Fungal amylases applied to the sweet potato starch for bioethanol production

Bioethanol is a sustainable energy source to help reducing the emission of pollutants into the global environment. In order to cope with that, the ethanol production technologies and use of efficient and low-cost substrates are developed. The objective of this research was to evaluate fungal amylases in sweet potato starch for bioethanol production by Saccharomyces cerevisiae in the laboratory. Endomelanconiopsis endophytica (1.40 U/mL), Neopestalotiopsis cubana (1.67 U/mL) and Fusarium pseudocircinatum (1.11 U/mL) with high enzymatic activities were selected and their amylases were tested for activity on sweet potato starch for bioethanol production. Simultaneous saccharification and fermentation was performed at 30° C and pH = 5.0. 17.3 - 88.1 (%) of bioethanol that was produced and compared to the expected theoretical yield. Therefore, amylases from these fungi simultaneously inserted on sweet potato starch and S. cerevisiae are potentially useful for bioethanol production. Endomelanconiopsis endophytica, Neopestalotiopsis


Introduction
In the future, non-renewable energy sources such as crude oil and its derivatives, natural gas and coal, which together account for 81.1% of the world's energy demand (International Energy Agency, 2021) will be of little use. Thus, the global community aims to reduce the dependence on fossil fuels and to find sustainable forms of energy, such as biofuels (Aditiya et al., 2016).
In Brasil, the contribution of renewable energy matrices is among the largest in the world (48.4%), with 33.1% coming from biomass (Ministério das Minas e Energia, 2021). In 2020, the sectors that presented the highest use of renewable sources were industry and transportation. The industrial sector presented renewability of 63% of its energy matrix. The fraction of renewable energy used in Brazilian transportation is currently 25%, with ethanol accounting for about 19.3% of fuel consumption (Ministério das Minas e Energia, 2021). These indices, which place Brasil in a prominent position on the world scene in the use of biomass as an alternative energy matrix with the use of fossil fuels, justify the need to seek the commercial use of bioproducts derived from plant material which are rich in carbohydrates for the production of biofuels. The Brazilian technology for producing bioethanol from sugarcane is one of the most successful and efficient worldwide. It's uses low-cost substrates, such as lignocellulosic material, which results in more competitive biofuels (Gonçalves et al., 2013). Second generation ethanol is derived from cellulosic or starchy biomass, rich in carbohydrates that can be hydrolyzed into glucose (Machado & Abreu, 2006), and it represents attractive renewable energy sources that can contribute widely to the implementation of sustainable and less polluting use of energy matrices.
In China, sweet potato (Ipomea batatas L.) was chosen as the main vegetable for bioethanol production, since the country produces about 92 million tons, i.e., more than 58% of the world's production. Asia with 66% of this total, followed by Africa (28.3%) and the Americas (4.6%). Brazil ranks 16th among the largest producers of sweet potato, with 805,4 tons (Empresa Brasileira de Pesquisa Agropecuária [Embrapa], 2021).
In this context, among biofuels, bioethanol is one of the sustainable alternatives for the global environmental issue.
Besides being environmentally friendly, it can replace gasoline (Gronchi et al, 2019). Aiming to produce bioethanol on a laboratory or industrial scale, starch (dry) or even fresh sweet potato can be used by simultaneous saccharification and fermentation (SSF) by Saccharomyces cerevisiae, however, this yeast is not able to degrade starch, so it is necessary to add enzymes wich are capable to hydrolyze starch in order to use this carbon source (Zhang et al., 2011).
Microorganisms producing α-amylases are desirable sources and of a wide interest because these enzymes exhibit structural and functional stability (Kumar et al., 2016). Among different microbial organisms, such as fungi and bacteria, few are capable of producing α-amylases, e.g., Lipomyces konenkoae, Streptomyces bovis, and glucoamylases, e.g., Rhizopus oryzae, Aspergillus awamori, and Aspergillus tubingensis, as well as they are efficient in degrading starch (Favaro et al., 2015). Among the micro-organisms, fungi are the most responsive to industrial demands (Beltagy et al., 2022).
Amylases are hydrolases that catalyze the hydrolysis of starch synergistically, producing dextrins and other smaller polysaccharides (Van Der Maarel et al., 2002). Thus, α-amylase hydrolyzes internal α-1,4 bonds of the polymer producing linear or branched oligosaccharides. In turn, β-amylases, glucoamylases break α-1,4 or α-1,6 bonds of non-reducing terminals of the amylose and the amylopectin chain; and de-ramifying enzymes hydrolyze α-1,6 bonds in the branches of the polysaccharide (Astolfi, 2019). Enzymatic hydrolysis of starch receives attention by researchers and the biofuels industry, as they can reduce energy consumption and simplify production (Favaro et al., 2015) In this context, in order to produce ethanol at the laboratory, in a pilot or industrial scale from sweet potato, starch (dry) or even fresh sweet potato can be used by simultaneous saccharification and fermentation (SSF) by Saccharomyces cerevisiae (Zhang et al., 2011). SSF is an operational process that, in a single step, combines saccharification and fermentation. In this assay, glucose monomers released by enzymatic hydrolysis of starch or lignocellulosic residues are rapidly converted into ethanol (Souza Filho et al., 2016;Schweinberger et al, 2016;Schweinberger, 2016). However, there are difficulties in optimizing the temperature and pH parameters for enzymes and microorganisms concomitantly (Schweinberger, 2016).
Aiming to try to circumvent these possible problems and to study the biotechnological potential of amylases from fungi in the digestive tract of plant material fragmenting insects, we tested three fungal strains: Endomelanconiopsis endophytica, Neopestalotiopsis cubana and Fusarium pseudocircinatum that produced the highest levels of amylases with potential to saccharify raw sweet potato starch. Thus, portions of raw starch were treated with enzymes from each of the selected fungi and Saccharomyces cerevisiae for efficient bioethanol production.

