Investigation of wheat cellulosic material as feedstock for bioenergy and biomaterials

Principal Investigators:
Dr. Sajid Alavi, Extrusion Processing, Grain Science and Industry, Kansas State University, Phone: 785-532-2403; salavi@ksu.edu
Dr. Buddhi P. Lamsal, Food Process Enzymologist, Grain Science and Industry, Kansas State University, Phone: 785-532-2875; Fax: 785-532-7010; lamsal@ksu.edu
Dr. Ron Madl, Director, BIVAP program, Grain Science and Industry, Kansas State University, Phone: 785-532-2875; rmadl@ksu.edu
Dr. Jon Faubion, Grain Science and Industry, Kansas State University, Phone: 785-532-5320; jfaubion@ksu.edu

Objectives:
1. To investigate and compare the efficacy of three mechanical pre-treatment processes, namely, size-reduction, extrusion and one proprietary milling technology, as pre-treatment methods for wheat cellulosic materials, namely, bran and wheat straw, to enable sugar recovery.
2. To investigate the feasibility of sugar recovery from mechanically pre-treated wheat bran and wheat straw by action of a combination of cellulosic enzymes.
3. To investigate the effects of extrusion processing, enzymatic hydrolysis and microbial fermentation on enhancement of antioxidant activity and recovery of phenolic compounds of wheat bran.

Procedures:
Objective #1: Grinding, extrusion and a proprietary milling technology are three physical pretreatments that will be evaluated for their efficacy in conditioning the wheat cellulosic materials for sugar release in downstream processing. Wheat bran and the straw for the study will be obtained from collaborators. They will be ground through a Wiley lab-scale roller mill and passed through screens to obtain fractions with at least three particle sizes distribution.
For extrusion treatment, ground wheat cellulosic materials will be fed at a rate of 20 g/min into a lab-scale twin screw extruder (Micro-18, American Leistritz, Somervile, NJ) with a die diameter of 8 mm. Simultaneously, sodium hydroxide (NaOH, 3% w/v) or potassium hydroxide (KOH, 3% w/v) will be injected by pump to the extruder barrel at a rate of 30 ml/min. This will result in a final hull concentration of 40% in the extruder. The alkali solution will serve to hydrate and soften the cellulosic feed and make it extrudable. Three extruder screw speeds (150, 300 and 450 rpm) will be investigated to achieve different levels of specific mechanical energy (SME) input.
For the third pretreatment procedure, wheat cellulosic materials will be ground with a proprietary milling technology into three particle sizes, by varying energy input level and milling time appropriately.
Effect of the above mechanical pretreatments (grinding, extrusion and a proprietary technique) on breaking down the less easily digested crystalline component of cellulose will be studied by using X-ray diffraction. Treated samples will be scanned at 1o/min from 2q = 10-26o with a step size of 0.05o. Crystallinity index of the samples will be determined as described by Laureano-Perez et al., 2005 (1).

Objective #2: Lignocellulosic enzymes cellulase, xylanase, and pectinase and their combinations will be evaluated for sugar release after the pretreatments named above. Enzymatic hydrolysis of the substrate will be at optimal pH and temperature for a given enzyme at three levels of concentration. Required amount of enzyme will be added once the slurry is adjusted to optimal temperature and pH of a given enzyme. Enzyme incubation will be for 1 h with constant shaking/stirring. Extraction of sugar will be done with 1:10 hull: water ratio at temperature of 60 °C and neutral pH. After centrifugation, sugar release in the extract will be determined with a suitable Nelson-Somogy or dinitrosalysilic acid (DNS) procedure. Enzyme-treated wheat cellulosic materials will be looked into for structural damage with scanning electron microscopy (SEM) images and compared with control.

Objective #3: Phenolic acid contents of the extruded wheat cellulosic materials will be determined with a colorimetric method, after extraction with a mixture of 1:1 acetone and methanol. Supecritical fluid extraction (SFE) using carbon dioxide as the solvent will also be evaluated as an alternative method for extracting antioxidant materials. Antioxidant capacity for the extract will be determined along with specific phenolic compounds known for their antioxidant activity, such as caffeic acid and chlorogenic acid (2). Extruded and enzyme-hydrolyzed wheat cellulosics will also be subjected to microbial fermention using lactic acid bacteria and the effect of the same on enhancement of phenolics and anti-oxidants will be investigated.

