lujan-fermentation of sbm

E:FoodEngineering&MaterialsScience

Fermentation of Soybean Meal Hydrolyzates withSaccharomyces cerevisiae and Zymomonas mobilisfor Ethanol ProductionDeivis E. Lujan-Rhenals, Ruben O. Morawicki, Edward E. Gbur, and Steven C. Ricke

Abstract: Most of the ethanol currently produced by fermentation is derived from sugar cane, corn, or beets. However,it makes good ecological and economic sense to use the carbohydrates contained in by-products and coproducts of thefood processing industry for ethanol production. Soybean meal, a co-product of the production of soybean oil, has arelatively high carbohydrate content that could be a reasonable substrate for ethanol production after fermentable sugarsare released via hydrolysis. In this research, the capability of Saccharomyces cerevisiae NRRL Y-2233 and Zymomonas mobilissubsp. mobilis NRRL B-4286 to produce ethanol was evaluated using soybean meal hydrolyzates as substrates for thefermentation. These substrates were produced from the dilute-acid hydrolysis of soybean meal at 135 °C for 45 min with0, 0.5%, 1.25%, and 2% H2SO4 and at 120 °C for 30 min with 1.25% H2SO4. Kinetic parameters of the fermentationwere estimated using the logistic model. Ethanol production using S. cerevisiae was highest with the substrates obtained at135 °C, 45 min, and 0.5% H2SO4 and fermented for 8 h, 8 g/L (4 g ethanol/100 g fresh SBM), while Z. mobilis reachedits maximum ethanol production, 9.2 g/L (4.6 g ethanol/100 g fresh SBM) in the first 20 h of fermentation with thesame hydrolyzate.

Keywords: ethanol, fermentation, hydrolysate, soybean meal, Saccharomyces cerevisiae, Zymomonas mobilis

IntroductionEthanol is 1 of the only 2 transportation fuels on the market

that is used primarily as part of a mixture with petroleum-basedgasoline. Currently, most ethanol is produced by fermentation ofsugars derived from cereals or sugar crops, which raises concernsabout the use of food crops for the biofuel industry. Therefore,production of bioethanol from nonhuman food sources is be-coming attractive, especially from coproducts associated with foodproduction (Neureiter and others 2002; Karmee and Lin 2014).

A potential source of fermentable sugars that has been over-looked is soybean meal (SBM), a coproduct of soybean oil pro-duction. Soybean meal, a premium protein source for animal feed,contains approximately 42% total carbohydrates and 55% protein(Baker and Stein 2009; Da Silva and others 2009). Of the total car-bohydrates, half are structural and the other half are comprised oflow-molecular weight sugars, oligosaccharides, and starch in rela-tively small quantities (Karr-Lilienthal and others 2005). Around17% of low-molecular-weight sugars consist of a mixture of glu-cose, arabinose, galactose, fructose, and sucrose (Grieshop andothers 2003). An additional merit of SBM as a potential source offermentable sugars for ethanol production is that after sugars areselectively extracted, a high-protein meal is left behind which canbe used for animal feed. Before SBM can be used as a substrate,fermentable sugars need to be released from the solid matrix, byhydrolysis with dilute acid or other methods.

MS 20150149 Submitted 1/26/2015, Accepted 4/17/2015. Authors Lujan-Rhenals, Morawicki, and Ricke are with Food Science Dept., 2650 N. YoungAve., Univ. of Arkansas, Fayetteville, AR 72704. Author Lujan-Rhenals is also withUniv. de Cordoba, Programa de Ingenierıa de Alimentos. Sede Berastegui. Km. 12Via Cerete-Cienaga de Oro, Cordoba, Colombia. Author Gbur is with AgriculturalStatistics Laboratory, Univ. of Arkansas Div. of Agriculture, W. Maple St., Fayetteville,AR 72701. Author Ricke is also with Univ. of Arkansas Center for Food Safety,2650 N. Young Ave., Fayetteville, AR 72704. Direct inquiries to author Morawicki(E-mail: [email protected]).

For this research, the capability of 2 microorganisms—Saccharomyces cerevisiae and Zymomonas mobilis—to ferment SBMhydrolyzates was tested. S. cerevisiae is one of the most commonorganisms used in industrial ethanolic fermentations. It is highlyrobust, resistant to toxic inhibitors, and highly tolerant of low pHlevels, which minimizes the risk of contamination (Weber andothers 2010). Z. mobilis, a gram-negative bacterium, is attractivefor ethanol production due to its high productivity. A downsideis that Z. mobilis has low resistance to toxic inhibitors and canferment only glucose, fructose, and sucrose (Rogers and others1982; Doran 1997; Weber and other 2010).