Fungal selection
Thirty-six fungi from the Carlos Rosa Collection (CRC) of the Universidade Federal do Tocantins, Palmas-TO, Brasil, were evaluated for their potential to produce amylases. The three strains with higher amylolytic enzyme activity were selected and tested for bioethanol production.

Growth media
The selected fungi were reactivated in Petri plates containing microbiological medium Merck® potato-dextrose agar (PDA) prepared at 1% (w/v) concentration. The fungi were then incubated in biochemical oxygen demand (BOD) -TECNAL 371 adjusted at 26 ºC for up to 10 days for growth. Subsequently, a disk of each fungal isolate, approximately 1 cm in diameter, was transferred to a 25 mL flask containing 3% malt extract solution on a rotary shaker (100 rpm) for 3 days at room temperature for mycelium growth, and then small aliquots of mycelium were stored in Eppendorf tubes (1.5 mL) and frozen at -20º C.

Fungal enzymatic biosynthesis
A mineral solution following that proposed by Papanikolaou and Aggelis, (2002) was prepared and enriched with sweet potato starch 1% (w/v). A disc of approximately 1 cm diameter was taken from the cultured microbiological medium (PDA) of each strain and inoculated into Erlenmayer flasks (125 mL) containing enriched mineral solution. Then, the flasks were placed under agitation on SL-180/DT SOLAB® shaker table (100 rpm) for 5 days for biosynthesis of amylolytic enzyme extract. In this study, the amylolytic enzyme activity and characterization assays were performed in triplicate.

Amilolytic enzyme assay
The amylolytic enzymatic extracts of each strain were submitted to the enzymatic activity assay, proposed by Ghose, (1987) adapted, to choose three fungal strains with higher amylolytic enzymatic activities.
The amylolytic activities were determined by mixing 0.5 mL of amylases from each fungal strain with 0.5 mL of 1% (w/v) substrate solution (0.5 g of sweet potato starch mixed to 50 mL citrate buffer pH= 4.8). Then the solution was heated in a water bath at 50 °C for 30 min. The amount of reducing sugar released was measured using 3,5-dinitrosalicylic acid (DNS) reagent (Miller, 1959). The potential (volumetric) amylolytic enzyme activity was calculated as micromol per min (µmol min-1) of reducing sugar in the enzyme solution (U/mL) according to equation 1 (Ghose, 1987).
Where: 0.37= estimated glucose content released upon hydrolysis of 1 U.

