Exploring the Utilization Potential of Spirogyra sp. Biomass for Ethanol Production: A Study on Sacchariﬁcation Optimization and High-Temperature Ethanol Fermentation

: Spirogyra sp. is one of the potential feedstocks for bioethanol production, owing to its high carbohydrate and low lignin contents. However, to date, its use has scarcely been reported, particularly in high-temperature ethanol fermentation. The present study investigated the use of Spirogyra biomass as a bioethanol feedstock by optimizing the conditions for biomass sacchariﬁcation, followed by ethanol fermentation via thermotolerant yeasts, i.e., Saccahromyces cerevisiae DBKKU Y-53, Kluyveromyces marxianus DBKKU Y-102, and Pichia kudriazevii RZ8-1. The optimization of the algal biomass hydrolysis using response surface methodology (RSM) showed that a maximum total sugar production of 14.75 ± 0.13 g/L was attained using 2.67% ( v / v ) sulfuric acid, 7.97% ( w / v ) of biomass loading, and 20 min of hydrolysis time. The fermentation of Spirogyra sp. hydrolysate containing 20 g/L of total sugar at 37 ◦ C showed that S. cerevisiae DBKKU Y-53, K. marxianus DBKKU Y-102, and P. kudriazevii RZ8-1 produced 4.05 ± 0.35 g/L, 4.48 ± 0.13 g/L, and 4.47 ± 0.19 g/L of ethanol, respectively. At 40 ◦ C, lower ethanol production of 1.07 ± 0.47 g/L, 3.93 ± 0.24 g/L, and 3.97 ± 0.19 g/L, respectively, were observed. Nevertheless, P. kudriazevii RZ8-1 exhibited a promising potential for the further development of a high-temperature ethanol fermentation process.


Introduction
Fossil fuels (coal, oil, and natural gas) have long been the major source of the global energy system, and their consumption has been increasing every year because of the global population rise and economic growth [1].Unfortunately, these fuels are non-renewable, and at the rate of current consumption, oil reserves have been projected to run out in 2052, gas in 2060, and coal in 2088 [2].Additionally, the consumption of fossil fuels causes serious concerns regarding air pollution, global warming, and health issues.Global CO 2 emissions have increased every decade, from 11 billion tons of CO 2 in the 1960s to 36.6 billion tons in 2022, 46% of which was from coal, 35% from oil, 15% from natural gas, 3% from the decomposition of carbonates, and 1% from flaring [3].Lelieveld et al. [4] recently reported that fossil-fuel-related emissions account for about 65% of the excess mortality rate attributed to air pollution.To mitigate this problem and replace fossil fuels with renewable energy by 2050, the production of renewable energy will have to increase by six-to eight-fold, assuming the energy demand is held constant at, or increased by 50% from, the 2020 energy demand [5].
One potential alternative energy to fossil fuels is bioethanol.It is widely regarded as an eco-friendly and renewable energy source because of its low toxicity, biodegradability, low emission of greenhouse gases during its combustion, and reproducibility [6][7][8].Bioethanol is currently utilized in the form of gasohol obtained after blending it with gasoline in various proportions [9].In the past decades, the consumption of bioethanol in many countries, including Thailand, has increased continually because of a worldwide energy policy that encourages the use of alternative energy in all sectors.The enforcement of the energy policy also leads to a reduction in petroleum imports, which helps reduce CO 2 emissions.In Thailand, bioethanol consumption of as much as 4.1 million liters per day was reported in 2020 [10].The major raw materials for bioethanol production in Thailand are sugarcane and cassava.However, these feedstocks are also used in the food industry, resulting in a competition for resources between food production and fuel production.To alleviate this problem, a search for potential alternative feedstocks for bioethanol production is needed.
