Evaluation of ethanol production from pito mash using Zymomonas mobilis and Saccharomyces cerevisiae

This study investigated the potential of pito mash (waste from sorghum brewing) as alternative and cost-effective feedstock for bioethanol production by means of fermentation using Zymomonas mobilis and Saccharomyces cerevisiae isolated from freshly tapped palm wine. Fermentation parameters such as pH, temperature and incubation period were studied. The fermentation microbes, Z. mobilis and S. cerevisiae were identified using API TM test kit and morphological characteristics, respectively. Analysis of reducing sugar residue was performed using dinitrosalicylic acid (DNS) method, while analysis of ethanol content was performed using gas chromatography. Pito mash recorded total starch content of 6.69%, reducing sugar content of 11.1 mg ml -1 and cellulose content of 0.41 mg g -1 . Saccharification by malting increased reducing sugar content by 77.9% (19.75 mg ml -1 ). The optimum fermentation conditions (pH, temperature and incubation period) for Z. mobilis and S. cerevisiae were 5.5, 35°C, 3 days and 6.0, 30°C, 4 day, respectively. The maximum ethanol yield of 3.03 g l -1 and efficiency of 62% were obtained for S. cerevisiae while yield of 3.63 g l -1 and efficiency of 74.2% were obtained for Z. mobilis. Z. mobilis conclusively may be better organism for ethanol production from pito mash.


INTRODUCTION
Energy consumption has increased steadily over the last century as the world's population increases, and more and more countries become industrialized.The traditional source of fuel, fossil fuel is continuously being depleted irrespective of the new geographical discoveries.There have also been concerns about the pollution and various health risks associated with the use of petroleum as fuel.In view of these, the importance of alternative energy source has become even more necessary not only due to the continuous depletion of the limited fossil fuel stock but also for safe and better environment (Chandel et al., 2007).The interest in biomass as the alternative source *Corresponding author.Email: cofosu.appiah@gmail.com.
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License of energy is gaining momentum more and more over the last century.Production of bioethanol from biomass is one of the alternative sources of fuel that has gained a lot of attention over the past years.Ethanol produced from renewable energy source is the most promising future biofuel (Marszalek and Kaminski, 2008).To be a viable substitute for a fossil fuel, Hill et al. (2006) contended that an alternative fuel should not only have superior environmental benefits over the fossil fuel it displaces, be economically competitive with it, and be producible in sufficient quantities to make a meaningful impact on energy demands, but it should also provide a net energy gain over the energy sources used to produce it.Bioethanol meets most of these criteria but the quantity of ethanol produced annually has not overtaken petroleum.The use of starchy crops such as cereals and tubers as substrates for the production of ethanol has however, been reported to be much costly and non-sustainable accounting for about 70% of ethanol production (Ramesh et al., 2004).This drawback has caused an emergence of alternative substrates such as agro-industrial wastes that are relatively abundant in the environment and serve as renewable energy resources.Some of these agroindustrial wastes are rice straw, sugarcane bagasse, corn cob, pito mash, rice husk, municipal wastes to mention a few (Ramesh et al., 2004).The problem, however, is the technology for conversion of the lignocellulosic part of these materials to bioethanol.The choice of the best technology for the conversion of lignocelluloses to bioethanol should be decided on the basis of overall economics (lowest cost), environmental (pollutants), and energy (higher efficiencies).Many investigations have been performed on the appropriate technology for the conversion of the lignocellulosic to ethanol as well as substrate with little or no lignin such as molasses (Chandel et al., 2007), but research work on the utilization of pito mash has been very limited.
Even though several microorganisms, including Clostridium species, have been considered as ethanologenic microbes, the yeast Saccharomyces cerevisiae and facultative bacterium Zymomonas mobilis are better candidates for industrial alcohol production (Castro, 2013).Traditionally, S. cerevisiae has been used for the production of ethanol; however, it has been associated with low alcohol tolerance and low productivity which for efficient ethanol production requires improvement.Z. mobilis, a gram negative bacterium possesses advantages over S. cerevisiae with respect to ethanol productivity and tolerance.Z. mobilis strain grown under anaerobic conditions can produce about 1.5 to 1.9 mol of ethanol from each mol of glucose, which is much better than ethanol produced by S. cerevisiae (Gonzalez-Sanchez et al., 2011).Zymomonas grows and ferments glucose very fast; its preference for low pH prevents contamination and grows in high glucose and ethanol concentration (He et al., 2014).The present study focuses on the potential use of pito mash as a substrate for both Z. mobilis and S. cerevisiae for bioethanol production under optimum fermentation conditions such as temperature, pH and incubation periods.