The objective of this research was to evaluate the ability ofS. cerevisiae and Z. mobilis to produce ethanol using acid hy-drolyzates of SBM as substrates. The kinetic parameters of thefermentation were determined using the logistic model.

Materials and Methods

Substrates for fermentationFive hydrolyzates of commercial SBM were used as substrates

for the fermentations. The hydrolyzates were obtained by treat-ing soybean meals with dilute sulfuric acid under the followingconditions: 135 °C for 45 min at 4 acid concentrations (0.0%,0.5%, 1.25%, and 2.0% H2SO4) and 120 °C, 1.25% H2SO4 for 30min. In previous research, these conditions have proven to producethe highest concentrations of fermentable sugars (Lujan-Rhenals2013).

Hydrolyzates were prepared by treating 50 g of SBM with250 mL of H2SO4 solution in 500-mL PYREX R© media stor-age bottles with screw caps (Lujan-Rhenals 2013). To avoid pres-sure buildup inside the bottles during the heat treatment, capswere not completely tightened. A Tuttnauer 2340E Steam Auto-clave (Tuttnauer U.S.A, Delran, N.J., U.S.A.) operated accordingto the preset program #1 (fast exhaust without drying) was usedfor the hydrolysis. Transient heating was not considered in the

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Fermentation of SBM hydrolyzates . . .

Table 1–Experimental design for the ethanolic fermentation ofsoybean meal hydrolyzates with S. cerevisiae NRRL Y-2233 andZ. mobilis NRRL B-4286.

Standard order Run order Microorganism Broth

1 7 S. cerevisiae SBMB02 14 S. cerevisiae SBMB03 6 S. cerevisiae SBMB14 18 S. cerevisiae SBMB15 2 S. cerevisiae SBMB26 9 S. cerevisiae SBMB27 1 S. cerevisiae SBMB38 12 S. cerevisiae SBMB39 11 S. cerevisiae SBMB410 16 S. cerevisiae SBMB411 3 Z. mobilis SBMB012 19 Z. mobilis SBMB013 4 Z. mobilis SBMB114 17 Z. mobilis SBMB115 8 Z. mobilis SBMB216 10 Z. mobilis SBMB217 5 Z. mobilis SBMB318 13 Z. mobilis SBMB319 15 Z. mobilis SBMB420 20 Z. mobilis SBMB4

SBMB0: 135˚C, 0% H2SO4, 45 min.SBMB1: 135˚C, 0.5% H2SO4, 45 min.SBMB2: 135˚C, 1.25% H2SO4, 45 min.SBMB3: 135˚C, 2% H2SO4, 45 min.SBMB4: 120˚C, 1.25% H2SO4, 30 min.

analysis. After the heat treatment, the reaction was stopped bycooling the mix in an ice-water bath followed by the modificationof pH to a range between 5.0 and 5.5 with NaOH pellets. Solidsand liquid were then separated by centrifugation at 3900 × g for35 min at 10 °C in an Allegra X-22R centrifuge with an SX4250Rotor (Beckman Coulter Inc., Brea, Calif., U.S.A.). Afterwards,the supernatants were filtered through Whatman #4 filter paper(Whatman Plc., Maidstone, Kent, U.K.), analyzed for the con-centration of fermentable sugars, and used as substrates for thefermentation. From this point forward, these substrates will bereferred to as soybean meal broth (SBMB) as listed in Table 1.

Microbial strainsStrains of S. cerevisiae NRRL Y-2233 and Z. mobilis subsp.

mobilis NRRL B-4286 were provided as lyophilized powders fromthe culture collection of the United States Dept. of Agriculture(USDA), Agriculture Research Service (Peoria, Ill., U.S.A.).

