Enzymatic digestibility and ethanol fermentability of AFEX-treated starch-rich lignocellulosics such as corn silage and whole corn plant

Hydrolysis of AFEX-treated starch and cellulose

CS and WCP contain a significant proportion of both starch and cellulose (Table 1) unlike typical lignocellulosics such as corn stover. Initial enzymatic hydrolysis studies were carried out using untreated and AFEX-treated cellulose (Avicel) and soluble starch to study the relative digestion kinetics as a function of enzyme loading. Percentage glucan conversion from cellulose and starch as a function of different protein loading (Accellerase™ and Stargen™) respectively, are shown in Figure 1. Avicel and starch were treated with AFEX under identical conditions as described in the Methods section. The glucose yield after hydrolysis of Avicel for 24 h (data not shown) and 72 h (Figure 1a) was largely comparable for untreated and AFEX-treated substrates for different protein loadings. These results are comparable with previous reports on the digestion kinetics of cellulose treated with ammonium hydroxide 28% w/w [20].

Components	Corn silage			Whole corn plant
	0X-CS	1X-CS	10X-CS
Glucan (cellulose + starch)	49.2 ± 1.4	45.9 ± 0.7	44.8 ± 2.5	64.7 ± 0.6
Cellulose	19.7 ± 1.2	19.1 ± 0.2	19.6 ± 0.3	15.5 ± 0.4
Starch	29.5 ± 0.2	26.8 ± 0.5	25.2 ± 2.2	49.2 ± 1.0
Xylan	11.4 ± 0.2	12.1 ± 0.4	11.6 ± 0.2	12.2 ± 0.4
Arabinan	2.7 ± 0.1	2.9 ± 0.5	2.4 ± 0.1	1.9 ± 0.1
Klason lignin	8.8 ± 0.7	8.6 ± 0.5	8.9 ± 0.6	7.1 ± 0.1
Crude protein	10.2 ± 1.8	8.2 ± 0.4	8.9 ± 0.4	7.1 ± 0.5
Crude fat	3.9 ± 0.4	3.0 ± 0.6	3.3 ± 0.8	ND
Water-soluble carbohydrates	3.1 ± 0.3	4.4 ± 0.2	4.1 ± 0.1	5.1 ± 0.3
Ash	3.7 ± 0.3	4.1 ± 0.1	6.3 ± 0.1	2.1 ± 0.1

X = the manufacturer recommended loading rate of 0.0015 g/g.

For AFEX-treated starch, the glucose yield after 24 or 72 h of hydrolysis was 2.5-4-fold higher than that obtained from untreated starch (Fig 1b). Hot concentrated ammonium hydroxide is thought to gelatinize starch through disruption of inter- and intramolecular hydrogen bonding, similar to that seen during treatment of crystalline cellulose with liquid ammonia [21, 22]. Disruption of the hydrogen bonds would create a more disordered ultrastructure and improve glucan chain hydration, greatly enhancing susceptibility to amylases. AFEX pretreatment and enzymatic hydrolysis of milled corn grain using Stargen™ (3 mg per gram starch) yielded 84% starch conversion within 12 h compared with 65% for untreated material (data not shown). However, after 72 h of hydrolysis, > 95% conversion was achieved for both substrates. The difference between the digestibility of soluble starch (typically isolated using acids) and corn grains may be due to differences in their ultrastructure.