Sample collection
The substrate for the fermentation, pito mash was obtained from local pito brewers in Kumasi in the Ashanti Region of Ghana.

Standard solid media
Five hundred milliliters of standard media was prepared by dissolving 2.5 g of yeast extract and 10 g of glucose in 500 ml conical flask containing 150 ml distilled water.Exactly 10 g of agar was then added, made up to mark and autoclaved at 121°C for 15 min.

Synthetic media
Synthetic media containing g/L: K2HPO4 1.0 g, (NH4)2SO4 1.0 g, MgSO4 0.5 and 20 g glucose was prepared in 1 L volumetric flask with 750 ml of distilled water, made up to mark and autoclaved at 121°C for 15 min.

Malt yeast peptone glucose media
Five hundred milliliters of MYPG was prepared by dissolving 1.5 g of yeast extract, 2.5 g peptone, 1.5 g malt extract, and 10 g of glucose in 500 ml conical flask containing about 150 ml distilled water.Exactly 10 g of agar (melted) was added and made up to mark and autoclaved at 121°C for 15 min.The pH of each medium was adjusted to the appropriate pH values using 1 N NaOH and 1 N HCl.

Sorghum base medium (SBM)
The SBM was prepared substituting glucose and malt extract with pito mash in the MYPG medium Isolation and identification of microorganisms Z. mobilis was isolated from palm wine using the method of Cheesbrough (2003).The inoculated plates were incubated at 30°C for 3 days.Pure colonies were obtained from re-cultivation in MYPG agar.Purified isolates from fresh plates of MYPG medium were identified as Z. mobilis using API TM test kit.S. cerevisiae was serially diluted and the sediment inoculated in standard media supplemented with chloramphenicol (0.05 mg/L) (Nwachukwu, 2001) and incubated at 28°C for 24 h.Colonies suspected to be yeast were purified and morphological and fermentable sugars were determined.

Malting and mashing of sorghum grains
Sorghum cultivar grains were washed and steeped in 0.2% sodium hydroxide and water for 8 and 16 h, respectively at room temperature.The steeped grains were allowed to germinate at room temperature for 4 days with daily sprinkling of water.The rootlets were broken and kept in oven for 24 h at 50°C.In the mashing process, a mixture of 20% malt and 80% mash was slurred and pre-heated to 45°C for 30 min after being raised to 100°C for 1 h.Complete saccharification was determined by iodine test.

Fermentation process
The procedures were adopted from Dowe and McMillan (2008).Fermentation was carried out in 500 ml Erlenmeyer flasks.The fermentation lock or bubble trap consisted of rubber stopper (with hole) through which a tube was inserted.A cotton plug was inserted in the tube and the tube was connected to silicone tubing.The other end was submerged in a test tube containing water.All mashes were cooled to a temperature between 27 and 30°C after liquefaction and saccharification and the pH adjusted with HCl.Saccharified mashes were then inoculated with 10 ml pre-culture S. cerevisiae and Z. mobilis in separate setups.Fermentation was performed in an incubator with intermittent shaking at optimized conditions.The fermentation process was monitored by measuring the sugar and ethanol contents.Ethanol concentration was estimated by Gas Chromatography and sugar content was measured using 3,5-dinitrosalicyclic acid (DNS) method.The expected ethanol amount was calculated after fermentation stoichiometry, assuming that 1.0 g of total sugars produced 0.511 g of ethanol.The efficiency of reducing sugar conversion into ethanol by both microorganisms (%) expresses the amount of produced ethanol relative to the theoretical quantity expected based on the sugar content of the malted sorghum, and it was calculated accordingly with the following equation: where TRSi is the initial sugar content before fermentation and TRSf is the final sugar content after fermentation (Carter, 2014)

Optimization of pH
Ten milliliters of the mashed sorghum was placed in different test tubes and pH adjusted to ranges of 4.0, 4.5, 5.0, 5.5 and 6.0 using 1 N HCl or 1 N NaOH.