Fermentable sugar analysisFermentable sugars in the hydrolyzates were analyzed with

High-Performance Size Exclusion Chromatography and Refrac-tive Index (HPSEC-RI) detection. The chromatograph was a Wa-ters, (Milford, Mass., U.S.A.) with a 515 HPLC pump, a manualinjector with a 50-μL sample loop, and a 2410 refractive in-dex detector set at 40 °C. Columns used for the separation ofsugars were a Shodex OH Pack SB-804 HQ (300 × 8 mm) fol-lowed by Shodex OH Pack SB-802 HQ (300 × 8 mm) (ShowaDenko America, Inc., New York, N.Y., U.S.A.) connected inseries. Columns were preceded by a Shodex OH pack SB-G(50 × 6 mm) guard column. Columns and guard column weremaintained at 55 °C in a column heater. A solution of 0.1 MNaNO3 with 0.2% NaN3 run isocratically at a flow rate of0.4 mL/min was used as the mobile phase. Sugars were quan-tified using 6-point calibration curves with fructose and glucoseas standards.

Cells reactivation and preparation of preinoculumand inoculum

Both S. cerevisiae and Z. mobilis were reactivated in 5 mL of yeastmalt (YM) broth (Dickinson and Company, Sparks, Md., U.S.A.)and incubated at 30 °C for 24 h in an orbital shaker (ThermoScientific Max Q 4450, Dubuque, Iowa, U.S.A.) set at 150 rpm.

The preinoculum consisted of 5 mL of sterile YM broth at 30 °Cwhere both S. cerevisiae and Z. mobilis were inoculated separatelyand grown overnight in the orbital shaker. For each respectivestrain, 0.2 mL of the culture was plated out with a loop over thesurface of 2 petri dishes containing YM agar (20 g/L of glucose,3 g/L of yeast extract, 5 g/L of peptone, and 3 g/L of malt extract)and grown overnight at 30 °C before adding to the final inoculum.The inoculum was prepared by adding all the colonies formed inboth dishes to 80 mL of SBMB and allowing the cells to adapt byincubating overnight at 30 °C in the orbital shaker.

FermentationFermentations were conducted at 30 °C for 36 h with an ini-

tial pH of 5 to 5.5, dissolved oxygen (%DO) less than 1% afterstabilization, and an initial biomass concentration between 7×106

and 1×107 cells/mL (Mullins and Nesmith 1987; Siqueira andothers 2008; Laopaiboon and others 2009). The fermentor was a1.3-LBioflo/CellinGen 115 Benchtop Fermentor & Bioreactor(New Brunswick Scientific, Edison, N.J., U.S.A.) with control sys-tems for temperature, pH, %DO, agitation, and foam level. Duringfermentation, samples were taken every 4 h to estimate fermen-tation kinetic parameters maximum specific growth rate (μmax),biomass yield from sugar (Yx/s), and ethanol yield from sugar(Yp/s)—along with volumetric ethanol productivity, rp (g/L/h).Cell density (nr. of cells/L) was determined with a hemacytometer(Double Neubauer Counting Chamber, 3110, Hausser Scientific,Horsham, Pa., U.S.A.) and a phase contrast microscope (NikonEclipse E400, Nikon Corporation, Tokyo, Japan). The cell den-sity was correlated with a calibration curve of cell dry weightobtained by drying aqueous solutions of known concentrations ofcells at 80 °C for 20 to 24 h (Alfenore and others 2002; Buhnerand Agblevor 2004). All kinetic parameters were calculated usingthe following equations:

Yx/s = −d xd s

Biomass yield (g biomass/g sugars) (1)

where x and s represent concentrations of cellular biomass (g/L)and substrate (g/L), respectively.

Y p/s = −d pd s

Ethanol yield from substrate (g ethanol/g sugars)

(2)

where P represents the concentration of ethanol (g/L)

r p = −(q p )x Volumetric ethanol productivity (g ethanol/Lh)

(3)

q p = d pd t

1x

Specific rate of product formation (4)

ms = (r s ) − r x/Yxs − r p/Ypsx

Maintenance coefficient

(g sugar/g cell h) (5)

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where rs = g sugar consumed/L, rx = g cell produced/L h, andrp = g ethanol produced/L h.

Growth kinetics and model development for the ethanolfermentation

The kinetic parameters of biomass production were determinedwith the logistic model (Eq (6)) (Wang and others 2004).

d x/d t = μmaxx[1 − x

xmax

](6)

where dx/dt is the rate of biomass during the time of fermentation(g cell/ h), μmax is the maximum specific growth rate (h-1), x is thebiomass concentration (g/L), and xmax is the maximum biomassconcentration (g/L).