To mimic the hydrolysis of matured whole plant-based substrates, a mixture of Avicel and starch (with and without AFEX treatment) was hydrolyzed with a combination of Accellerase™ and Stargen™-based enzymes (Figure 2). The mixed substrate containing Avicel and starch at a ratio of 1:1 (dry weight basis; DWB) was pretreated with AFEX and hydrolyzed with a mixture of Accellerase™ (10.7 mg/g of cellulose) and Stargen™ (7.5 mg/g of starch). The AFEX-pretreated Avicel-starch mixture had higher glucan conversion compared with the untreated control. There was a 25-30% increase in glucan conversion after AFEX pretreatment. The maximum glucan conversion (at 72 h) of the AFEX-pretreated Avicel-starch mixture was essentially comparable with that of the independently hydrolyzed substrates. This result indicates that both starch and cellulose, within whole plants, can be pretreated using AFEX to maximize overall glucan yield. However, an interesting observation during these experiments was the marginal reduction in overall yields by 5-10% for the mixed substrate, suggesting that nonspecific binding of enzymes to cellulose and starch could result in slightly lower conversions. Further support for this hypothesis was found by conducting sequential hydrolysis of starch (by Stargen™) followed by cellulose (by Spezyme CP™) in WCP (Figure 3). Interestingly, the overall glucan conversions for both untreated and AFEX-treated WCP were 15-20% higher if amylases were added 12 h before addition of cellulases. Novozyme 188™ was also found to have significant amylase activity, giving close to 60-70% glucose yield within 12 h when used for hydrolysis of AFEX-treated WCP. It was also found that deactivating (by thermal denaturation) the amylases before the addition of cellulases resulted in marginally higher conversions (data not shown). Interestingly, hydrolysis of AFEX-treated WCP with Spezyme CP™ yielded higher glucose yield (> 400 g/kg biomass at 168 h) than that from untreated WCP (< 100 g/kg biomass). This suggests that trace amylase activity in Spezyme CP™ was able to digest AFEX-pretreated WCP more effectively than the untreated substrate during prolonged incubation (similar to that in Figure 1b comparing untreated and AFEX-treated starch). By contrast, the extent of glucan (both starch and cellulose) hydrolysis for both untreated and AFEX-treated WCP was comparable in the presence of only Novozyme 188™ (which is abundant in amylase activity; 5300 IU/mL [23]). These results suggest that in enzyme-limiting conditions, the beneficial role of pretreatment becomes much more evident. Spezyme CP™ is typically supplemented with Novozyme 188™ to prevent cellobiose inhibition due to lack of sufficient β-glucosidase activity in the former (unlike Accellerase 1000™, which has sufficient β-glucosidase activity). For the sake of simplicity and to help accentuate differences between untreated and AFEX-treated substrates, all further experiments were conducted using Accellerase 1000™ and Stargen™ added simultaneously (at time t₀) at significantly lower protein loadings (typically < 10 mg/g starch or cellulose).

Effect of AFEX pretreatment conditions on CS digestibility

The effects of AFEX pretreatment conditions on enzymatic digestibility of uninoculated CS (0X-CS) were explored. Ammonia loading was varied between 0.1 and 3 g per g biomass at 90°C, 60% moisture (DWB) and 5 min residence time. The glucan conversion increased with increased ammonia loading up to 1 g/g biomass (Figure 4a). Further increases in ammonia loading beyond this point did not significantly improve glucan hydrolysis. When moisture content was varied (20-200%, DWB) under similar conditions, maximum glucan conversion was achieved at lower moisture loadings (20%) (Figure 4b). At higher moisture levels, glucan conversion decreased marginally. Untreated CS that was dried (UT) before hydrolysis gave the lowest glucan conversion. However, it was found necessary to dry the samples (0X-CS) before AFEX pretreatment at lower moisture loadings. Temperature was varied between 50 and 130°C at fixed moisture content (60%), ammonia to biomass loading (1:1 w/w) and residence time (5 min). There was no major effect of temperature on glucan conversion within the range tested (Figure 4c). However, xylan conversion increased by 2-3-fold at temperatures > 90°C compared with 50°C. This is probably due to extensive cleavage of LCC ester complexes at higher temperatures, which enhances xylan digestibility [12]. In addition, there was less variability in glucan conversion at higher temperatures (90-130°C). Therefore, the AFEX conditions used for further experiments on dried CS (0X-CS, 1X-CS or 10X-CS) were 90°C, 1:1 ammonia loading, 60% moisture and 5 min residence time to maximize glucan and xylan conversions.