Strain identity
Mycelium samples from the 3 strains with best amylolytic enzyme activity were sent for molecular identification, amplification, and gene sequencing to the laboratory of Applied Molecular Biology at Instituto Biológico, São Paulo-SP, Brasil (Romão et al., 2024).

Sweet potato starch
The sweet potato starch was obtained by donation of a sealed 400 g package with nutritional label stating 85% starch, by a local merchant.

Enzyme characterization -pH and thermal stability
Portions of sweet potato starch were added to flasks containing citrate buffer at pH= 4.0; 4.5; 5.0; 5.5; 6.0; 6.5 to form 1% (w/v) substrate solutions. Then 0.5 mL of amylases from each of the fungi were added separately to 0.5 mL of substrate solution at each pH and the release of reducing sugars (Miller, 1959) was quantified at each pH for determination of amylolytic activity as a function of pH.
Thermal stability of the amylases was verified by standardized assay of enzyme activity at temperatures ranging from 20 °C to 90 °C. Samples (50 mL) of enzyme solution were incubated in an ultratermostatic bath (SL 152 SOLAB ® ) for 1 hour (h) at temperatures of 20, 30, 40, 50, 60, 70, 80 and 90 °C, and aliquots (0.5 mL) were taken at each temperature to measure the amylolytic enzymatic activity as previously described according to Ghose (1987) and adapted from it

Determination of ethanol concentration
Triplicate aliquots (1 mL) of SSF solution from each sample were withdrawn at each time interval (t) and placed in Eppendorf tubes (1.5 mL), then frozen at (-20 ºC) and sent to Instituto Federal de São Paulo, Matão, São Paulo, Brasil, for quantification of ethanol in Trace GC Ultra Thermo Scientific gas chromatography equipment with FID detector and TRIPLUS Headspace automatic injector, Agilent DB-5 column (30 mm 30.25 mm).
The assay was performed by injecting 1 µL of the sample, keeping the column at 35 °C for 7 minutes. Then a heating ramp of 15 °C/min was performed until 220° C was reached, and it was kept at 220° C for 1 minute. The injector temperature was maintained at 250 °C. After the gas chromatographic assay, the concentrations (% v/v) of bioethanol were determined using the standard calibration curve equation.

Statistical analysis and caculation
The results obtained were expressed as means and standard deviation, and the bioethanol means were compared by Tukey's test at 95% confidence level (p < 0.05).

Fungi identification
The filamentous fungi identified and analyzed in this study are: Endomelanconiopsis endophytica, Neopestalotiopsis cubana and Fusarium pseudocircinatum. The molecular sequences and morphological structures were described by Romão et al. (2024).
Using the Web of Science ™ and PubMed ® databases, as well as using as keywords "fungal name" and "amylases" and "biothanol", no scientific articles were found in the last 5 years that reported application of the fungi selected in this study for bioethanol production, which highlights the originality of this study.

Activity enzyme
The fungi E. endophytica, N. cubana and F. pseudocircinatum showed 1.40; 1.67 and 1.11 U/ mL as the best averages of amylolytic enzyme activity under the conditions of the present study, T= 50° C and pH= 4.8 (Ghose, 1987), respectively.
The average amylolytic activity (U/mL), estimated total protein content (mg/mL), and average specific activity (U/mg) are described in Table 1.