Macro-and microalgae are considered high-potential feedstocks for third-generation bioethanol production.These biomasses contain high contents of insoluble carbohydrates, particularly cellulose and hemicellulose, which are easier to convert into fermentable sugars and, subsequently, ethanol through biorefinery platforms compared to lignocellulosic materials [11,12].Moreover, the algae can be grown on non-arable land or in fresh water, wastewater, and saline water with zero nutrient input [13][14][15].The content of macropolymers, particularly cellulose and hemicellulose, varies, depending on the algal species.Thus, algae containing high amounts of cellulose and hemicellulose and low levels of lignin are desirable for ethanol production.To date, several algal species have been investigated as feedstocks for ethanol production, including green algae (Ulva rigida, U. fascista, Cholrella vulgaris) [16,17] and red algae (Gracilaria verrucosa, Eucheuma spinosum) [18,19].However, there have been only a handful of reports investigating bioethanol production from Spirogyra sp.
Spirogyra sp. is a filamentous green alga that is found ubiquitously in bodies of stagnant water and slowly flowing freshwater rivers all over the world [20].It can also thrive in wastewater and is a widely known indicator of pollution and eutrophication in aquatic ecosystems [21].Owing to its ability to remove nutrients from water bodies and its ability to outgrow other algae species, Spirogyra sp. has been recognized as a potential species for bioremediation processes [22].Moreover, this alga can accumulate carbohydrates to up to 64% of its dry mass [23].Its cell wall comprises mainly cellulose, with low lignin content, and the cellular compartment contains starch that can be hydrolyzed and converted into ethanol [24].Therefore, the growing and utilization of Spirogyra sp.biomass have two explicit advantages, i.e., the improvement of water quality and the algal biomass's potential use as a source of easily fermentable feedstock.Despite this potential, the use of Spirogyra sp.biomass as an ethanol feedstock has scarcely been reported in the literature.This could be due partly to the necessity of pre-treatment and saccharification to break down the algae structures for the subsequent fermentation process.Several processes have been used, including physical, chemical, physico-chemical, and biological methods.However, the chemical approach, particularly acid hydrolysis, is more accessible and cost-effective than the others, as it is a simple method that can be carried out under mild conditions [25,26].However, the use of unsuitable acid hydrolysis conditions may lead to the formation of microbial inhibitors, e.g., acetic acid, formic acid, furfural, and 5-hydroxymethylfurfural (HMF), which negatively affect microbial growth and metabolism.Therefore, it is important to optimize the hydrolysis conditions to maximize sugar production from the biomass while avoiding the excessive formation of inhibitors.
Alongside the use of potential feedstock and a suitable pretreatment approach, the use of effective ethanol producers is equally important.Thermotolerant yeast is known to be highly efficient in ethanol production.Apart from producing a high ethanol titer, its use can help reduce cooling costs and the risk of contamination via mesophilic microorganisms [27].To date, several thermotolerant yeasts have been isolated, and their ethanol production efficiencies using different feedstocks have been reported [28][29][30][31][32].However, a literature survey revealed that there have been only a handful of studies reporting ethanol production from Spirogyra sp.biomass, particularly at a high temperature.Therefore, the present study investigated the use of Spirogyra sp.biomass as a feedstock for bioethanol at a temperature of up to 40 • C. Acid hydrolysis conditions, i.e., hydrolysis time, the concentration of acid, and biomass loading, were statistically optimized, and the resulting hydrolysate was used to assess the bioethanol production efficiency of several thermotolerant yeasts, i.e., Saccharomyces cerevisiae DBKKU Y-53, Kluyve-romyces marxianus DBKKU Y-102, and Pichia kudriazevii RZ8-1.