Considering the following boundary conditions: t = 0, x = xo,S = So, and P = 0, Eq (6) was integrated and Eq (7) was obtainedas a result, which relates biomass production (X) and fermentationtime (t) and was used to fit the experimental data.

x = xo xmaxeμmax

t

xmax − xo + xo eμmax

t(7)

For the calculation of the kinetics parameters, only glucose andfructose were considered as growth-limiting-substrates becausethese sugars were consumed in the highest and most consistentquantities by the microorganisms according to the HPLC analysis.

Experimental designFor the fermentation, the experimental design was a complete

randomized block design with microorganism type and broth asfactors (Table 1). At each level, experiments were run twice.

Statistical analysisData was analyzed with SAS Version 9.2 software (SAS Inst. Inc.,

Cary, N.C., U.S.A.) and the kinetic parameters μmax, xmax, and xo

determined. Analysis of covariance (ANCOVA) was used to fitthe data to the logistic model using the Gauss-Newton nonlinearregression method and comparisons of each regression coefficientacross the treatments. For the minimum inhibitor concentrationsand kinetics parameters, data were analyzed with JMP R© version9.0.0 (SAS Inst. Inc.). To analyze the kinetic parameters (Yp/s,Yx/s, ms, and qp), an analysis of variance (ANOVA) was used anddifferences in the mean of the kinetic parameters were analyzedusing the Tukey–Kramer test (α = 0.05).

Results and Discussion

Ethanol fermentation with S. cerevisiae and Z. mobilisS. cerevisiae exhibited optimal responses for the production of

ethanol, sugars consumption, and cell growth in all the SBMbroths used in this study (Figure 1). However, in most fermen-tation broths, Z. mobilis was not as efficient (Figure 2). Thisbacterium exhibited no growth with the hydrolyzate SBMB3(135 °C, 2% H2SO4, 45 min), but with the SBMB1(135 °C, 0.5% H2SO4, 45 min) displayed its maximum ethanolproduction. S. cerevisiae, on the other hand, demonstrated rapidsugar consumption, likely because it is a robust microorganismthat was not critically affected by the inhibitors present in mostof the hydrolyzate SBMB (Lujan-Rhenals and others 2014). Zy-momonas mobilis was affected by inhibitory compounds of theSBMB ((Lujan-Rhenals and others 2014) and also by the pres-ence of salts coming from the hydrolyzate, such as sodium acetate

(CH3COONa) which is the sodium salt of acetic acid formed dur-ing the reaction and sodium sulfate (Na2SO4) formed during theneutralization of the sulfuric acid with sodium hydroxide. (Yangand others 2010) demonstrated that the combination of elevatedNa+ and acetate ions led to a synergistic inhibitory effect on strainZM4.

Kinetics of substrate consumption during fermentationS. cerevisiae exhibited its most rapid sugar consumption of

15.6 g/L during the initial 12 h of fermentation with the SBMB4(120 °C, 1.25% H2SO4, 30 min) and 15.1 g/L during the 1st 12 hof fermentation with the SBMB1 (135 °C, 0.5% H2SO4, 45 min)(Figure 1e). On the other hand, the SBM broths from the most se-vere hydrolysis treatments appeared to result in a low rate of sugaruse from the fermentation broth by S. cerevisiae (Figure 1e). Thislow rate of sugar consumption occurred due to toxic compoundsformed during the hydrolysis process that reduces the yeast growth(Lujan-Rhenals and others 2014).

Z. mobilis also expressed its most rapid sugar uptake of 15.0 g/Lper 16 h of fermentation with the SBMB1 (135 °C, 0.5% H2SO4,45 min) (Figure 2b). Similar to S. cerevisiae, Z. mobilis had lowrates of apparent sugar consumption during growth in the broths(Figure 2c and 2d) resulting from the most severe pretreatments,particularly SBMB2 (135 °C, 1.25% H2SO4, 45 min), which isprobably due to the inhibitory effect of acetic acid and salts aspreviously discussed.

Overall, the maintenance coefficient ms (g sugar consumed/g cell h) for both microorganisms did not yield statistically signifi-cant differences except for S. cerevisiae with SBMB2 and SBMB3,which gave higher values (2.7 and 2.8 g sugar/g cell h, respectively)than the other coefficients (Table 2). However, numerically, all ms

coefficients for Z. mobilis were lower than the ms coefficients cal-culated for S. cerevisiae which means that the bacteria apparentlyrequired less carbon substrate per cell than the yeast. This result hasalso been previously reported by (Dien and others 2003). There-fore, Z. mobilis utilizes less substrate per gram of cells producedthan S. cerevisiae and has more carbon available for the productionof ethanol (Dien and others 2003).

