Bioethanol Production from Tofu Waste by Simultaneous Saccharification and Fermentation (SSF) using Microbial Consortium

Tofu waste can be used as a raw material for bioethanol production due to its high carbohydrate content in the form of starch. A microbial consortium, consisting of Aspergillus niger and Saccharomyces cerevisiae. The study’s first objective wasto capture the amount of sugar produced from starch hydrolysis using single cultures of Aspergillus niger.The study’s second objective wasto determine the amount of ethanol produced by the SSF technique. Aspergillus niger was used to produce an amylase enzyme that hydrolyzes starch into simple sugar.Then, Saccharomyces cerevisiae was used to produce bioethanol from the sugar produced earlier.The synthesis of bioethanol consists of two main stages, hydrolysis and fermentation. In previous studies, the hydrolysis and fermentation processes were performed separatelyusing a separated hydrolysis and fermentation (SHF)technique. This studyprocesses via a simultaneous saccharification and fermentation (SSF) technique which produced higher substrate efficiency, cell yield, and product yield compared to the SHF process.The characterization process showed that tofu waste flour was mainly composed of carbohydrates, which comprised 52.82±0.01% (dw) and had a starch content of 35.1±0.2% (dw). Sugar from the starch of the tofu waste was produced by batch system cultivation for 84 hours using Aspergillus niger. The highest sugar production (14.48 g/L) was achieved during the 48^th hour. Then, Saccharomyces cerevisiae was used to convert the produced sugar into bioethanol. The production of bioethanol by SSF using a microbial consortium for 72 hours was 7.69 g/L of bioethanol, with a yield of bioethanol per substrate use (Yp/s) of 0.23 g ethanol/g substrate and a substrate conversion efficiency of 88%.

Aspergillus niger; Bioethanol; Saccharomyces cerevisiae; Simultaneous saccharification and fermentation; Tofu waste

renewable energy resource.It can be produced from biomass that is categorized into sugar, starch, and lignocellulose-based material (Kelly et al., 2009). Tofu waste is a potential bioethanol feedstock since 59.95% of its contents are carbohydrates (Yustina& Abadi, 2012) and starch (Sudaryati et al., 2013). Tofu waste is abundantly available in Indonesia and has low economic value. Currently, tofu waste’s utility is limited to feed material.

Starch-based material requires a hydrolyzing agent to convert the starch into simple glucose. Based on Zambare (2010), Aspergillus niger mold has significant potential to produce amylase and glucoamylase enzymes that can hydrolyze starch into sugar.The amylase family has two major classes: (1) dextrin, fructose, glucose, lactose, and maltose; and (2) starch enzymesconsisting of amylase (EC 3.2.1.1) and glucoamylase (EC 3.2.1.3). Amylase can hydrolyze starch into maltose and glucose,while glucoamylase (GA) can produce single glucose units.

Saccharomyces cerevisiae can potentially be used as a fermentation agent to convert glucose into ethanol.Based on Hossain et al. (2017), utilization of agricultural waste using Saccharomyces cerevisiaewith SSF technique can increase bioethanol production.

SSF can provide the following advantages: (1) increase the speed of the hydrolysis process by sugar conversion; (2) reduce the enzyme requirement; (3) increase product yield; and (4) reduce the need for sterilization (Zhang et al., 2011).SSF technique using both Aspergillus nigerand Saccharomyces cerevisiae can produce ethanol simultaneously from the accumulation of sugar that was produced from the starch-based material.

The synthesis of bioethanol from starch consists of two main stages, hydrolysis and fermentation. In previous studies, the hydrolysis and fermentation processes were performed separatelyusing a separated hydrolysis and fermentation (SHF)technique. According to Ali et al. (2011), an SSF technique using a microbial consortium is more effective than SHF (Separated Hydrolysis and Fermentation) technique. SSF can produce a higher ethanol yield which ismore productive than SHF (Dahnum et al., 2015).

Based on the study by Arnata and Dewi (2013), using a microbial consortium such as Trichoderma spp., Aspergillus spp., or Saccharomycescerevisiae in a medium of cassava starch at the beginning of the cultivation process can increase the ethanol content by 11% (w/v) and increase efficiency by 40% (w/v) compared to a monoculture of Saccharomyces cerevisiae.

Currently, producing bioethanol as biofuel is not competitive compared to conventional fuel due to its higher production cost. There are at least two strategies to reduce the cost of production: (1) usingcheap and abundant substrates; and (2) improvingprocess technology to increase yield and productivity.A microbial consortium, consisting of Aspergillus nigerand Saccharomyces cerevisiae,was used instead of commercial enzymes to minimize production costs. In this study, the synthesis of enzyme derived from microorganisms without the addition of synthetic enzyme.

This study were to find (1) the pattern of Aspergillus niger growth; (2) determine the highest sugar production time during cultivation; and (3) analyze tofu waste as a potential medium of bioethanol production using an SSF technique and a microbial consortium.

