3.1 Performance of MFC
Combination of fermentation and electricity generation demonstrated a promising performance in the MFC. Both the yeast S. cerevisiae and P. fermentans grew rapidly under experimental setup (membrane less single chambered MFC). All the setup depicted a typical behaviour of MFC in terms of its electrochemical responses. In the absence of exogenous electron mediator, the intracellular proteins of the model organisms play a major role in electron transfer located inside the cell. This could possibly overcome by adherence of yeast cells to the carbon fibre surface through force driven interaction such as electrostatic or physical adsorption (Rossi et al. 2015).
3.2 Electrochemical response
Electrochemical performance of both the MFC setup containing either P. fermentans or S. cerevisiae, gradually increased after 24 h of inoculation, which was almost stable upto 15 days of the incubation. Maximum OCV for P. fermentans 0.318 ± 0.0039 V was recorded on 15th day (Fig. 1a).
Current density also increased gradually from 4.848 mA m− 2 (1st day) to 57.348 mA m− 2 (15th day) (Fig. 2c and d). Power density of the cell also followed similar pattern with maximum power density of 8.299 mW m− 2 was recorded on 15th day. For S. cerevisiae maximum recorded OCV was 0.287 ± 0.009 V on 15th day (Fig. 1a) with increasing current density from 1.66 mA m− 2 on 1st day to 43.63 mA m− 2 on 15th day (Fig. 2a and b). Power density was also recorded with same increasing pattern out of which maximum is 4.47 mW m− 2 on 15th day (Fig. 2a).
The internal resistance (Rint) was very high against different Rex from 1000 Ω-82 KΩ for both the organisms on 1st day which drastically decreases from 2nd day onwards (Fig. 3a and b). Combination of carbon fibre anode and stainless steel cathode has been explored well in the setup. Thus indicates the present setup was intact without any physical damage or corrosion and can be reused efficiently after sterilization. MFC appears to be an ecological approach to achieve the cost-effective electricity generation. The maximum power density for P. fermentans was 8.299 mW m− 2/100mL while the current density was 57.348 mA m− 2/100mL on 15th day and 0.318 ± 0.0039 V power output for the same day. From past years, Pichia has been explored in the field of MFC such as Pichia pastoris, Pichia stipitis, Pichia kudriavzevii etc., for power generation with genetic modifications and less interest was focused on efficient bioethanol formation (Pal and Sharma 2019). Whereas the S. cerevisiae was measured at power density of 4.473 mW m− 2/100mL on 15th day while the current density was 43.636 mA m− 2/100mL on 15th day and power output was of 0.287 ± 0.0094 V.
Amongst them P. fermentans was resulted in better power, current density and OCV than S. cerevisiae. This is because of low resistance and large surface area of carbon brush electrode. Makes them ideal features as anodes from small as well as large scale applications of MFCs (Yuan et al. 2020a). Yuan et al recently published a report on simultaneous power generation system and bioethanol formation. In this, they used S. cerevisiae as model organism for dual and single chambered MFC setup with or without Mediator for power generation and ethanol production. This study resulted in a high power output (5.2 ± 0.5 W/m3) and high ethanol yield (92.5 ± 2%). There are many factors affecting electron transport such as cell metabolism, pH value, mediator type, yeast cell growth, substrate. But excessive addition of MB for electron transfer could affect the activity of yeast and lasts in unbalancing and blocking of electron transmission.
3.3 Glucose consumption and ethanol production
The quantifiable reducing sugar content was carried out using Dinitrosalicylic acid (DNSA) method. Basic principle involves for the method is interaction of an alkaline solution of DNSA with reducing sugars such as glucose and fructose wherein the aldehyde group of reducing sugar is oxidized to the carboxylic acid and the 3-nitro group (NOO−). The change in colour was measured at 540 nm (Jain et al. 2020). The variation of the orange-red color index was the indication of presence of reducing sugar to high/low extent.