Kinetics of ethanol productionS. cerevisiae exhibited its maximum ethanol production of

8 g/L during the 1st 8 h of fermentation of the SBMB1 (135 °C,0.5% H2SO4, 45 min) and SBMB4 (120 °C, 1.25% H2SO4,30 min). In contrast, the lowest ethanol production (5.67 g/Lethanol) occurred with the SBMB3 (135 °C, 2% H2SO4, 45 min)after 28 h of fermentation, where possibly some inhibitor com-pounds (salts, acetate from acetic acid, and other unidentified toxicsubstances) could have reduced the ethanol productivity.

Ethanol production by Z. mobilis peaked at 9.2 g/L ethanol af-ter 20 h of fermentation with the SBMB1 (135 °C, 0.5% H2SO4,45 min). The SBMB0 (135 °C, 0% H2SO4, 45 min) allowed amaximum ethanol rate of 3.9 g/L ethanol after 20 h of fermen-tation. However, the lowest ethanol production levels—1 g/L at20 h—were attained with the SBMB2 (135 °C, 1.25% H2SO4,45 min) and SBMB4 (120 °C, 1.25% H2SO4, 30 min), whichis likely caused by inhibitors (acetate from acetic acid, other salts,and unknown compounds). There have been previous reports thatethanol production with Z. mobilis and its growth can be reducedsubstantially even with low concentrations (2 to 8 g/L) of aceticacid (Wang 2008).

The ethanol yield (Yp/s, g ethanol/g of sugars) reported inTable 2 did not show significant statistical differences between the

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2 microorganisms and the respective SBM broths used. However,numerically both microorganisms showed similar ethanol yieldsto the theoretical values, which is 100% for 0.51 g ethanol/g ofsugars (Doran 1997). Fermentations in the SBM0 and SBMB1gave the highest ethanol yields, 96% of the theoretical yield forboth microorganisms, which are comparable or even higher thanvalues reported in previous research (Siqueira and others 2008;Zhang and Feng 2010; Da Cunha-Pereira and others 2011; Lettiand others 2012; Romao and others 2012). Additionally, thehighest ethanol volumetric productivity (rp) for S. cerevisiae wasachieved with the SBM1 (1.01g ethanol/L h) and SBMB4 (1.00g ethanol/L h), with no significant difference between the 2, butdiffered with respect to the other broths used in this research(Table 2).

These values for S. cerevisiae are comparable to, and some higherthan those reported by other researchers (Da Cunha-Pereira andothers 2011). However, Z. mobilis reached a maximum ethanolvolumetric productivity (rp) of 0.61 g ethanol/Lh only with theSBM1, which is lower than the values, 1.4 to 1.8 g ethanol/Lh withZ. mobilis during the production of ethanol using SBM molasses(Letti and others 2012). Also, it is lower than 2.8 g ethanol/Lhobtained for the production of ethanol with Z. mobilis fromsweet potato (Zhang and Feng 2010); however, it is higher than0.59 g/Lh using lignocellulosic material as substrate (Mohagheghiand others 2002). These results demonstrate that the applicationof Z. mobilis for ethanol fermentation is not feasible when thesubstrate is SBM broth obtained under the conditions applied inthese experiments. This is probably due to the high concentration

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Figure 1–Fermentation profiles of SBM broths with S. cerevisiae NRRL Y-2233. Acronyms defined in Table 1.




Table 2–Means of kinetic parameters of ethanol fermentation in batch culture with S. cerevisiae NRRL Y-2233 and Z. mobilissubspecies mobilis NRRL B-4286 in hydrolyzed soybean meal broths ± SE. Acronyms defined in Table 1.

