incubation temperature. The lowest alcohol production rate recorded under the same conditions using the untreated potato and cucumber media (2.15% v/v) by the isolate DJ1 (Figure 3B; Dataset 2). Fermented media with the highest percentage of alcohol (14.17% v/v) was distilled by a fractional distillation set. This highest percentage was achieved by the yeast isolate SC1 in fermentations condition of 30°C, pH 6 at 120 rpm. The cellulosic media pretreated with Bacillus subtilis was distilled after fermentation and the distilled product (one-time distillation) had an ethanol percentage of 52% v/v. In contrast, cellulosic media which was not treated with Bacillus subtilis had an ethanol percentage of 12% v/v after the first distillation. Fermentation in other media was recorded highest at 6.23% or 62.3 gm/L (w/v) alcohol production at pH 6 by SC1 isolate after 24-hrs fermentation (Table 3).

Figure 3. Alcohol production from vegetable peels by yeast isolates SC1 and DJ1 at pH 6.

Media treated with Bacillus subtilis (A) and No pre-treatment (B).

Discussion

Despite the availability of several industrial strains of yeasts, local isolates are usually more adapted to their own climatic condition. In this study, yeasts were isolated from local resources to serve the economic purpose. Utilization of isolated yeasts is an important strategy for the production of bioethanol¹². On the basis of the white and creamy appearance of selected isolates on solid media with butyrous colony texture, polar budding and oval cellular shape it can be assumed that isolates are members of Saccharomyces spp. from the method described by Boekhout and Kurtzman (1996)¹³. Fermentation of different sugars by the selected yeast isolates was observed. Yeast isolates from sugarcane (SC1) utilized glucose, maltose, fructose, galactose, starch, sucrose, and arabinose but failed to grow on sorbitol, melibiose, mannitol, trehalose, inositol, xylose and lactose. Yeast isolates from date juice (DJ1) utilized glucose, maltose, fructose, galactose and starch, but failed to grow in trehalose, xylose, sucrose and lactose. The conclusion was further reinforced by biochemical tests performed using bioMérieux’ API® 20C kits. Kit results for SC1 and DJ1 indicated that all of the isolates are Saccharomyces cerevisiae. As previous studies by Ramani et al. (1998)¹⁴ indicate that API® 20C kits have a statistical accuracy of 97% for common yeasts, the conclusions were assumed to be correct. Furthermore, nitrate assimilation tests for all isolates yielded negative results which confirming our hypothesis. Thermotolerance tests also indicated that all isolates (SC1 and DJ1) grew best at 30°C within a 48-hour incubation period; this is also the optimum growth temperature of Saccharomyces cerevisiae described by Alexopoulos (1962)¹⁵. As for ethanol tolerance, the general trend observed was a decrease in terms of tolerance of all isolates above 10% ethanol concentration signified by a slowdown in growth rate with a near growth stunt at 20%. Teramoto et al. (2005)¹⁶ demonstrated that members of Saccharomyces spp. can tolerate ethanol concentrations of up to 16.5%. However, since the isolates are wild-type Saccharomyces yeasts, an average maximum tolerance of 10% ethanol does not mean that they cannot be members of Saccharomyces spp. Different growth factors affect the pH tolerance of yeast. It was reported by Ivorra et al. (1999)¹⁷ that the optimum pH range for ideal growth varies from 4–6 depending on the strain. The cellular structure of yeast has a diverse mechanism to endure pH. In this experiment, yeast isolates SC1 and DJ1 had a variable growth result from pH 2–10. Both of the isolates had excellent growth from pH 4 to 6. However, those isolates were able to grow at all the pH condition, but pH lower than 3 and higher than 7 was not suitable for a good growth. Overall, pH 5 and 6 were optimum growth conditions where isolate SC1 had its best growth at pH 6 and isolate DJ1 had its best growth at pH 5. The ethanol production rate was recorded from the fermentation of different cellulosic media after 24 and 48-hours fermentation. The production rate ranged from 2.15% or 21.5 gm/L to 14.17% v/v or 141.7 gm/L (w/v). Isolate SC1 had the highest rate of ethanol production (14.17% v/v), and isolate DJ1 had the lowest rate of ethanol production (2.15% v/v) in shaking condition at 30°C with a media pH of 6. Ethanol production rate was also observed in shaking condition at 30°C with a media pH of 5. In this condition, isolate SC1 had the highest rate of ethanol production (9.42% v/v) and isolate DJ1 had the lowest rate of ethanol production (2.17% v/v), which surpassed the previous reports^18–20. Ethanol production using kitchen waste media has exceeded the earlier works²¹. In a study, Nofemele et al. (2012)²² demonstrated 7.8% percent ethanol production from sugarcane molasses using Saccharomyces cerevisiae. Maximum 9.48% ethanol yield resulted in a similar 2012 study²³.

In Bangladesh, five yeast isolates were reported²⁴ to be used for the similar experiments where those isolates (TY, BY, GY-1, RY and SY) had alcohol production rate of 12.0%, 5.90%, 5.80%, 6.70% and 5.80%, respectively at 30°C after 48 hours of incubation. Arapoglou et al. (2010), Wantanee and Sureelak (2004) and Yamada et al. (2009) reported 7–20 gm/L ethanol yield from potato peels²⁵, which is exceeded (46.6–82.7 gm/L) in the present study. Significant elevation of ethanol production rate was observed in the co-fermentation process where cellulosic media were inoculated with cellulolytic bacteria previously. Overall, the method proves the efficiency of the co-fermentation^26–28. The economic advantage of using vegetable peels media over molasses is the recycling process of abundant garbage. On the other hand, molasses has a purchasing value. Addition of nutrition supplements in future endeavors is also recommended¹⁸.

Conclusions

The present study allowed the isolation and characterization of two Saccharomyces cerevisiae isolates (SC1 and DJ1) with potential for ethanol production. Yeast isolated from sugarcane juice (named SC1 for this study) showed the highest percentage of alcohol production from cellulosic substrates. Vegetable peels pretreated with cellulolytic bacteria are detected as a suitable fermentation substrate. If the fermentation conditions are optimized, this procedure may be used for large-scale bioethanol production from cellulosic wastes. Scaling up of the experiment can be beneficial for power generation, bioethanol can be used as an alternative to fossil fuels. The raw materials required for the production of bioethanol are cheap and available. It will decrease environmental pollution, pave the pathway towards a proper waste management system and also fertilizers can be produced from the wasted substrate. This study was limited to vegetable peels media without diversification. In future, municipal organic waste may be considered in this regard. Recombinant strains may be employed to optimize the alcohol production rate, too.

Data availability

Dataset 1: Alcohol production from vegetable peels by yeast isolates SC1 and DJ1 at pH 6. Media pre-treated with Bacillus subtilis. 10.5256/f1000research.13952.d195639²⁹

Dataset 2: Alcohol production from vegetable peels by yeast isolates SC1 and DJ1 at pH 6. Media without Bacillus subtilis pre-treatment. 10.5256/f1000research.13952.d195640³⁰