CONCLUSIONS AND RECOMMENDATIONS FOR
FUTURE STUDIES
The goal of this dissertation was to understand how protease can be used more effectively in corn dry fractionation for ethanol production. In line with the four specific objectives stated in the Introduction, the following are our major conclusions.
1. Protease had a greater impact on dry fractionation than on wet fractionation, in terms of increasing fermentation rates and reducing residual starch in endosperm fiber recovered at the end of fermentation.
2. As far as conventional liquefaction is concerned, protease incubation neither affected liquefaction outcomes nor subsequent saccharification of liquefied mash. Two qualifications regarding this finding need to be stated: (1) starch hydrolyzing enzymes (ie, amylases) were used at recommended loadings, and therefore, our conclusion cannot extend to lower enzyme loadings and (2) saccharification extended only to 20 hr, at which point product inhibition (by glucose and maltose) likely had taken effect, and therefore, our conclusion cannot be extended to longer saccharification periods (>20 hr) expected during fermentation.
3. Under any level of urea supplementation, the addition of protease to generate free amino nitrogen (FAN) from endosperm increased fermentation rates. When urea was limiting (<2.5 mg N/g glucose), FAN supplementation increased ethanol yields, but not when urea was sufficient. With FAN supplementation at the highest level (480 mg FAN/L in the mash or 140 mg FAN/100 g ds), ethanol yield was less (by 2 g/L) compared to a urea control during fermentation using
conventional liquefaction. This yield reduction was correlated with a higher amount of unutilized maltose at the end of fermentation. No such ethanol yield reduction occurred during fermentation using a granular starch hydrolyzing
4. In the case of germ derived FAN, the optimal level for improving ethanol yields and production rates was 80 to 90 mg FAN/100 g ds. Germ FAN resulted in lower unutilized maltose, around half the amount resulting from endosperm derived FAN (on similar FAN level basis).
From this study, we identified four potential issues that would benefit from further investigation.
1. On the issue of using a protease to facilitate starch hydrolysis (ie, liquefaction and saccharification), study should be made whether lower enzyme loadings (of amylases) would favor protease treatment over control. Expressed in question form: would optimal amylase loadings be lower with protease treatment? In addition, how would such a response to lower amylase loading with protease treatment be different between conventional and GSHE process?
2. How is maltose utilization by yeast during fermentation tied to free amino nitrogen levels? Maltose consumption occurs late in fermentation when glucose is depleted because of catabolite repression in yeast (ie, high glucose
concentrations inhibit maltose uptake and utilization). On the other hand, preferred amino acids are consumed quickly (first 24 hr) during fermentation, while leaving less preferred amino acids (especially proline and glycine) in the media. Could the timing suggest more than coincidence and could the
accumulation of nonpreferred amino acids actually influence the efficiency of maltose utilization?
3. Related to issue (2) above is the observation that fermentation using GSHE
process did not result in unutilized maltose even with high FAN supplementation. It would be interesting to test the hypothesis that lower steady state glucose
concentrations during GSHE process prevents catabolite repression of maltose in yeast, thereby negating the effect of high FAN on maltose utilization.
4. For the same FAN level, supplementing with germ derived FAN resulted in smaller amounts of unutilized maltose remaining after fermentation than with
endosperm derived FAN. This points to differences between amino acid compositions of the two sources of protease generated FAN. It would be interesting to investigate whether such a composition can be optimized, and whether we could select for proteases based on this optimal amino acid composition.
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APPENDIX A
STARCH HYDROLYZING ACTIVITY IN PROTEASES AND ITS
EFFECT ON GRANULAR STARCH HYDROLYZING ENZYME
(GSHE) PERFORMANCE
We tested amylolytic side activities of various commercial protease preparations by incubating (in 45ºC, pH 4.2) proteases in 5% w/v starch solution (Fisher Chemical, Fairlawn, NJ) and then measuring the reducing sugar liberated after 30 min using DNS assay (Miller 1959). We observed starch hydrolyzing activities in these proteases (Table A.1).
Table A.1. Reducing sugar liberated from soluble starch after incubation with various proteases. a,b,c
Protease Reducing Sugar (g/L)
NS5000P NS50045 GC106 No Enzyme Control 0.53 ± 0.04 0.68 ± 0.10 0.24 ± 0.01 bdl a
Protease loading was 0.0001% v/v. b
Values are mean±1 average deviation (2 replicates); bdl: below detection limit. c
NS5000P and NS50045 (Novozymes, Franklinton, NC); GC106 (Genencor, Palo Alto, CA)
We assessed the significance of proteases’ intrinsic amylolytic activities when applied with granular starch hydrolyzing enzymes (GSHE) to dry grind corn. GSHE NS50040 (0.2 mL/100 g slurry) was added to 25% solids slurry made up of ground corn (hammer milled through 1-mm opening). Incubation was conducted for 3 hr at 45C, pH 4.2, and samples collected for reducing sugar measurements. Contributions from protease side activities were negligible (Figure A.1 and A.2).