2.1.    Preparation of Tofu Waste Flour as Media

One kilogram of wet tofu waste was squeezed to obtain a starch suspension and dried at  80^oCfor 9 hours in an oven. Then the tofu waste was milled using a food processor, and a flour of waste tofu was then tested in dry weight form. The tofu waste flour was characterizedthroughproximate analysis (AOAC, 1995) ofthe starch, amylose, and amylopectin using the Anthrone method (Sattler and Zerban, 1948).

2.2.    Preparation Cultures of the Microbial Consortium

The isolated cultures utilizedin this study (Saccharomyces cerevisiae(IPBCC.Y. 05.544)andAspergillus niger(IPB.93.265.CCBS420.64)) were obtained from the Institut Pertanian Bogor Culture Collection (IPBCC) at Bogor Agriculture University. Isolates of Aspergillus niger were refreshed on a Potato Dextrose Agar (PDA) medium (Bratachem, Indonesia). The culture was incubated at 25^oC, for 5–7 days before inoculation.Isolates of Saccharomyces cerevisiae were refreshedon a PDA medium and incubated for 3 days. Isolates were grown on 50 ml of Yeast Malt Broth (YMB)propagation medium (Bratachem, Indonesia) consisting of 5 g/l yeast extract, 5 g/l malt, 10 g/l glucose, and 5 g/l peptone in a 200 ml Erlenmeyer flask. Incubation was performed in a 125 rpm shaker at room temperature (±30^oC) for 24 hours before inoculation.

2.3.    Cultivation of Aspergillus niger

Cultivation of Aspergillus niger was carried out to determine the microbial growth curve. Biomass yield, residual starch, total sugar, and total plate count (TPC) from the cultivation process were used to identify the pattern of growth and the required time for highest sugar production. Tofu waste flour witha concentration of 10% (w/v) was dissolved in 1.2 liters of distilled water and then 10% (v/v) inoculum was added to the substrate. The cultivation process was carried out at room temperature by using 150 rpm agitation and 1 vvm aeration. A sample was taken every 12 hours for 84 hours.

3.1.    Characterization of Tofu Waste Flour

Drying was carried out to reduce the wet tofu waste’s high water content. Reducing thesize of the tofu waste increases the surface area of the material, facilitating enzyme hydrolysis performance. In this study,1 kg of wet tofu waste produced 200–300 g of tofu waste flour. Tofu waste flour containsnutrients needed by microbes such as protein, fat, and glucose. The characteristics of tofu waste flour in proximate analysisare shown in Table 1.

Table 1 Proximate analysis of tofu waste flour

These data have been processed. The mean ± standard deviation (n = 2); dw: dry weight

^aYustina& Abadi (2012), ^bSudaryati et al. (2013),^cJenie et al. (2004), ^dAnalysis Result.

Tofu waste is a perishable product due to its high water content. Environmental pollution will occur if the tofu waste is disposed of carelessly or without being processed. Tofu waste should be dried to reduce the water content, crushed, and sifted into aflour so it has more surface area and therebyinteract better with water and facilitate enzyme hydrolysis.

Tofu waste contains 39.23% of starch, a much higher proportion than the 11.49% of starch in soybean waste reported by (Sudaryati et al., 2013). This difference may be attributable to the duration of soybean harvesting. Starch content decreasesasharvest time increases. The plant enzymes able to hydrolyze starch into simple sugar also change over time (Winarno, 1997).

The amylose and amylopectin levels in the tofu waste flour in this study were 28.09±0.07% and 73.00±0.1%, respectively. The proportion of amylose and amylopectin from different sources vary depending on variety and growth location (Winarno, 1997). Protein was detected in about 17.02±0.02% (dw) of the tofu waste starch. Protein, one of the elements composing the cell membrane,  is expected to be the main source of nitrogen for microorganism growth(Moore 1982). Nitrogen is an essential macronutrient for the growth and formationof enzymes (Reed & Rehm, 1983). Protease enzymesare produced by Aspergillus niger (Parathaman et al., 2009).

3.2.    Cultivation of Aspergillus niger

Figure 3 shows the growth of Aspergillus niger based on dry weightbiomass.The early phase of Aspergillus nigerstarted at the 0 hourwhen the new culture was inoculated into the cultivation media.Aspergillus niger entered into the adaptation phase soon after inoculation. The microbial growth was low due to the new media environment during the adaptation phase. The lag phase followed the adaptation phase. The exponential phase occurredbetweenthe 24^th and 48^th hours. During this exponential phase, the microbes grew at their maximum growth rate.

Figure 3 Growth pattern of Aspergillus niger during cultivation

Figure 4 Cultivation results by the batch system of tofu waste using Aspergillus niger

During hours 0–12 (the adaptation phase), sugar production inAspergillus nigerincreased slowly. During this adaptation phase, the ?-amylase enzyme formed as a catalyst for starch hydrolysis (Ezugwu et al., 2015). Sugar production increasedrapidly along with the growth of Aspergillus niger from the 12^th hour until the 48^th hour.

The amylase and glucoamylase enzymes wereproduced by Aspergillus nigerand cut off the polymer chains of starch into simplemonomer units. Starch concentration decreased along with mold growth as sugar production increased.Aspergillus nigergrowth reached the end of the exponential phase at the 48^th hour. The maximum concentration of sugar achieved was 14.42 g/L.Aspergillus nigeralso consumes fats (Falony et al., 2006) and protein (Srinubabu et al., 2007).If polysaccharide content in the cultivation media is low, residual starch is reduced drastically.