Microorganisms and Culture Conditions
S. cerevisiae DBKKU Y-53 [28], K. marxianus DBKKU Y-102 [33], and P. kudriazevii RZ8-1 [30] were used in this study.The activation of the yeasts was conducted by transferring one single colony of the yeasts growing on yeast extract-malt extract (YM) agar into 10 mL of YM broth and incubating it at 30 • C in a rotary incubator shaker at 150 rpm for 24 h.Inocula were prepared by transferring 5 mL of the activated culture into 45 mL of YM broth and cultured under the same conditions for 24 h.

Biomass of Spirogyra sp.
The biomass of Spirogyra sp. was harvested from November to December 2020 from Nong Sam Muen Lake, located in Phu Khiao, Chaiyaphum, Thailand.After washing thoroughly with tap water to remove dirt and sand, followed by drying in a hot-air oven at 60 • C for 48 h, the algal biomass was ground using a kitchen blender and sieved through a 30-mesh sieve.The biomass was stored in a plastic container at 30-35 • C until use.The spirogyra sp.biomass was analyzed at the Central Laboratory (Thailand) Co., Ltd., Khon Kaen, Thailand, using the standard methods of AOAC [34].

Acid Hydrolysis and the Concentration of Spirogyra sp. Hydrolysate
The conditions for the acid hydrolysis of the algal biomass were optimized using the response surface methodology (RSM) with Box-Behnken design (BBD).The effects of 3 variables, i.e., the concentration of sulfuric acid (A, 1-3%, v/v), biomass loading (B, 4-8%, w/v), and hydrolysis time (C, 10-20 min), were investigated with total sugar production as the response.The design matrix consisted of 15 experimental runs (Table 1).The calculated amount of the biomass was transferred into a 50-mL screw-cap glass bottle containing 30 mL of appropriately diluted acid solution.Then, it was heated at 121 • C in an autoclave for the designated time.After cooling to room temperature, the hydrolysate was collected via centrifugation at 10,000 rpm for 10 min.The supernatant was analyzed for total sugar, acetic acid, furfural, and HMF concentrations.The total sugar concentrations obtained from all the experimental runs were used to optimize the hydrolysis conditions.A confirmation test was subsequently performed to validate the predicted optimum conditions.The hydrolysate obtained after the hydrolysis under the optimum conditions was concentrated via boiling to raise the total sugar concentration to around 20 g/L.The microstructures of the biomass before and after the hydrolysis under optimum conditions were also examined.The ability of the yeasts to grow at high temperatures was examined using the method described by Techaparin et al. [29].The yeasts were streaked on YM agar and incubated at 30 • C, 37 • C, 40 • C, and 45 • C for 48 h before their growth was observed via visual inspection.The ethanol tolerance ability of the yeasts was tested by inoculating ca. 1 × 10 6 cells/mL of the yeasts into 50 mL of YM broth containing 7%, 10%, and 13% (v/v) ethanol, which was equivalent to ca. 55, 79, and 103 g/L of ethanol, and incubating at 35 • C and 150 rpm for 48 h.After that, the cell concentration was determined and used to calculate the percentage of cells' survival (Equation ( 1)).

Cell survivals (%) =
The number of viable cells × 100 The number of total cells (1)

Ethanol Fermentation
The production of ethanol by the three yeast strains at 37 • C and 40 • C was examined.The Spirogyra sp.hydrolysate containing ca. 20 g/L of total sugar was supplemented with 1 g/L of urea, 0.5 g/L of Na 2 HPO 4 , 2.5 g/L of KH 2 PO 4 , 1 g/L of MgSO 4 , 1 g/L of (NH 4 ) 2 SO 4 , and 0.001 g/L of FeSO 4 [35].Then, 100 mL of the hydrolysate was transferred into 250-mL Erlenmeyer flasks and inoculated with the yeast at an initial cell concentration of ca. 1 × 10 6 cells/mL.The flasks were closed with air-lock stoppers before they were incubated at 37 • C or 40 • C and 100 rpm for 48 h.Samples were collected at 12-h intervals to determine cell growth and pH, as well as ethanol and sugar concentrations.

Analytical Methods
The total sugar concentration was determined using the phenol-sulfuric method using glucose as the standard [36].Concentrations of acetic acid, furfural, HMF, and individual sugars in the algal hydrolysate were determined using high-performance liquid chromatography (HPLC), following the method of Chamnipa et al. [30].The microstructure of the Spirogyra sp.biomass was observed under a scanning electron microscope (Hitashi-SU3800) at the Faculty of Dentistry, Khon Kaen University, Thailand.Cell concentration was determined using a hemocytometer and the methylene blue staining technique [37].Ethanol concentration was analyzed using gas chromatography (GC) via the method of Laopaiboon et al. [38].The samples were filtered through a syringe filter with a pore size of 0.45 µm before the analyses.
All the experiments on ethanol production were performed in triplicate, and the standard deviation (SD) values were calculated using MS Excel 2019 software.Multiple comparison tests for each experimental treatment were carried out using Duncan's multiple range test (DMRT) at a 95% confidence level using the SPSS program.

Spirogyra sp. Biomass Compositions
The biomass of Spirogyra sp.comprises 64.0% total carbohydrates, 12.9% protein, 1.6% fat, and 11.4% ash with a moisture content of 10.0%.Compared with the ranges of carbohydrate, protein, and fat contents reported by Ge et al. [22], i.e., 41.5-55.0%,16.7-19.5%,and 2.8-10.0%,respectively, the carbohydrate content of the biomass used in the present study was higher, while lower protein and fat contents were observed.This could be attributed to the differences in the species and growth conditions.However, this was considered beneficial since more fermentable sugars could be obtained with a higher carbohydrate content in the biomass.