0 10 20 30 40 50 60 70 80 90 100 0 0.5 1 1.5 2 2.5 3 3.5 Time (hr) Pt -P o ( R e du c ing S ug a r, g/ L) 0% v/w 0.1% v/w 0.2% v/w Added NS5000P Concentrations
Figure A.1. Reducing sugar liberated from 25% w/w dry grind corn with addition of GSHE NS50040 (0.2% v/w) and protease NS5000P (0, 0.1 and 0.2% v/w). Pt-Po : reducing sugar at
0 10 20 30 40 50 60 70 80 90 100 0 0.5 1 1.5 2 2.5 3 3.5 Time (hr) Pt -P o ( R e du c ing S ug a r, g/ L) 0% v/w 0.1% NS50045 0.1% GC106 Added Protease Concentrations
Figure A.2. Reducing sugar liberated from 25% w/w dry grind corn with addition of GSHE NS50040 (0.2% v/w) and proteases NS50045 (0.1% v/w) and GC106 (0.1% v/w). Pt-Po :
APPENDIX B
ENDOSPERM FERMENTATION USING REDUCED PROTEASE
LOADINGS
In Chapter 4, the minimum FAN supplementation level used to assess effectiveness as a nitrogen source for endosperm fermentation was 340 mg FAN/L. We found from the FAN consumption profile (Figure 4.3) the average FAN consumed during fermentation was close to 200 mg FAN/L. We examined whether a much lower protease (NS50045) loading of 0.05 mL/100 g slurry (generating 16212 mg FAN/L) could support complete fermentation without urea supplement. We also examined whether the protease could be added at the start of
fermentation (or simultaneous proteolysis (SP), rather than pretreating the samples (PP)). Except for the lower protease loading and the latter modification (SP), the procedure followed exactly Section 4.1.5 (Chapter 4).
Ethanol production trends (SP and PP) are shown in Figure B.1, along with 0.2 mL protease/100 g slurry treatment and urea control (112 mg urea/100 g slurry) for comparison. Fermentations were complete in 72 hr (no remaining glucose detected by HPLC) and final ethanol yields for the lower protease loading (SP and PP) were comparable to that of urea only (1271, 1281 and 1271 g/L for PP, SP and urea only, respectively). Therefore, the use of a lower protease loading (0.05 mL/100 g slurry) had not resulted in a similar reduction in ethanol yields as had higher protease loadings (0.40 mL/100 g slurry). However, the values for
fermentation extent at 48 hr (881 and 842% for PP and SP, respectively) were lower than those obtained with higher protease loading or with urea control.
0 2 4 6 8 10 12 14 16 0 10 20 30 40 50 60 70 80 Time (hr) Ethano l (%v /v) SP 0.05 PP 0.05 PP 0.20 Urea
Figure B.1. Ethanol production during fermentation of DF corn endosperm treated as follows: SP 0.05, simultaneous proteolysis at protease loading of 0.05 mL/100 g slurry (ie, added right
before fermentation); PP 0.05 and 0.20, pretreatment at protease loadings of 0.05 and 0.20 mL/100 g slurry, respectively; and urea supplemented control (112 mg urea/100 g slurry).
APPENDIX C
EFFECT OF PROTEASE TREATMENTS ON ENDOSPERM
FERMENTATION USING GRANULAR STARCH
HYDROLYZING ENZYMES (GSHE)
We examined the effect of protease incubation on endosperm fermentation using a granular starch hydrolyzing process. A similar range of FAN (≤430 mg/L, Table C.1) used during conventional fermentations in Chapter 4 was obtained by protease incubation.
Fermentation conditions were similar to those in Section 4.1.5 (Chapter 4), but instead of going through a liquefaction step after protease incubation, slurry was adjusted directly to pH 4 and fermented after adding 0.05 mL GSHE/100 g slurry (NS50086 from Novozymes, Franklinton, NC). Ethanol product profiles were indicative of faster fermentation rates with protease treatment compared to urea control (Figure C.1). Final maltose concentrations were below detection, including at the highest FAN level (Table C.1). At the highest FAN level, ethanol yield was not different from the urea control; whereas, at the intermediate FAN level, ethanol yield was higher than the control (Table C.1).
0 20 40 60 80 100 120 140 0 12 24 36 48 60 72 Time (hr) Ethanol (g/L) 0.0 mL 0.1 mL 0.4 mL Protease Loading (per 100 g slurry)
Figure C.1. Ethanol production profile of endosperm fermentation using granular starch hydrolyzing enzyme (GSHE) NS50086.
Table C.1. Initial free amino nitrogen (FAN) and final maltose and ethanol concentrations during endosperm fermentation using granular starch hydrolyzing enzyme (GSHE) NS50086. a,b,c
Protease (mL/100 g slurry) FAN (0 hr) (mg/L) Maltose (72 hr) (g/L) Ethanol (72 hr) (g/L) 0 (urea control) 711 bdl 126.70.9 a 0.1 24414 bdl 130.20.3 b 0.4 43530 bdl 127.40.8 ab a
bdl: below detection limit b
Values reported as Mean1 SD (2 replicates) c
APPENDIX D
POLYPEPTIDE AND AMINO ACID PROFILES GENERATED
BY PROTEASE TREATMENT OF ENDOSPERM AND GERM
Product profiles from protease incubation of both dry fractionated endosperm and germ were characterized using SDS-PAGE (Figure D.1) and amino acid analyzer (Figure D.2). A brief description of the procedure is as follows. Slurry was prepared from ground endosperm and intact germ (25 and 18% ds, respectively) in acetate buffer (pH 5, 50 mM). Protease NS50045 and subtilisin (Subtilisin A, Type VIII, Sigma-Aldrich, St. Louis, MO) were loaded at 0.1 mL/100 g slurry and 50 U/100 g slurry, respectively. Slurry was incubated in water bath (50C) for 3 hr. For amino acid analysis, hydrolyzate supernatant samples were filtered through 0.2 µm filters (Waters, Milford, MA) into HPLC vials and stored frozen. Amino acid analysis was accomplished using Zorbax Eclipse-AAA column in Agilent 1100 HPLC (Agilent Technologies, Santa Clara, CA). For SDS-PAGE, supernatant samples were 6.25 times diluted before mixing with SDS denaturing buffer (1:1 volume ratio). Samples were boiled for 10 min before loading into 4 to 20% Tris-HCl gel (Bio-Rad Laboratories, Hercules, CA).