Optimization of temperature
Ten milliliters of the mashed sorghum was placed in different test tubes at varying initial temperature values of 30, 35, 40 and 45°C.

Optimization of incubation period
Ten milliliters of the mashed sorghum was placed in different test tubes and incubated at 30°C, pH 4.5 and periods of 24, 48, 72, 96 and 120 h.

Chemical analysis
The amount of reducing sugars was estimated by dinitrosalicylic acid (DNSA) using methods described by Negrulescu et al. (2012).Ethanol concentration was determined using a Perkin Elmer, Autosystem XL, Gas Chromatograph (USA) equipped with a flame ionization detector (FID), coupled to a Yokogawa 3021 Pen recorder.

RESULTS AND DISCUSSION
Ethanol produced from spent sorghum using S. cerevisiae and Z. mobilis separately S. cerevisiae and Z. mobilis were employed in fermenting pito mash hydrolysate containing 19.75 g ml -1 reducing sugar.In both organisms, a continuous increase in ethanol yield was accompanied with decreased reducing sugar concentration during the whole period of fermentation (Figures 1 and 2).The fermentation with Z. mobilis proceeded very rapidly and was essentially completed in three days with maximum yield of 3.63 g l -1 . Fermentation with S. cerevisiae required three days to complete with a yield of 3.03 g l -1 .In all cases, the sugar utilization was faster in Z. mobilis than in S. cerevisiae.Ttest analysis showed significant difference between the amounts of sugar utilized by Z. mobilis and S. cerevisiae on each day at 95% confidence interval.This indicates that the utilization of reducing sugar on each day is dependent on the microorganism used.In both cases, the percentage yield of ethanol produced at each fermentation time examined using Z. mobilis was higher compared to that using S. cerevisiae.Bacteria are known to multiply faster than yeast thus Z. mobilis might reached the lag phase faster than S. cerevisiae and therefore utilized its substrate faster.The ethanol yield for Z. mobilis was higher than that of S. cerevisiae at all fermentation periods.S. cerevisiae is known to employ the EMP pathway to metabolize glucose producing 2 moles of ATP from 1 mole of glucose whereas Z. mobilis employing the E-D pathway produces 1 mole of ATP from 1 mole of glucose (Wang et al., 2014).Ming et al. (2014) reported that a significant amount of the carbon source is converted into biomass as a result of the E-D pathway used by this microorganism.All the enzymes involved in fermentation are expressed constitutively, and fermentation enzymes comprise as much as 50% of the cells' total protein (Ming et al., 2014).Z. mobilis maintains a high level of glucose flux through the pathways to compensate for its low yield (Rutkis et al., 2013).Clark et al. (2013) reported that there is linearity between maximum cell growth) and ethanol production with S. cerevisiae strains.However, Z. mobilis perform less biomass formation and efficient production of ethanol compared to S. cerevisiae (Sootsuwan et al., 2013).The low ethanol conversion efficiency by S. cerevisiae might therefore be due to the fact that a portion of the substrate was converted to cell mass and other products.Although liquefaction and saccharification might probably kill some microorganism that might cause contamination, both organisms were able to metabolize their substrate faster  thus competitively inhibiting the growth of other microorganisms.They can therefore be used to produce ethanol using non-sterile substrate.This could reduce energy cost involved in sterilizing the substrate.
According to Tao et al. (2005) and Aggarwal et al. ( 2001), cheap raw material, low processing cost and high productivity are the main considerations for most ethanol production.This work therefore shows that under appropriate conditions pito mash can be used as alternative and cost-effective feed stock for the production of bioethanol without supplementing the fermentation broth with other nutrients.The substrate was able to support the growth of both organisms without the addition of nutrients indicating that pito mash has high carbohydrate (starch) and protein content.The products of saccharification of the pito mash also did not inhibit ethanol production by Z. mobilis and S. cerevisiae as indicated by the high fermentation efficiencies.This is advantageous compared to lignocellulosic materials which require pretreatment that sometimes leads to production of inhibitory compounds that, in turn, decrease ethanol production by the organisms (Chandel et al., 2007).