Acid Hydrolysis of Spirogyra sp. Biomass
The acid concentration, biomass loading, and hydrolysis time were evaluated as independent variables for the acid hydrolysis of Spirogyra sp.Biomass.Table 1 shows that the total sugar production varied from 7.4 to 14.9 g/L, depending on the hydrolysis conditions.An analysis of variance (ANOVA) of the results showed that the acid concentration (Factor A, p-value 0.0008) and biomass loading (Factor B, p-value <0.0001), as well as their interaction (p-value 0.0355), had significant effects on the total sugar production (Table 2).A quadratic equation (Equation ( 2)) was developed based on the results shown in Table 2 and found to have a p-value smaller than 0.0001 with a p-value of the lack of fit of 0.0982.These, together with the high regression coefficient (R 2 ) and adjusted R 2 of 0.9952 and 0.9864, respectively, as well as a low coefficient of variable (CV) of 2.46%, indicated that the equation was adequate for the prediction of total sugar production within the ranges of the variables tested.Note that the interaction term BC, with a coefficient of 0.018, was omitted from Equation ( 2), as this did not significantly affect the total sugar production.Three-dimensional surface plots were generated to visualize the interaction effects between the variables tested on total sugar production.Figure 1A shows that the total sugar production was highly dependent on the acid concentration and biomass loading.Increasing the acid concentration and biomass loading increased the total sugar production, as more substrate and catalyst were available for the hydrolysis reaction [18].However, using too high of an acid concentration could have led to higher degrees of sugar degradation [39], leading to a low sugar yield and the formation of microbial inhibitors [13].This phenomenon was recently reported by Soliman et al. [40]; the use of sulfuric acid at higher than 3% led to decreased reducing sugar production from macroalgal biomasses (Sargassum latifolium and U. lactuca).On the other hand, increasing the biomass loading beyond a certain level could result in increased medium viscosity, which, in turn, impedes mass and heat transfers during the process, leading to a low hydrolysis yield [41].This effect has been reported in several studies, including that by Harchi et al. (2018) [16], in which increasing the U. rigiga biomass loading from 10% to 15% led to lower sugar yields.To further confirm the effects of acid hydrolysis on the biomass, SEM was examine the surface of the biomass before and after the reaction.Figure 2A shows untreated Spirogyra sp.biomass had a relatively smooth surface and was almost The interaction between acid concentration and hydrolysis time did not significantly affect sugar production (p-value 0.0792) (Table 2).Figure 1B shows that, despite the total sugar production rising sharply when the acid concentration was increased, prolonging the hydrolysis time from 10 to 20 min exerted only subtle effects on the biomass hydrolysis.Based on these results, it was considered that the hydrolysis time investigated in the present study might be too short, resulting in a low degree of biomass hydrolysis [42].However, a longer reaction time was not investigated due to the possibility of extensive sugar degradation [43], which would result in a low sugar yield.Furthermore, the acid hydrolysis of hemicellulose present in Spirogyra biomass [44] could lead to the generation of organic acids, e.g., acetic acid [45], which is inhibitory to cells.As shown in Table 3, besides monomeric sugars, acetic acid was produced under all conditions tested, so prolonging the hydrolysis time would increase the acid production, adversely affecting the subsequent fermentation process.For the interaction between biomass loading and the reaction time, this was also found to insignificantly affect the total sugar production (p-value 0.2443) (Table 2).As shown in Figure 1C, the total sugar concentration increased sharply when the biomass loading was increased, regardless of the hydrolysis time used.Using Equation (2), numerical optimization revealed that the maximum total sugar production of 14.92 g/L would be obtained under the optimum conditions of 2.67% (v/v) sulfuric acid, 7.97% (w/v) biomass loading, and a hydrolysis time of 20 min.A confirmation experiment conducted under these conditions yielded 14.75 ± 0.13 g/L of total sugar.This was within the 95% confidence intervals (CI) of 14.17 to 15.63 g/L, indicating the validity of the predicted conditions.After comparing with other studies reported in the literature, it was found that the optimum conditions obtained in the present study differed greatly from the others.Nevertheless, this was not surprising since the conditions for the hydrolysis reaction could be highly dependent on the species of algae, more specifically their compositions.For instance, Dawei et al. [46] reported that the optimum conditions for the dilute acid hydrolysis of 5% (w/w) Enteromorpha at 121 • C were 1.8% (w/w) sulfuric acid and a hydrolysis time of 60 min.In another study, Hong et al. [47] reported the optimum conditions for Gelidium amansii, Laminaria japonica, and Codium fragile hydrolysis to be a temperature of 150 • C, a reaction time of 60 min, and 5% (w/w) sulfuric acid.Biomass of G. amansii was also used in a study by Sivagurunathan et al. (2017) [48], and the highest reducing sugar production was achieved at 121 • C with a 5% (w/v) macroalgal biomass, 1% (v/v) sulfuric acid, and a hydrolysis time of 30 min.Recently, Hessami et al. (2019) [49] optimized the dilute acid hydrolysis of G. elegans for bioethanol production.The highest hydrolysis yield of 39.42% was obtained using 2.5% (w/v) sulfuric acid at 120 • C for 40 min.To further confirm the effects of acid hydrolysis on the biomass, SEM was used to examine the surface of the biomass before and after the reaction.Figure 2A shows that the untreated Spirogyra sp.biomass had a relatively smooth surface and was almost free of trenches.After the dilute acid hydrolysis, the appearance of the biomass dramatically changed.The biomass surface was found to be much rougher, as shown in Figure 2B.These changes were thought to be due mainly to the degradation of carbohydrates (hemicellulose and cellulose) on the surface of the algal biomass.The results given in Table 3 demonstrate that glucose and xylose were the main reducing sugars in the hydrolysate, while acetic acid was the only inhibitor detected in the hydrolysate.The yield of sugars and inhibitors obtained using acid hydrolysis of various algal biomass are compared in Table 4.
Processes 2023, 11, x FOR PEER REVIEW 8 of 16 and inhibitors obtained using acid hydrolysis of various algal biomass are compared in Table 4.