Yp/s Yx/s Ms RpSBM broth Microorganism (g ethanol/g sugar) (g cell/g sugar) (g sugar/g cell h) (g ethanol/Lh)

SBMB0 S. cerevisiae 0.49 ± 0.021 a 0.23 ± 0.014 abc 0.69 b 0.67 ± 0.028 bSBMB0 Z. mobilis 0.49 ± 0.007 a 0.23 ± 0.057 ab 0.44 b 0.37 ± 0.007 cdSBMB1 S. cerevisiae 0.49 ± 0.014 a 0.10 ± 0.014 cd 1.19 b 1.00 ± 0.028 aSBMB1 Z. mobilis 0.48 ± 0.028 a 0.08 ± 0.014 d 0.97 b 0.61 ± 0.007 bcSBMB2 S. cerevisiae 0.46 ± 0.007 a 0.04 ± 0.007 d 2.73 a 0.61 ± 0.113 bcSBMB2 Z. mobilis 0.26 ± 0.042 a 0.15 ± 0.021 bcd 0.65 b 0.11 ± 0.007 deSBMB3 S. cerevisiae 0.40 ± 0.057 a 0.02 ± 0.007 d 2.78 a 0.29 ± 0.092 deSBMB4 S. cerevisiae 0.49 ± 0.007 a 0.11 ± 0.007 bcd 1.13 b 1.01 ± 0.021 aSBMB4 Z. mobilis 0.29 ± 0.113 a 0.32 ± 0.014 a 0.17 b 0.07 ± 0.007 e

Means followed by the same letters in each column did not have significant differences. (P = 0.05). Z. mobilis exhibited no growth with the SBMB3.

of acetic acid, salts, and other toxic unknown compounds formedduring the acid hydrolysis in the broths obtained under severetreatments.

Kinetics of biomass growth and model developmentfor the fermentation

Data reported in Figure 1a indicate that S. cerevisiae yielded itsmaximum biomass concentration (2.3 g/L) after 12 h of anaerobicfermentation when exposed to the SBM0 (135 °C, 0% H2SO4,45 min) followed by enzymatic treatment with cellulase. Like-wise, this maximum concentration of biomass (2.3 g/L) was alsoreached with SBMB4 (120 °C, 1.25% H2SO4, 30 min) at 24 h.

However, SBMB3 (135 °C, 2% H2SO4, 45 min) exhibited a ma-jor inhibition of yeast growth since the biomass yielded no morethan 0.5 g/L after 32 h of fermentation. Overall, production ofbiomass by S. cerevisiae appeared to be directly associated withethanol production.

Z. mobilis exhibited its highest biomass concentration, 3.9 g/L,after 20 h of fermentation (Figure 7.2a) with the SBMB0 (135 °C,0%, H2SO4, 45 min), followed by enzymatic treatment with cel-lulase. Likewise, the biomass produced (3.3 g/L) when fermentingthe SBMB1 (135 °C, 0.5% H2SO4, 45 min) was very close to theformer but required 24 h to reach this level. In contrast, SBM2(135 °C, 2% H2SO4, 45 min) exhibited the primary inhibitoryeffects on Z. mobilis growth due to higher concentrations of salts,

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Figure 2–Fermentation profiles of SBM broths with Z. mobilis NRRL B-4286. Acronyms defined in Table 1.


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Table 3–Means of logistic model parameters of ethanol fermentation in batch culture with S. cerevisiae NRRL Y-2233 and Z. mobilissubspecies mobilis NRRL B-4286 in hydrolyzed soybean meal broths ±SE. Acronyms defined in Table 1.

SBM broth Microorganism µmax (h-1) xo (g biomass/L) xmax (h-1)

SBMB0 S. cerevisiae 0.63 ± 0.12 a 0.17 ± 0.08 a 2.28 ± 0.05 aSBMB0 Z. mobilis 0.55 ± 0.05 a 0.18 ± 0.05 a 3.79 ± 0.06 f lSBMB1 S. cerevisiae 0.45 ± 0.09 afgh 0.32 ± 0.09 a 1.81 ± 0.05 bSBMB1 Z. mobilis 0.16 ± 0.02 bk 0.18 ± 0.04a 5.1 ± 0.92 g lSBMB2 S. cerevisiae 0.21 ± 0.11 bdfi 0.16 ± 0.08 a 0.66 ± 0.10 cejSBMB2 Z. mobilis 0.45 ± 0.25 aijkl 0.18 ± 0.1 a 0.65 ± 0.05 hjkSBMB3 S. cerevisiae 0.19 ± 0.15 begj 0.14 ± 0.08 a 0.49 ± 0.12 dekSBMB4 S. cerevisiae 0.60 ± 0.11 a 0.24 ± 0.09 a 2.23 ± 0.05 aSBMB4 Z. mobilis 0.29 ± 0.03 cdehl 0.26 ± 0.06 a 2.97 ± 0.09 i

Means followed by the same letters in each column did not have significant differences (p = 0.05). Z. mobilis exhibited no growth with the SBMB3.

acetic acid, and other inhibitors in the broth that restricted itsgrowth (Doran 1997; Yang and others 2010). In general, Z. mo-bilis also showed growth-associated ethanol production with theexception of those broths where the bacteria were considerablyinhibited.