Temperature and pH
The influence of temperature and pH are determining factors in the enzymatic activity and, in effect, influence the release of glucose molecules useful for bioethanol production. Our study presented as optimal temperature (T) and pH points, respectively, T= 50° C ( Figure 1) and pH= 5.0 ( Figure 2).
We observed that at 60° C, the enzymatic activities remained between 48% and 60% of the maximum enzymatic activities and more than double the activity at the initial T (20° C). However, after 60° C there is a progressive loss of fungal enzymatic activities, i.e., the losses of amylolytic activities (LAA), in relation to the activities at initial T, reach, 62.9%, 74.8%, and 51.8% at 90° C, as described in Table 2.
We further note, that small variations around the optimum pH value cause reduction or loss of amylolytic enzyme function ( Figure. 2). Table 2. Enzymatic activity (U/mL) as a function of temperature and loss of amylolytic activity.
Amylase/Fungi 20° C 30° C 40° C 50° C 60° C 70° C 80° C 90° C LAA (%) where they still maintained about 96.1 % of the initial activity, and at temperatures of 60°C and 70 °C the α-amylases still maintained more than 51.2 % of the initial activity. Silva et al. (2018) when analyzing the thermal stability and pH of enzymes from Trichoderma reesei observed that temperatures around 55 °C and pH= 2 -11 showed higher enzyme activities, but at temperatures above 60 °C the enzyme activity is strongly reduced, indicating thermal degradation of the enzymes. Marco et al. (2015) studied the characterization of the thermostability of β-mannanase from Aspergillus foetidus and determined the optimal temperature and pH, respectively, 60° C and pH variable between 3.0 -9.0.
We found in our study that the combination of temperature 50 °C and pH = 5.0 showed the maximum for amylolytic enzyme activity and therefore favor the release of glucose in the bioreactor medium in SSF. Figures 1 and 2 show the behavior of the enzymatic activity of each fungus as a function of T and pH, respectively.

Bioethanol production
For SSF and thus bioethanol production, the temperature was kept at 30 °C for optimization of fermentation by S.
cerevisiae as performed by Gronchi et al. (2019) and pH= 5.0 in the fermentation bioreactors. However, Zhang et al (2013) adjusted in the bioreactors pH= 4.0 and temperature at 30 °C for SSF assay for ethanol production from sweet potato root. Figure   3 shows the average determined concentration (mg/mL) of glucose throughout the SSF process.   Beltagy et al. (2022) produced bioethanol from free amylases from Aspergillus flavus and S. cerevisiae incubated simultaneously, whose actual yield achieved was 0.07 g/ g sugar/L. However, amylases from Fusarium oxyparum were used on starch-rich foods for conversion into bioethanol with the help of S. cerevisiae and thus yield of 20.6 g/L performed in SSF (Prasoulas et al., 2020).
Furthermore, using factorial strategy to evaluate bioethanol production from Solanum lycicarpum fruit starch and Saccharomyces bayanus yeast, Morais et al. (2019) obtained the highest bioethanol production when 1 % glucose equivalent was fermented in 48 h. Under these conditions the production yield was 92.13 %.
Using optimized cultivation factors for Candida albicans, the conversion rate of sweet potato starch to bioethanol was 0.426 g/g, and of potato starch was 0.437 g/g (73 %) (Aruna et al., 2014). However, Zhang et al. (2013)   possibly demonstrates a stable amylolytic enzymatic action, therefore maintaining the fermentative process during this period.
There was no statistically significant difference in bioethanol concentrations of the fungi in 48 hours, by Tukey test at 95% confidence level.

Conclusion
The amylases from the fungi Endomelanconiopsis endophytica, Neopestalotiopsis cubana and Fusarium pseudocircinatum are moderately tolerant to temperature variations and poorly tolerant to pH variations. However, this research showed that fungal amylases applied simultaneously with industrial S. cerevisiae on sweet potato starch showed performance in SSF and thus are potentially useful for ethanol production, as good yields could be obtained.
Further studies should be performed to evaluate other conditions as well as starch-rich substrates. It is suggested to perform enzyme purification studies and associate fermentative yeasts as well as thermophilic microorganisms simultaneously, or studies of immobilization of different amylases applied to SSF and comparative analysis of costs and energy yields in different conditions of fermentative processes for bioethanol production.