Growth and Ethanol Tolerance of the Yeast Strains at High Temperatures
There are many factors to consider for ethanol production, such as temperature and ethanol concentrations that increase during a fermentation process.The explicit advantages of high-temperature ethanol fermentation are the low risk of bacterial contamination and the reduced cost associated with cooling [51].However, too high of a temperature can affect enzyme activity, as well as metabolism and yeast growth [52].In the case of ethanol concertation, it affects the enzyme structure and reduces enzyme activity [53].
The growth performance of S. cerevisiae DBKKU Y-53, K. marxianus DBKKU Y-102, and P. kudriazevii RZ8-1 on YM agar at different incubation temperatures (30 • C, 37 • C, 40 • C, and 45 • C) are shown in Figure 3.All the thermotolerant yeasts grew well at temperatures up to 40 • C.However, only K. marxianus DBKKU Y-102 and P. kudriazevii RZ8-1 could thrive at 45 • C, while S. cerevisiae DBKKU Y-53 showed obviously low growth.This finding was in agreement with other studies.For instance, Sree et al. [54] investigated the growth of S. cerevisiae isolated from soil at a thermal power plant in India and demonstrated that all the yeasts grew well below 40 • C, and only two strains were able to grow at 44 • C in a liquid medium but not on a solid medium.Edgardo et al. [55] reported that 11 strains of thermostable S. cerevisiae could grow at 35 • C and 40 • C; however, none of these grew at 45 • C. On the other hand, a study by Techaparin et al. [29] showed that P. kudriazevii KKU-TH43 and KKU-TH33 isolated from soil samples could grow at a high temperature of 45 • C. Likewise, Pongcharoen et al. [56] demonstrated that P. kudriazevii NUNS-4, NUN-5, and NUNS-6 isolated from soil in sugarcane plantation areas in Thailand could grow at a high temperature up to 45 • C. A study by Banat et al. [57] reported that K. marxianus was able to grow at temperatures as high as 52 • C.
45 °C.On the other hand, a study by Techaparin et al. [29] showed that P. kudriazevii KKU-TH43 and KKU-TH33 isolated from soil samples could grow at a high temperature of 45 °C.Likewise, Pongcharoen et al. [56] demonstrated that P. kudriazevii NUNS-4, NUN-5, and NUNS-6 isolated from soil in sugarcane plantation areas in Thailand could grow at a high temperature up to 45 °C.A study by Banat et al. [57] reported that K. marxianus was able to grow at temperatures as high as 52 °C.The ability of the yeast strains to tolerate ethanol, as a component in the fermentation medium, in the range 7 to 13% (v/v) at 35 °C is shown in Figure 4.The ability to tolerate ethanol was divided into three categories: highly tolerant (more than 50% of cells survived), moderately tolerant (25-50% of cells survived), and slightly tolerant (less than 25% of cells survived) [58].As shown in the figure, all yeasts tested were highly tolerant to ethanol with up to 10% (v/v).Further increasing the ethanol concentration to 13% led to a further reduction in cell survival.However, S. cerevisiae DBKKU Y-53 and P. kudriazevii RZ8-1 were still highly tolerant, while the survival of K. marxianus DBKKU Y-102 dropped below 50%, so it was considered moderately tolerant to ethanol at 13% at 35 °C.Overall, S. cerevisiae DBKKU Y-53 exhibited the best performance in ethanol tolerance, followed by P. kudriazevii RZ8-1 and K. marxianus DBKKU Y-102, respectively.The present results agreed well with other studies, such as that of Luong [59], who reported an ability of S. cerevisiae to tolerate high ethanol concentrations in the range 115 to 200 g/L, and Kumar et al. [60], who reported the ethanol tolerance of Kluyveromyces sp.IIPE453 (MTCC 5314) at 82 g/L.Similarly, a study by Techaparin et al. [29] demonstrated that S. cerevisiae KKU-TH33, KKU-TH43, KKU-TH200, and KKU-TH200 and P. kudriazevii KKU-VN35 exhibited a high ethanol-tolerant ability at 13% (v/v) ethanol with over 50% cell survival.Studies by Ndubuisi et al. [61] and Pongcharoen et al. [56] also demonstrated that P. kudriazevii LC375240 and P. kudriazevii NUPHS33 and NUPHS34 were able to grow in YPD containing an ethanol concentration of 15% (v/v).The ability of yeast strains to withstand different stresses depends on several factors, including the yeast species, genetic background, and culture The ability of the yeast strains to tolerate ethanol, as a component in the fermentation medium, in the range 7 to 13% (v/v) at 35 • C is shown in Figure 4.The ability to tolerate ethanol was divided into three categories: highly tolerant (more than 50% of cells survived), moderately tolerant (25-50% of cells survived), and slightly tolerant (less than 25% of cells survived) [58].As shown in the figure, all yeasts tested were highly tolerant to ethanol with up to 10% (v/v).Further increasing the ethanol concentration to 13% led to a further reduction in cell survival.However, S. cerevisiae DBKKU Y-53 and P. kudriazevii RZ8-1 were still highly tolerant, while the survival of K. marxianus DBKKU Y-102 dropped below 50%, so it was considered moderately tolerant to ethanol at 13% at 35 • C. Overall, S. cerevisiae DBKKU Y-53 exhibited the best performance in ethanol tolerance, followed by P. kudriazevii RZ8-1 and K. marxianus DBKKU Y-102, respectively.The present results agreed well with other studies, such as that of Luong [59], who reported an ability of S. cerevisiae to tolerate high ethanol concentrations in the range 115 to 200 g/L, and Kumar et al. [60], who reported the ethanol tolerance of Kluyveromyces sp.IIPE453 (MTCC 5314) at 82 g/L.Similarly, a study by Techaparin et al. [29] demonstrated that S. cerevisiae KKU-TH33, KKU-TH43, KKU-TH200, and KKU-TH200 and P. kudriazevii KKU-VN35 exhibited a high ethanol-tolerant ability at 13% (v/v) ethanol with over 50% cell survival.Studies by Ndubuisi et al. [61] and Pongcharoen et al. [56] also demonstrated that P. kudriazevii LC375240 and P. kudriazevii NUPHS33 and NUPHS34 were able to grow in YPD containing an ethanol concentration of 15% (v/v).The ability of yeast strains to withstand different stresses depends on several factors, including the yeast species, genetic background, and culture conditions.For instance, P. kudriavzevii exhibited higher thermal tolerance than S. cerevisiae [32].The ability of the yeast cells to withstand ethanol stress might be associated with the expression of genes involved in heat-shock protein synthesis, ATP production, trehalose, and glycogen production [28].As demonstrated by Phong et al. [62], the overexpression of the ATP6, OLE1, and ERG8 genes improved the growth and ethanol production of S. cerevisiae HG1.1 under heat and ethanol stresses.Further study is needed to clarify this phenomenon.[32].The ability of the yeast cells to withstand ethanol stress might be associated expression of genes involved in heat-shock protein synthesis, ATP production, tr and glycogen production [28].As demonstrated by Phong et al. [62], the overex of the ATP6, OLE1, and ERG8 genes improved the growth and ethanol product cerevisiae HG1.1 under heat and ethanol stresses.Further study is needed to cla phenomenon.