Effect of microbial inoculation on pretreatment efficacy and enzymatic digestibility of ensiled corn plants (CS)

The effect of microbial inoculation on the composition and glucan digestibility of CS (before and after AFEX pretreatment) was studied. Although the cellulose and hemicellulose contents were not altered significantly, starch content was reduced by 10-15% upon inoculation of CS (Table 1). AFEX pretreatment of dried CS samples (with/without inoculation) resulted in 15% lower hydrolysis yields for 10X-CS compared with 0X-CS (Figure 5). These results indicate that microbial inoculation before the ensiling process was not beneficial to overall glucan hydrolysis yields. Previous work has shown that biological pretreatment of lignocellulosic cell walls with fungi (for example, white rot Pleurotus ostreatus) before conventional thermochemical processing enhances enzymatic digestibility of cellulose by cleaving LCC linkages [24]. The mechanisms by which lignin restricts cell wall degradation and hydrolysis of cellulose are not well understood. Enteric fermentation studies suggest that the influence of lignin on microbial deconstruction of plant cell walls may be primarily due to physical mechanisms such as shielding of cellulose but the effect of lignin may also involve more specific molecular interactions [25, 26]. However, the microbes (for example, Lactobacillus, Pediococcus) present during conventional ensilation lack the enzymatic activities required to cleave LCC linkages and improve overall substrate digestibility.

High solid loading-based CS and WCP enzymatic hydrolysis

Untreated and AFEX-treated (90°C, 1:1 ammonia loading, 60% moisture and 5 min residence time) CS (0X-CS) samples were hydrolyzed under high solid loading (that is, 6% w/w glucan loading) conditions to increase sugar concentration in the hydrolyzate and maximize ethanol titer (Figure 6). To further increase xylose yield, the cellulase (Accellerase™) and amylase (Stargen™) cocktails were supplemented with hemicellulases (Multifect Xylanase) during high-solid loading-based hydrolysis. As expected, AFEX pretreatment of silage significantly enhanced both glucan and xylan conversions. The glucose and xylose yields from AFEX-treated silage were 1.8- and 4.4-fold higher than untreated silage (at 1% glucan loading; data not shown). The glucan conversion was 70% and was about 10% lower at 6% glucan loading than at 1% glucan loading, because of the higher concentration of monomeric sugars in the hydrolyzate, which are known to inhibit cellulase activity [27, 28]. The enzymatic digestibility of WCP at 6% glucan loading was also tested before and after AFEX pretreatment. However, it was found that the overall hydrolysis yield at 6% glucan loading for AFEX-treated WCP was significantly lower that that reported previously (Figure 3). This is probably due to the significantly lower protein loading we used (10-15-fold difference), compounded by end-product inhibition of hydrolytic enzymes at 6% glucan loading compared with previously reported experiments.

Ethanol fermentation of CS- and WCP-based hydrolyzates

High solid loading-based enzymatic hydrolyzates of untreated and AFEX-pretreated 0X-CS and WCP were used for ethanol fermentation. Figure 7a depicts the consumption of glucose and xylose during production of ethanol from the 6% glucan loading-based AFEX-treated 0X-CS hydrolyzates. The hydrolyzate of untreated silage contained 45.4 g/L of total sugar (94.1% glucose and 5.9% xylose; data not shown). Total sugar concentration increased to 62.0 g/L (87.8% glucose and 12.2% xylose; Figure 7a) during hydrolysis of AFEX-pretreated 0X-CS. During fermentation of AFEX 0X-CS hydrolyzate by S. cerevisiae 424A (LNH-ST), glucose was almost completely consumed within 24 h, but, about 27% of the xylose remained unused even after 72 h (Figure 7). Ethanol concentration in the broth was 28 g/L after 72 h, which corresponds to 93% theoretical yield based on total sugar consumption. In untreated silage, 23 g/L ethanol with a metabolic yield of 98% was produced at 72 h (data not shown).