The sugar consumption and ethanol production by these yeasts was studied in 100 mL single chambered cell setup in sugar medium containing 10% (Glucose) 100 mL Erlenmeyer flasks, supplemented with 0.25% yeast extract and incubated as described above. The glucose content was calculated from the standardization curve of D-glucose (y = 6.5333x + 0.0613, R² = 0.9909) and expressed as mg/mL. The glucose was efficiently consumed during initial days of incubation period; about 15–20% of glucose was consumed in one day. After 24 h, the glucose concentration reduced from 10% (w/v) to 7.8 ± 0.0004% (w/v) in the reactors inoculated with P. fermentans and 8.4 ± 0.0003% (w/v) for S. cerevisiae, while the glucose concentration was only 0.2 ± 0.00002% (w/v) at the end of the experiment. A detectable amount 0.025% (v/v) of ethanol was observed on day 1 for P. fermentans, which was maximum on 12th day 4.7% (v/v) and remain constant on 13th day and decreased gradually. As, Pichia utilizes hexoses slowly than pentose sugar as a carbon source and ethanol formation during fermentation process (Tahir and Mezori 2020).
Whereas, in the case of S. cerevisiae the ethanol concentration measured on day 1 was 0.03 % (v/v) with remaining glucose concentration of 8.4 % (w/v). S. cerevisiae produced maximum ethanol on 12th day 5.6 % (v/v) and the remaining glucose concentration was left with 0.23 % (w/v) at the end of experiment (Fig. 4a and b). It shows alcohol production by yeast cells P. fermentans and S. cerevisiae on day wise fermentation analysis.
The 24 h old cells subjected to fermentation in a 10 % glucose solution (10g/100mL) and have ability to ferment glucose to ethanol. The cell setup of P. fermentans showed maximum 4.7 % (v/v) ethanol production of theoretical yield (6.41 % v/v for 10 % glucose) on day 12 and continued to remain constant upto day 13 and gradually decreased on consecutive days (Nandal et al. 2020). Since 1 mol of glucose produces 2 mol of ethanol and 2CO2. This infers that the power generation in MFC setup is independent of fermentation process. In point of fact, ethanol and power generation occur concurrently with the process of glucose metabolization through yeast. It been reported from several studies that under anaerobic conditions, 1 mol of glucose can be converted to give 2 mol of ethanol and 2 mol of electrons. It is evident that the under anaerobic conditions, yeast-MFC cannot utilize all the electrons from complete oxidation of glucose. Another major reason is the direct electron transfer by a yeast cell is very limited, as compared to Shewanella and Geobacter (Christwardana et al. 2018). Therefore, this study focused on the simultaneous production of both bioethanol and electricity generation so that the substrate can be fully utilized for significant benefit.
Based on the stoichiometric information, glucose concentrations were theoretically converted to electron amounts. It was used to calculate the amount of electrons passed through the MFC circuit during glucose oxidation (Flimban et al. 2019). The CE According to the formula for CE, CE = (CEX × 100/CTh), one can calculate how many electrons were involved in electron transfer through the MFC external circuit (Yuan et al. 2020b). Over 24 h, the CE of the proposed MFC setup for P. fermentans was 0.012% and increased upto 0.89% gradually along with the increase in the current density (Fig. 1b). Whereas, the CE efficiency after 24 h for the S. cerevisiae was 0.002% and increased to 0.47% on concluded day. Still, most of the electrons may possibly involve in the ethanol production process. This indicates that MFC setup had little effect on fermentation efficiency (Walker and Stewart 2016; Yuan et al. 2020a). The carbon fibre of electrode provides massive surface area for yeast adsorption on its electrode surface. Hence, the application of yeast cells on carbon fibre electrode improved the immobilization effect onto the electrode (Yuan et al. 2020a). A higher glucose concentration would yield more electrons for high electricity generation and ethanol (dos Passos et al. 2019; Zhao and Zhang 2019). Whereas, a massive literature survey suggests the ongoing efforts for energy generation and biofuel formation (Moradian et al. 2021). Still there is lot to unravel about the efficient mechanism behind the electron transfer and different substrates conversion to energy generation requires further consideration.