During the cultivation process, the level of residual starch decreasesdue to the Aspergillus niger’s metabolic activity,which produces hydrolysis enzymes such as amylase (Bedan et al., 2014),glucoamylase (Parbat &Barkha, 2011), and invertase (Veanna et al., 2011). Starchof tofu waste, therefore, can be used as a carbon source for the growth of Aspergillus nigerto producesugar. In addition,Aspergillus nigercan also produce cellulase enzymes (Jayant et al., 2011). Hence, the fiber in tofu waste can also be used by Aspergillus niger to produce sugar.

3.3.    Simultaneous Saccharification and Fermentation (SSF)

Bioethanol was produced from tofu waste starch via an SSF technique that used a microbial consortium consisting of Aspergillus niger and Saccharomyces cerevisiae. The Aspergillus nigerfunctioned as a saccharification agent to convert starch into sugar; this sugar is subsequently converted through fermentation into bioethanol by the Saccharomyces cerevisiae. According to Nadir et al. (2009), using starch and an SSF technique with a mixture of bacteria is more effective at producing bioethanol than replacing the microbes or adding enzyme at each stage of the process. Bioethanol production from abundant raw materials can be obtained throughout the cultivation time, with lower costsand shorter processing times that increase productivity.

Figure 5 Growth of microbial consortium biomass during SSF

The cultivation process was carried out with full aeration of 185e vvm (Jagani et al., 2010). Aeration is needed for a group of molds and yeasts to produce cells. Oxygen in aerobic cultivation is the main factor affecting a microorganism’s survival. The aeration process cannot be separated from the agitation process. The air flow from the compressor entered the medium to support aeration and agitation.

The exponential phase of microbial consortium occurredbetween the 24^th and 48^th hours. This mold phase is important because cell activity increases significantly.Enzymes can be harvested at the beginning of the exponential phase (Bedan et al., 2014). The results prove that enzyme accumulation occurred in this phase.

Figure 6 Cultivation resultsby SSF using a microbial consortium

The maximum bioethanol concentration, which was obtained during the 72^nd hour, was 7.69 g/L. Saccharomyces cerevisiaecell numbers increased significantly until the 24^th hour. Saccharomyces cerevisiaeconverted sugar into bioethanol by fermentation in addition to respiration. Sugar concentration from starch hydrolysis increased until the 12^th hour, after which it began to decline, while yeast growth began to increase. These changes indicate that the sugar substrate had been consumed by the mold and yeast for cell production and by the yeast for bioethanol formation. The ethanol concentration increased from 2.81 g/L at the 12^th hour to 6.82 g/L at the 24^th hour.

Starch hydrolysis activity was high because mold also needs sugar as a carbon and energy source for growth. Energy needs decreased since mold experiences sporulationat the end of fermentation. However, microbes are able to survive by consuming the remains of substrated and dead microbes because mold cell walls contain cellulose and chitin (Sharp, 2013), while yeast contains protein and manan (Huang, 2008). According to Moore (1982), the hyphae of mold can absorb simple molecules such as sugars, while the more complex forms of polymers such as cellulose, starch, and protein will be processed outside the cells using their extracellular enzymes.

The TPC results show the growth of molds and yeasts in the microbial consortium, which proves that both microbes can grow when tofu waste starch is used as a cultivation media. The cell doubling time of the mold was every 2–6 hours, while the yeast doubled every 20–120 minutes(Laskin, 1977). The maximum growth of molds was 5.4 log cell, which occurred at the 48^th hour, while the maximum growth of the yeast was 6.90 log cell, which occurredat the 60^th hour.

A microbial consortium consisting ofAspergillus niger andSaccharomyces cerevisiaewas used for bioethanol production.Saccharomyces cerevisiaecannot produce hydrolase enzymes that canbreak down starch into glucose (Zhang et al., 2006). Hence, Aspergillus niger is required to produce amylolytic enzymes (Itelima et al., 2013). The amylase and glucoamylase enzymesin Aspergillus nigercan be used to hydrolyze starch. Saccharomyces cerevisiaeis required to convert the resulting sugar into ethanol.

This study’s results clearly show thattofu waste is a viablemedium for bioethanol production.Tofu waste has a high carbohydrate content of 54.04±0.01% (dw), with 39.23±0.2% (dw) in the form of starch.Aspergillus nigerachieved a peak sugar productionsof 14.42 g/L during the 48^th hour of cultivation.

The processes of bioethanol production from starch was previously carried out in three steps: hydrolysis, saccharification, and fermentation.But in SSF technique, the steps were only one, so the time required for cultivation is shorter, hence increase productivity. Beside that, the SSF technique can reduce the number of enzymes neededand reduce the need for sterilization. The SSF technique has proven to be superior in productivity and process time reduction compared to the SHF technique. The implementation of SSF technique usingAspergillusniger as the saccharification agent and Saccharomyces cerevisiae as the fermentation agent simultaneously produced 7.69 g/Lethanol.

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