Ethanol Fermentations
Bioethanol productions from the acid hydrolysate of Spirogyra sp.via the yea °C and 40 °C were investigated.At 37 °C, the maximum ethanol production via S. c DBKKU Y-53, K. marxianus DBKKU Y-102, and P. kudriavzevii RZ8-1 were 4.05 ± 4.48 ± 0.13 g/L, and 4.47 ± 0.19 g/L, respectively (Table 5).On the other hand, w fermentations were performed at 40 °C, lower ethanol production was observed.anol production via S. cerevisiae DBKKU Y-53, K. marxianus DBKKU Y-102, and P. evii RZ8-1 decreased to 1.07 ± 0.47 g/L, 3.93 ± 0.24 g/L, and 3.97 ± 0.19 g/L, resp This could be due to the inhibition effect on cell growth at a higher temperature accumulation of toxic compounds, including ethanol, in the fermentation broth temperatures, the ribosomes and enzyme structures of the yeast cells are affected ing the yeast metabolism [63].Nevertheless, a close inspection of the results revea the ethanol yield obtained at 40 °C was only slightly lower than that at 37 °C fo strains tested, being between 58.8% and 62.7% and between 62.7% and 70.6% of t retical yield (0.51 g-ethanol/g-glucose)), respectively.This implied that the abilit yeasts to convert a carbon source into ethanol was only subtly affected, so thes have potential for use in high-temperature ethanol production.
The temperature commonly used for bioethanol production in tropical cou 37 °C, and this could rise to ca. 40 °C during the processes due to yeast metabol Although the monosaccharides typically found in Spirogyra sp. are glucose, xylos tose, and arabinose [65], S. cerevisiae and some strains of P. kudriazevii cannot utiliz for ethanol production.On the other hand, K. marxianus has been reported to be consume xylose and arabinose for ethanol production under some circumstan under shaking conditions, but it was unable to produce ethanol from these suga static conditions [66].Our results showed the ability of the yeasts to consume sug °C was not significantly different, except for arabinose consumption via P. kudriaze 1 (Table 6).At 40 °C, however, S. cervisiae DBKKU-53 consumed much less sugar co