Optimization of pH
Generally, ethanol concentration increased with increased pH in both S. cerevisiae and Z. mobilis.However, the increase was more pronounced in S. cerevisiae than Z. mobilis.For S. cerevisiae, ethanol concentration began to increase with increased pH till it reached maximum at pH of 6, and then decreased at pH 6.5 (Figure 3).In the case of Z. mobilis, fermentation took place at pH of 4 and gave higher ethanol concentration compared to ethanol concentration produced by S. cerevisiae at the same temperature.Ethanol concentration reached maximum at pH 5.5, beyond which it began to decrease.In the case of S. cerevisiae, there was significant (p<0.05)difference between the ethanol produced at all pH values.Optimum pH for ethanol was between 6.0 and 6.5 with pH of 6.0 producing the maximum ethanol volume of 0.948 mgml -1 for S. cerevisiae.For Z. mobilis, there was significant difference in ethanol produced all pH values.The optimum pH was between 5.0 and 5.5 with 5.5 producing the highest ethanol of 1.85 mgml -1 .In all cases, ethanol produced by Z. mobilis was higher compared to S. cerevisiae.The result agrees with observation by Hwang et al. (2004) who reported that the activities of ethanol producers are slightly suppressed at pH below 4.5.

Optimization of temperature
In Z. mobilis, there was initial increase in ethanol concentration with temperature increase from 30 to 35°C; however, beyond 35°C increasing temperature became inhibitory to ethanol production (Figure 4).The decrease was more pronounced at 45°C. S. cerevisiae produced maximum amount of ethanol at 30°C and further increase in temperature (35 to 45°C) was inhibitory to its ethanol production ability.Analysis of variance indicated that for S. cerevisiae, there was significant (p<0.05)difference in the ethanol produced at each temperature.However, there was no significant (p<0.05)difference in ethanol produced at the temperature of 35 to 45°C.The highest concentration (0.951 mg l -1 ) was produced at temperature of 30°C for S. cerevisiae, followed by 0.849 mg l -1 at 35°C.The lowest volume (0.323 mg l -1 ) was produced at 45°C.In the case of Z. mobilis, there was significant (p<0.05)difference between ethanol produced at all temperatures.However, there was no significant (p<0.05)difference between ethanol produced at 30 to 35 and 40 to 45°C.The highest concentration of 1.951 mg l -1 was produced at temperature of 35°C followed by 1.889 mg l -1 at the temperature of 30°C.At all temperature values, the concentration of ethanol produced at each fermentation examined using Z. mobilis was significantly different from using S. cerevisiae.Similar observations were made by Panesar et al. (2007).It was also indicated that, decrease in the membrane phospholipids content may be responsible for the unique thermal sensitivity of Z. mobilis cells grown at higher temperature (Panesar et al., 2007).

Optimization of fermentation period
As shown in Table 1, maximum ethanol production of 0.855mgml -1 for S. cerevisiae was observed on the fourth day of fermentation whereas maximum ethanol production of 1.269mgml -1 was observed for Z. mobilis on the third day of fermentation.In both organisms, there was a sharp increase in ethanol concentration within the first two days of fermentation period (Figure 5).Generally, there was a decline of ethanol production after the optimum for both organisms (Figure 5) which could be attributed to the build-up of inhibitory toxins produced in the fermentation medium as reported previously by Zakpaa et al. (2009).

Conclusion
Pito mash (waste from sorghum brewing) was a suitable substrate for bioethanol production by Z. mobilis and S. cerevisiae since high yield of ethanol was produced.However, Z. mobilis (3.63 g l -1 ) demonstrated higher biomass conversion efficiency, hence higher ethanol concentration compared to S. cerevisiae (3.03 g l -1 ). Pito mash could therefore be used for large scale bioethanol production, hence reducing its threat to the environment.

Figure 3 .Figure 3 .
Figure 3.Effect of pH on ethanol production by S. cerevisiae and Z. mobilis using pito mash as substrate.

Figure 4 .Figure 4 .
Figure 4. Effect of temperature on ethanol production by S. cerevisiae and Z. mobilis using pito mash as substrate.
followed by different superscript are significantly different at P<0.05.

Figure 5 .Figure 5 .
Figure 5.Effect of time duration on ethanol production by S. cerevisiae and Z. mobilis using pito mash a substrate.

Table 1 .
Effect of fermentation period on ethanol production.