Data of cell growth responses fitted to a logistic model duringthe 1st 24 h of fermentation are shown in Table 3. The maxi-mum specific growth rates (μmax) shows that in the SBM0 andSBMB4, μmax for S. cerevisiae (0.63 and 0.60 h-1, respectively)were higher but not significantly different than μmax for Z. mobilis,0.55 h-1, in the substrate T3C0t3. Wang and others (2004), alsousing the logistic model, reported a μmax of 0.1 h-1 for the ethano-lic fermentation of apple juice with S. cerevisiae ( Huang and Wang2010) obtained a μmax of 5.2 h–1 using Saccharomyces diastaticuswith mixed sugars as a substrate. Mohagheghi and others (2002)reported for Z. mobilis a μmax of 0.34 h-1 for ethanolic fermenta-tion of hydrolyzed lignocellulosic biomass. In general, μmax for S.cerevisiae was higher for substrates obtained with pretreatments ofless severity; Z. mobilis μmax did not show the same pattern due tothe effect of inhibitors present in some of the SBM broths.

The initial concentration of biomass (x0) was not significantlydifferent among all the broths for both microorganisms, whichis an indication that the inoculum was homogeneously preparedand introduced to each SBM broth. The other kinetic parameterderived by fitting the logistic model to the 1st 24 h of fermentationwas the maximum concentration of biomass (xmax). For S. cerevisiae,xmax was 2.28 and 2.23 g/L for SBMB0 and SBMB4, respectively,while the maximum xmax for Z. mobilis was 3.79 g/L and 5.1 g/Lfor the SBMB0 and SBMB1, respectively.

The biomass yield from substrate (Yx/s) listed in Table 2 wassignificantly higher (P = 0.05) for S. cerevisiae (0.1 g cell/g sugarconsumed) than Z. mobilis (0.08 g cell/ g sugar consumed) whenfermenting the SBMB1. Higher values with S. cerevisiae thanZ. mobilis have also been reported by other researchers (Dien andothers 2003, Aitabdelkader and Baratti 1993). This is a function ofthe better resistance of the yeast to the inhibitors present in thesesubstrates (Weber and others 2010). However, with SBM2 the ef-fect was the opposite, the value for Z. mobilis was 0.15 g cell/gsugar and for S. cerevisiae was 0.04 g cell/g sugar. Nonetheless, mostof the values for S. cerevisiae were higher than the previous reports(Siqueira and others 2008) for the production of bioethanol fromsoybean molasses and similar to values (0.14 g/g) reported in otherwork (Ahmad and others 2011).

ConclusionsS. cerevisiae exhibited good qualities in the production of

ethanol, sugars consumption, and cell growth for all acid-hydrolyzed soybean meal broths tested without any additional

nutrients, while Z. mobilis had a lower performance for mostbroths. The highest ethanol production of S. cerevisiae, 8 gethanol/L, (4 g ethanol/100 g fresh SBM) was reached duringthe 1st 8 h of fermentation with the broth obtained at 135 °C,0.5% H2SO4, 45 min and the one at 120 °C, 1.25% H2SO4, and30 min. However, Z. mobilis yielded maximum ethanol produc-tion, 9.2 g/L ethanol, (4.6 g ethanol/100 g fresh SBM) only afterthe 1st 20 h with the hydrolyzate obtained at 135 °C, 0.5% H2SO4,45 min.

AcknowledgmentsThis research was partially supported by the Arkansas Soybean

Promotion Board. Deivis E. Lujan-Rhenals was supported inpart by COLCIENCIAS of Colombia, LASPAU, and Univ. deCordoba (Colombia).

Author ContributionsDeivis Lujan-Rhenals performed the experiments, analyzed the

data, and wrote the paper. Ruben Morawicki designed the exper-iments, analyzed the data, and wrote the paper. Edward Gburprovided technical details in the experimental design and statisti-cal analysis. Steven C. Ricke provided technical support with thefermentation.

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