Biomass, chemicals and enzymes

Corn plants, either in the immature state to be used for ensiling (CS) or as mature WCP were obtained from Michigan State University Farms (East Lansing, MI, USA). The corn hybrid used was NK 49-E3 (Syngenta, Basel, Switzerland) which is a typical CS hybrid used in the Great Lakes Region. The corn plants used in this study were planted on 8 May 2008 and harvested on 19 September 2008 for ensilation. The WCP were harvested after the plant reached physiological maturity, which occurred approximately 6 weeks after harvest for ensilation. WCP was harvested as stover and grain separately (moisture content < 15% DWB). WCP-based samples were milled using a Wiley mill (Christy and Morris, Chelmsford, UK) (10 mm sieve attachment) followed by mixing of the grain and stover fractions at a mass ratio of 1:1 (w/w). Ensiling was accomplished by sealing 500 g immature entire corn plant samples in plastic bags using a commercial grade vacuum seal food machine (CG-15; Cabela, Sidney, NE, USA). The sealed bags were stored at 21°C for 30 days to imitate a typical on-farm ensiling process. We also evaluated the effect of a commercially available microbial inoculant product (Silo-King, Agri-King Inc., Fulton, IL, USA) on ensiled corn digestibility at a 0X, 1X and 10X loading (X = the manufacturer recommended loading rate of 0.0015 g/g). The inoculant product is composed of lactic acid-producing organisms, such as Lactobacillus plantarum, Pediococcus pentosaceus and Enterococcus faecium, and is used by farmers to enhance the feed quality of ensiled corn [29]. The CS samples were frozen using liquid nitrogen, milled using a laboratory blender (Hamilton Beach, Washington, NC, USA), and passed through a 10 mm screen sieve. The milled CS samples were stored in sealed Ziploc Storage Bags (SC Johnson, Racine, Wisconsin, USA) at -20°C for long-term storage. The moisture content of CS was between 63 and 67% (DWB). The CS samples were dried to < 10% moisture (DWB) using a 50°C oven, to allow suitable adjustment of the water loadings used during pretreatment.

Avicel PH101 and soluble starch S5160599 (lot #054261) were purchased from Fluka (Tokyo, Japan) and Fisher Scientific (USA), respectively. Commercial enzymes used for degrading cellulose were Spezyme CP™ (88 mg/ml) and Accellerase 1000™ (84 mg/ml; lot #1600844643) (both gifts from Genencor Division, Danisco US Inc., Rochester, NY, USA), The enzymes used for degrading starch were Novozyme 188™ (149 mg/ml) (Sigma-Aldrich (Sigma, St. Louis, MO, USA) and Stargen 001™ (62 mg/ml; lot #4900851951) (gift from Genencor Division). The enzyme used for degrading hemicellulose was Multifect Xylanase™ (35 mg/ml) (gift from Genencor Division). The concentrations of these enzymes were estimated using a Kjeldahl-based method (Dairy One Feed Stock Analyzing Co., Ithaca, NJ, USA).

Compositional analysis

Crude protein, starch, crude fat and water-soluble carbohydrate content of CS (0X, 1X and 10X) and WCP were determined at the Forage Testing Laboratory (Dairy One Inc.). In addition, acid and neutral detergent fiber values were determined for WCP. Polysaccharide (cellulose, xylan and arabinan), Klason lignin, extractive and ash content were determined based on the standard National Renewable Energy Laboratory protocols [30]. Glucan content refers to total cellulose and starch composition of the substrate. WCP was composed of 49.2% starch and 15.5% cellulose (total glucan 64.7%).

AFEX pretreatment

AFEX pretreatment was carried out as described previously [11]. After charging liquid ammonia into the reactor containing the biomass at the appropriate moisture content, the reactor temperature was raised rapidly to the desired level and held constant for 5 min. Subsequently, ammonia was rapidly released through the exhaust valve. The treated biomass was removed from the reactor and air-dried overnight in a fume hood to remove residual ammonia. AFEX was carried out on CS at different moisture loadings (20 to 200% DWB), temperatures (50°C to 130°C) and ammonia loadings (0.1-3 g ammonia per gram dry weight of biomass). WCP, starch and Avicel samples were pretreated with AFEX at 90°C for 5 min reaction time (total residence time in the reactor after injection of ammonia was ~ 25-30 min), 60% moisture (DWB) and 1:1 (w/w) ammonia to biomass loading.

