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Bioethanol production from banana stem

The feasibility of using banana sap as a feedstock to produce ethanol is evaluated in this study. Banana sap is obtained by crushing the pseudostems and concentrated ten times and supplementing with other industrial byproducts such as corn steep liquor(CSL), spent wash (SW), and yeast extract (YE) for ethanol production. Acid and alkali hydrolyzes are performed to enhance the sugar levels of the sap before fermentation. Two different strains of Saccharomyces cerevisiae (MTCC170 and MTCC180) are used for fermentation. In general, supplementation of banana sap with industrial byproducts significantly enhanced the ethanol production. The maximum ethanol production (2.5 g/L) is observed with concentrated banana sap supplemented with 25% SW (v/v) with MTCC170, which is 16-fold higher than banana sap alone. The ethanol content is also higher in alkali-hydrolyzed banana sap supplemented with 25% SW compared to control. These results suggest that banana sap can be used as a renewable source to produce ethanol by supplementing with other industrial byproducts.

Biological Materials

Saccharomyces cerevisiae strains (MTCC170 and MTCC180) used in this study. These cultures were maintained on YEPD medium (3 g yeast extract (YE), 10 g peptone, 20 g dextrose, 1 L distilled water, and 15 g agar). Banana stems were crushed through sugarcane presser and filtered through a cheese cloth to remove solid particles. The clarified liquid was collected and concentrated ten times using a rotary evaporator and was further stored at 4 °C.

Analysis of Samples

The banana sap was analyzed for the different physicochemical properties such as pH, total organic carbon, fatty acids, COD, and BOD. Banana sap (ten times concentrated), SW, and CSL were then analyzed for COD, BOD, total suspended solids (TSS), total dissolved solids (TDS), total solids (TS), sugar content, phosphorus content, and nitrogen levels according to the standard methods.

Hydrolysis of Banana Sap

The ten times concentrated banana sap was subjected for acid and alkali hydrolyses to enhance the sugar level. Banana sap was hydrolyzed with concentrated hydrochloric acid (HCl). Briefly, HCl was added to banana sap samples to reach a final concentration of 0.5, 1.0, and 1.5 N (v/v). Each suspension was then autoclaved at 121 °C for 30 min. After hydrolysis, the pH of the sample was adjusted to 5.6 with 0.1 N NaOH. Alkali hydrolysis was performed by adding 0.5, 1.0, and 1.5 N NaOH to banana sap (v/v) and autoclaved at 121 °C at 30 min, cooled, and pH was adjusted to 5.6. The sugar content was estimated in hydrolyzed samples.

Ethanol Fermentation

In the present study, ethanol fermentation of the ten times concentrated hydrolyzed banana sap was evaluated by mixing other industrial byproducts. Two strains S. cerevisiae MTCC170 and MTCC180 were selected as biocatalysts for fermentation as these strains were well established for fermentation and known to ferment glucose and fructose. Batch fermentations were carried out in triplicate in sterilized 250-mL Erlenmeyer flasks with capped with screw cap. The working volume of each flask was 50 mL. To optimize the time period for maximum fermentation, 50 mL of the banana sap supplemented with 0.3% YE (v/w) was taken in 250-mL Erlenmeyer flasks and the pH was adjusted to 5.6. Then the two yeast strains S. cerevisiae MTCC170 and MTCC180 were inoculated separately and the flasks were incubated at 30 °C in a shaker at 150 rpm. The samples were analysed for ethanol production at different time intervals, i.e., at days 1, 2, 3, 4, and 5. The acid and alkali hydrolyzed samples mixed along with CSL, SW, and YE resulted in samples such as sap, sap + 1% CSL, sap + 3% CSL, sap + 5% CSL (v/v), sap + 1% YE, sap + 3% YE, sap + 5% YE (v/w), sap + 25% SW, sap + 50% SW, and sap+ 75% SW (v/v). The sugar content in these samples was determined by dinitrosalicylic acid method. Banana sap supplemented with the above-mentioned byproducts (50 mL) was taken in 250-mL Erlenmeyer flasks and the pH was adjusted to 5.6. Then the two yeast strains S. cerevisiae MTCC170 and MTCC180 were inoculated separately and the flasks were incubated at 30 °C in a shaker at 150 rpm. The samples were harvested after 4 days and analyzed for ethanol production. The ethanol content in the fermentation medium was estimated using the chromic acid method. Briefly, 10 mL of fermented broth was distilled, approximately 5 mL of distillate was collected and mixed with 25 mL of potassium dichromate and kept in water bath at 80 °C for 15 min. The samples were cooled and their optical density (600 nm) was recorded by UV-vis spectrophotometry. Ethanol standards were made by using ethanol/water (v/v) solution. All the experiments were carried out in triplicate.