Ethanol Fermentations
Bioethanol productions from the acid hydrolysate of Spirogyra sp.via the yeasts at 37 • C and 40 • C were investigated.At 37 • C, the maximum ethanol production via S. cerevisiae DBKKU Y-53, K. marxianus DBKKU Y-102, and P. kudriavzevii RZ8-1 were 4.05 ± 0.35 g/L, 4.48 ± 0.13 g/L, and 4.47 ± 0.19 g/L, respectively (Table 5).On the other hand, when the fermentations were performed at 40 • C, lower ethanol production was observed.The ethanol production via S. cerevisiae DBKKU Y-53, K. marxianus DBKKU Y-102, and P. kudriazevii RZ8-1 decreased to 1.07 ± 0.47 g/L, 3.93 ± 0.24 g/L, and 3.97 ± 0.19 g/L, respectively.This could be due to the inhibition effect on cell growth at a higher temperature and the accumulation of toxic compounds, including ethanol, in the fermentation broth.At high temperatures, the ribosomes and enzyme structures of the yeast cells are affected, inhibiting the yeast metabolism [63].Nevertheless, a close inspection of the results revealed that the ethanol yield obtained at 40 • C was only slightly lower than that at 37 • C for all the strains tested, being between 58.8% and 62.7% and between 62.7% and 70.6% of the theoretical yield (0.51 g-ethanol/g-glucose)), respectively.This implied that the ability of the yeasts to convert a carbon source into ethanol was only subtly affected, so these might have potential for use in high-temperature ethanol production.
The temperature commonly used for bioethanol production in tropical countries is 37 • C, and this could rise to ca. 40 • C during the processes due to yeast metabolism [64].Although the monosaccharides typically found in Spirogyra sp. are glucose, xylose, galactose, and arabinose [65], S. cerevisiae and some strains of P. kudriazevii cannot utilize xylose for ethanol production.On the other hand, K. marxianus has been reported to be able to consume xylose and arabinose for ethanol production under some circumstances, e.g., under shaking conditions, but it was unable to produce ethanol from these sugars under static conditions [66].Our results showed the ability of the yeasts to consume sugars at 37 • C was not significantly different, except for arabinose consumption via P. kudriazevii RZ8-1 (Table 6).At 40 • C, however, S. cervisiae DBKKU-53 consumed much less sugar compared with its consumption at 37 • C and that of the others at the same temperature.A different letter in the same row indicates a significant difference at a 95% confidence level.
Previously, Diaz-Nava et al. [67] studied the effect of carbon sources on the growth performance and ethanol production of P. kudriazevii ITV-S42 and reported that the yeast did not use xylose or sucrose.Talukder et al. [68] reported a similar observation that S. cerevisiae Dj-1 and P. kudriazevii Dj-2 could not grow on an agar medium containing xylose (yeast extract-peptone-xylose (YPX) medium), but a naturally mutated strain of S. cerevisiae Dj-3 grew well.Ndubuisi et al. [61] reported that P. kudriazevii LC375240 was able to consume pentoses, e.g., xylose and arabinose, but it did not convert these to ethanol.In the present study, the xylose utilization of the three thermotolerant strains was tested by incubating the yeasts in a liquid YPX medium at 37 • C for 48 h.Interestingly, P. kudriazevii RZ8-1 was observed to convert xylose to ethanol.The ethanol yield of K. marxianus DBKKU Y-102 and P. kudriazevii RZ8-1 was 0.25 and 0.08 g-ethanol/g-xylose, respectively.The xylose utilization ability of P. kudriazevii was very weak with xylose as the sole carbon source, and the concentration of glucose enhanced the xylose consumption of this yeast [69].Naturally, yeast strains capable of xylose fermentation possess xylose reductase and xylitol dehydrogenase.After the uptake of xylose into the cells, xylose is converted into xylitol via xylose reductase and NAD(P)H and then to xylulose via xylitol dehydrogenase and cofactor NAD + before being added to a phosphate group to form xylulose-5-phosphate (X5P), which enters the pentose phosphate pathway (PPP), yielding ethanol [70,71].A study by Rahman et al. [72] demonstrated that P. Kudriazevii UniMAP 3-1 produced ethanol from xylose with a yield of 0.019 g-ethanol/g-xylose at 40 • C, and xylitol was detected.This indicated that P. kudriazevii have enzymes to convert xylose to ethanol (xylose reductase and xylitol dehydrogenase) and the transporter needed to uptake xylose into the yeast cells.However, its ability to utilize xylose depends on the glucose concentration.
It should be noted that the ethanol yields obtained in the current study were slightly lower than the theoretical ethanol yield.This might be because the yeasts could not convert pentoses into ethanol.Although xylose could be transported into the cells via facilitated diffusion when the glucose concentration in the medium is low [73-75], the yeasts lack the necessary metabolic pathway to convert this into ethanol [76].Another possibility was that there were low yeast cell and sugar concentrations in the media due to several samplings during the fermentation.Under this condition, low ethanol production could be expected, as a high cell concentration has been reported to be necessary for high ethanol production [77].The ethanol concentration produced in this study was lower than in other studies [28,30,33], possibly due to the difference in the feedstocks used.
Acetic acid is an inhibitor that affects microbial activities during the fermentation process.It could be generated as a result of a hydrolysis reaction of the acetyl groups in hemicellulose during sulfuric acid pretreatment [78].This acid could also be detected during cell division during ethanol fermentation [79].The toxicity of acetic acid depends on its concentration and the pH of the culture medium.This effect is pronounced when the concentration of the acid is high and the pH of the medium is lower than its dissociation constant (the pKa of acetic acid is 4.75).Under these conditions, the protein and lipid structures of the cell membrane are adversely affected, inhibiting yeast metabolism [74].A study by Favaro et al. [80] showed that the growth of S. cerevisiae on a yeast nitrogen base (YNB) medium containing 1.80 g/L of acetic acid reduced to 89% of that observed in a medium containing no acid.Pattanakittivorakul et al. [81] pointed out that acetic acid at a concentration higher than 5 g/L can inhibit the growth and ethanol fermentation ability of S. cerevisiae at 37 • C. Acetic acid concentrations higher than 5 g/L also inhibited the growth of P. kudriazevii RZ8-1 [30].In contrast, K. marxianus could grow in a medium containing as high as 15 g/L of acetic acid [82].In the present study, an acetic acid concentration was present in the range of 0.38 to 0.93 g/L (Table 6).At these low concentrations, it might exert only subtle effects on the yeast activities.
Statistical analysis showed an insignificant difference in ethanol production at 37 • C.However, at 40 • C, significantly higher ethanol production was obtained by K. marxianus DBKKU Y-102 and P. kudriazevii RZ8-1 as compared to S. cerevisiae DBKKU Y-53 (Table 5).These results, coupled with the reports that P. kudriazevii could produce ethanol at a temperature of up to 45 • C [30,83] and the ability of P. kudriazevii to produce ethanol from xylose [69,84,85], suggested that P. kudriazevii was the most potential ethanol producer among the strains tested for ethanol production from Spirogyra sp.biomass at high temperatures.