3.4 Effect of pH
In industrial ethanol production, yeast tolerates wide range of pH, thus making the whole process less susceptible to contamination. It was observed that higher acidic conditions produces larger amount of ethanol (Mohd Azhar et al. 2017). At the time of yeast fuel cell setup for P. fermentans and S. cerevisiae, the initial pH was maintained at 7. In the cells inoculated with P. fermentans, the pH decreases to 6.5 after 24 h and here ethanol was measured is 0.3% (v/v), which was further decreased to 5 and was constant from day 4 to day 7 and during these days the concentration of ethanol ranged from 0.8–2.3% (v/v). A decrease in pH and increase in ethanol yield was observed during further incubation. In S. cerevisiae after 24 h the pH decreased upto 6.6 and a detectable volume of ethanol was recorded 0.3% (v/v). The pH decreased upto 5 during further 9 days of incubation and increasing ethanol concentration upto 5.6% (v/v). As per the results obtained P. fermentans produced maximum ethanol 4.7% on 12th day with pH 4.3, while S. cerevisiae produced maximum ethanol 5.6% on 12th day of incubation with 4.8 pH (Figure S1). The yeast S. cerevisiae strains are considered to be pillars of fermentation industry since then dominated ethanol fermentation due to their low pH tolerance for ethanol formation, organic acids, and low oxygen availability. It is evident that in P. fermentans the correlated effect of respiratory and fermentative pathways supports growth and product formation. This yeast ferments glucose or xylose under oxygen-limited conditions (Kwak et al. 2019).
3.5 EPS production
EPS production was observed in both the yeast P. fermentans and S. cerevisiae along with their growth and colonization on anode surface. Growing biofilm were observed under scanning electron microscopic images (Fig. 5).
The biofilm was developed on the anode surface as well as on the top of the medium. A significant correlation between EPS production and yeast growth was observed as analysed on different days (day 1, 5, 10 and 15). The cells grew rapidly along with the biomass accumulation on different time intervals and resulted in gradual increase in biomass, protein, EPS and carbohydrate (Fig. 6a and b).
Both the yeast P. fermentans and S. cerevisiae produced EPS along with a dense biofilm on carbon fibre anode, which showed efficient direct electron transfer. However, its EPS may have boosted the electron transfer via the indirect mechanism (Pal and Sharma 2019). Evident studies suggested that the combination of yeast attached anode improves electron transfer directly creating synergistic effect (Chung et al. 2016). EPS composed of polysaccharides, extracellular DNA, glycoproteins, glycolipids and proteins. This EPS plays some significant roles such as microbial cell to cell communication, protection from external and specially extracellular electron transfer (Flemming et al. 2016). It is evident that presence of carbohydrate in EPS carry out specific functions in mat formation.
The protective glycocalyx acts as a mediator in yeast for electron transfer and may be involved in oxidation and reduction reactions. The presence of EPS matrix was confirmed by infrared (IR) spectrum. After 24 h (day 1), the EPS production for Saccharomyces cerevisiae and Pichia fermentans was minimum but increased gradually during further incubation on day 5, day 10 and day 15. The spectrum of purified EPS showed numerous peaks from 3585 − 502 cm⁻¹ (Figure S2a and b). The EPS absorption frequency from 3585 to 3174 cm⁻¹ and 3688 − 3071 cm⁻¹ showed the presence of alcoholic (O-H) group, primary and secondary amine group confirms the polysaccharide nature of EPS produced by S. cerevisiae and P. fermentans, respectively. Peaks ranging from 2969 − 2763 cm⁻¹ and 2956 − 2763 cm⁻¹ represent saturated aliphatic (alkene/alkyl) with methyl C-H (− CH3) asymmetrical/symmetrical stretch and ether and oxy compound with methoxy, C-H stretch (CH3-O-) in the case of S. cerevisiae and P. fermentans, respectively (Abid et al. 2021). The absorption peak at 1628 cm⁻¹ was due to the stretch vibration of carboxyl group (C = O) in S. cerevisiae. Peaks ranging from 1838 − 1221 cm⁻¹ represents primary amide C = O, -CONH2 and secondary amide with N-H stretch bend in EPS produced by P. fermentans.
The absorption peaks ranging from 1011 to 1036 cm⁻¹ showed alkyl halide and stretching vibrations of pyranose ring (Li et al. 2014). The peaks between 904 − 509 & 604 cm⁻¹ showed stretch of alkyl halides and d (C-O-C) glyosidic linkage respectively (Pal and Sharma 2019).