Enzymatic hydrolysis

AFEX-treated substrates were used without washing with water before hydrolysis. Enzymatic hydrolysis of substrates was carried out based on the National Renewable Energy Laboratory (NREL) protocol [30] at a total volume of 15 ml using screw-capped vials. The substrate was hydrolyzed in 50 mM sodium citrate buffer (pH 4.8) at various enzyme loadings (as mg protein per gram cellulose, starch or xylan). Tetracycline (40 mg/L) and cycloheximide (30 mg/L) were added to prevent microbial growth. Hydrolysis was conducted at 50°C with mild agitation (150 rpm). Sampling was carried out at 12, 24, 72 and 168 h.

High solid loading-based enzymatic hydrolysis

High solid loading hydrolysis was based on 6% glucan (cellulose + starch) loading for each substrate. The pretreated substrate was hydrolyzed in fed-batch mode in two stages (3% glucan loading for each stage) separated by a 24 h time interval. The hydrolysis was carried out in a 2000 ml conical flask (500 ml reaction volume) with 50 mM sodium citrate buffer (pH 4.8) and incubated at 50°C with shaking at 250 rpm. After 24 h, a second batch of solids and appropriate quantity of enzymes were added to the flasks and incubated under identical conditions for an additional 48 h. Tetracycline at 40 mg/L was added to avoid microbial growth during hydrolysis. The hydrolyzates were centrifuged at 10,000 rpm (10, 100 × g) for 30 min, and the supernatants were sterilized by filtration for subsequent ethanol fermentation.

Analytical methods

Separation and quantification of monomeric sugars was conducted using a high performance liquid chromatography (HPLC) machine equipped with an automatic sampler ( LC2010; Shimadzu Scientific Instruments, Columbia, MD, USA) and refractive index detector (Waters RI Detector, 410; Waters Corporation, Milford, MA, USA). For acidic-based hydrolyzates, a HPX-87H Aminex column (Bio-Rad, Hercules, CA, USA) maintained at 65°C using a 5 mM sulfuric acid-based mobile phase (flow rate of 0.6 mL/min) was used for monosaccharide analysis, and a HPX-87P Aminex column maintained at 85°C using water as the mobile phase (0.6 ml/min) was used for analysis of enzymatic hydrolyzates. The concentrations of glucose, xylose and ethanol in the fermentation broths were simultaneously estimated using the HPX-87H column.

Fermentation culture and media

Genetically engineered S. cerevisiae 424A (LNH-ST) was obtained from Dr Nancy Ho (Purdue University, West Lafayette, IN, USA). This strain contains xylose-metabolizing genes integrated into the host chromosome [31]. This strain was cultured routinely in YEPX (1% yeast extract, 2% peptone and 2% xylose) medium at 30°C with shaking at 150 rpm. The culture was maintained on YEPX-agar plates at 4°C for regular use.

Ethanol fermentation

The seed culture was prepared by inoculating YEP-glucose medium with cells from the plate culture followed by incubation at 30°C with agitation at 150 rpm. After 48 h, the cells were harvested by centrifugation. The supernatant was discarded and cells were transferred to 100 ml of fresh fermentation medium in 250 ml Erlenmeyer flasks. The flasks were closed with rubber stoppers pierced with a thin surgical needle to allow release of the carbon dioxide formed during fermentation. The inoculated flasks were incubated at 30°C with agitation (100 rpm) in a temperature-controlled orbital shaker. The culture growth was monitored by measurement of optical density at 600 nm. The initial OD₆₀₀ of all cultures was about 0.1. During fermentation, 1 ml culture samples were removed at regular time intervals and analyzed for glucose, xylose and ethanol. The metabolic ethanol yield (Y_p/s) was calculated as the mass of ethanol produced per unit mass of sugar utilized during fermentation. The theoretical yield of ethanol for glucose or xylose is 0.51 g ethanol per gram sugar. Volumetric ethanol productivity (Q_v) of fermentation was calculated as the amount of ethanol (g/L) produced per unit time (h) of fermentation.