Conclusions
The biomass of Spirogyra sp. was shown to be a feasible substrate for ethanol production via three thermotolerant yeasts, i.e., S. Cerevisiae DBKKU Y-53, K. Marxianus DBKKU Y-102, and P. Kudriazevii RZ8-1.The acid hydrolysis of the algal biomass under optimum conditions yielded 14.75 ± 0.13 g/L of total sugar.All the strains tested performed equally well in ethanol fermentation at 37 • C. Nevertheless, K. Marxianus DBKKU Y-102 and P. Kudriazevii RZ8-1 exhibited better performance at a high temperature (40 • C), yielding 3.93 and 3.97 g/L of ethanol at 40 • C, respectively.Because of this, P. kudriazevii RZ8-1 was considered the best thermotolerant ethanol producer tested in this study.However, as it can be seen that the total sugar yield attained from the biomass was relatively low, further study is required to enhance the yield, which, in turn, would enhance ethanol production.

Figure 1 .
Figure 1.Response surface plots showing interaction effects between acid concentration mass loading (A), acid concentration and hydrolysis time (B), and biomass loading and hy time (C) on total sugar production from Spirogyra sp.Biomass.

Figure 1 .
Figure 1.Response surface plots showing interaction effects between acid concentration and biomass loading (A), acid concentration and hydrolysis time (B), and biomass loading and hydrolysis time (C) on total sugar production from Spirogyra sp.Biomass.

Figure 2 .
Figure 2. Scanning electron microscope images at 1000× of Spirogyra sp.biomass before (A) and after (B) dilute acid hydrolysis under the optimum conditions.

Figure 2 .
Figure 2. Scanning electron microscope images at 1000× of Spirogyra sp.biomass before (A) and after (B) dilute acid hydrolysis under the optimum conditions.

Table 1 .
Experimental design for optimizing the acid hydrolysis of Spirogyra sp.biomass.

Table 2 .
ANOVA of the quadratic model (Equation (2)) for total sugar response.

Table 3 .
Sugars and inhibitors present in Spirogyra sp.Hydrolysate.

Table 3 .
Sugars and inhibitors present in Spirogyra sp.Hydrolysate.

Table 4 .
Comparison of sugar production via acid hydrolysis with various algae.

Table 6 .
Sugar utilization and acetic acid formation of thermotolerant yeasts at 37 • C and 40 • C.