Results

The physicochemical characteristics of the banana sap

pH: 5.6, 6.5 g/L total organic carbon, 0.9 g/L fatty acids, 6000 mg/L BOD, and 24 000 mg/L COD. The highest BOD was observed in SW followed by concentrated banana sap and CSL, which indicates that in SW aerobic biological organisms need more amount of dissolved oxygen to break down the organic material. TS, TDS, and TSS were high in CSL and lower in concentrated banana sap. Phosphorus levels were also estimated, which are less in banana sap as compared to the CSL and SW. The nitrogen content was higher in CSL compared to other samples. The sugar content was higher in concentrated banana sap followed by SW.

Optimization Conditions

To optimize the time period for maximum ethanol production, concentrated banana sap supplemented with 0.3% YE was inoculated with S. cerevisiae strains MTCC 170 and MTCC 180. The ethanol production was increased with increase in time period up to 4 days and decreased thereafter. Maximum ethanol production was observed at day 4 of fermentation in both yeast strains. Based on these results, the fermentation time was optimized as 4 days for further experiments.

Ethanol Fermentation

Acid and alkali hydrolyses were performed to enhance the sugar levels in concentrated banana sap. The maximum sugar levels were obtained when the concentrated banana sap was hydrolysed with 1 N HCl or 1 N NaOH compared to other treatments and, hence, these conditions were selected for further studies. The

reducing sugar levels increased from 7.1 to 7.93 g/L(11% increase) in acid hydrolyzed sample, while it was from 7.1 to 7.5 g/L (5% increase) in alkali hydrolyzed sample.

In acid hydrolyzed samples, the supplementation of banana sap with industrial byproducts, such as CSL, SW, or YE, and fermented with S. cerevisiae strains MTCC170 and MTCC180 enhanced the ethanol production. The maximum ethanol production was observed with concentrated banana sap supplemented with 25% SW (v/v) with MTCC170 where the ethanol content was 2.5 g/L (16-fold higher) followed by MTCC 180 with 1.85 g/L (eightfold higher) compared with banana sap alone.

The two strains showed about sevenfold higher ethanol production than banana sap alone.

In alkali hydrolysis, concentrated sap alone showed lower amount of ethanol production with S. cerevisiae strainsMTCC170 and MTCC180. The ethanol content increased when the sap was supplemented with other industrial byproducts such as CSL, SW, and YE.  Compared to control, CSL also increased the ethanol production and the maximum ethanol content was observed when sap was supplemented with 5% CSL where it increased about one- and 1.5-fold higher compared to banana sap alone. When compared to acid and alkali hydrolyzed banana samples, acid hydrolysed samples supplemented with CSL, SW, and YE yielded more ethanol than alkali hydrolyzed samples.

Conclusions

In the present study, banana sap supplemented with other industrial byproducts was explored for bioethanol production. The ethanol content increased with supplementation of byproducts, such as CSL, SW, and YE, compared to the sap alone. The maximum ethanol content was recorded when the sap is mixed with 25% SW (v/v) with both S. cerevisiae strains in acid hydrolysed samples compared to sap alone. Supplementation of banana sap with 5% YE also increased the ethanol production by eight- and six fold in acid hydrolyzed sample treated with MTCC170 and MTCC180, respectively. The results suggest that the banana sap disposed into the environment can be used as a potential source for bioethanol production. However, studies are required to optimize the hydrolysis of the banana sap, as well as further up concentration of the sap to 20 or even 50 times, which could be economically viable and also select a suitable yeast strain for its maximum fermentation.

Reference:

Gupta, G., Baranwal, M., Saxena, S. and Reddy, M.S., 2019. Utilization of banana stem juice as a feedstock material for bioethanol production. CLEAN–Soil, Air, Water, 47(9), p.1900047.