Justification: High-value commodities like corn and sorghum have been predominant feedstock for starch-based bioenergy (ethanol) or biomaterials. As of May 2006, the US had 97 ethanol plants in operation with a capacity of 4.5 billion gallons per year, utilizing about 1.6 billion bushels of grains (3). Dramatic increase in ethanol production using grain starch-based technology may not be practical, as grain production for ethanol will compete for limited agricultural land needed for food and feed production (4). Lignocellulosic materials from crop residues, agricultural, and industrial by-products are potential sources for low-cost ethanol production (4). One such source of biomass is wheat cellulosic materials. Current predominant usage for wheat bran is as low-value ruminant dietary fiber/protein supplement, and for straw is chopped and mixed back into the field. Straw has found some use in fiberboard preparation.

Extrusion technology has been used to a limited degree in the past as a continuous bio-reactor mechanism for pre-treatment of lignocellulosic material (5). Preliminary work has also been done in the extrusion lab at Kansas State University for pre-treatment of corn-based cellulosic material with encouraging results. However, a systematic study needs to be conducted with wheat cellulosic materials as the raw material for extraction of fermentable sugars, utilizing extrusion and other mechanical pre-treatments in combination with enzymatic treatments.

The proposed study intends to investigate the suitability of mechanical pretreatment techniques to make wheat cellulosic materials amenable to breakdown for higher sugar recovery. Recovered sugar could be utilized as a feedstock for bioethanol or biomaterial, thus adding value. This research will serve to establish a protocol for determining the value of various wheat cellulosic materials when used as feedstock for ethanol production.

In addition to sugar, wheat bran consists of high-value biological components like phenolics. It would be worthwhile to evaluate the antioxidant capacity of wheat bran and to study the effect of microbial fermentation on enhancing the same. Demonstrated presence of such bio-active components with antioxidant capacity and the ability to extract them using a relatively simpler technique like supercritical fluid extraction will add tremendous value to wheat bran.

Project location: The project will be conducted at KSU Grain Science and Industry department. Primary laboratory activities will be carried out using the faculties available in the Department of Grain Science and Industry including the Extrusion Lab, Enzyme Applications Lab, and Analytical lab.

Duration: The proposed study is for one year duration. Follow up work are expected and will be requested in the form of proposal for the coming years.

Progress Report
Period: 2nd Quarter FY08: October 1st to December 31st, 2007

Objectives (for 2nd Quarter):

1. Compare mechanical pre-treatment (extrusion) with chemical pre-treatment (acid hydrolysis) for degradation of wheat bran to achieve improved enzymatic action; and

2. Use cellulase enzyme to achieve enhanced sugar recovery from pre-treated wheat bran.

Progress Report:

In the last quarter (Quarter 1) it was demonstrated that mechanical treatments like grinding, extrusion and sonication could be used to modify the lignocellulosic structure of wheat bran and boost enzymatic hydrolysis for production of generic C6 (glucose) and C5 (xylose and arabinose) sugars. In the present study we compared the efficacy of extrusion processing as a pre-treatment method for wheat bran with acid hydrolysis, which is a traditional chemical method. Results were quantified in terms of total sugar release after enzymatic treatment following the initial mechanical or chemical pre-treatment.

Dilute acid hydrolysis has been extensively studied in the literature as a pre-treatment method and has also been commercialized to a certain extent for production of ethanol from lingnocellulosic feedstocks. It is important to quantify sugar release using acid pre-treatment in order to establish a bench-mark for comparison with any new method, and to establish the relative efficiency and economic feasibility of the latter.

In the last quarter, grinding was established as a prerequisite for any further pre-treatment and also was optimized. Grinding helps in increasing the surface area and reducing the crystallanity of cellulose, thus facilitating better enzymatic action. Thus wheat bran was ground before acid pre-treatment. For extrusion pre-treatment grinding was carried out following extrusion. Two ground controls were also taken one with particles size (PS) <132μm and other with PS >132μm.

Dilute acid Treatment

Dilute acid treatment was carried out with 2% sulfuric acid. Two and half grams of ground wheat bran was suspended in 25ml of 2% H2SO4 for 30 minutes at 60oC. Following acid treatment mixture was centrifuged at 5000g for 10 minutes and supernatant was assayed for reducing sugars using standard DNS assay. Residue obtained after centrifugation was washed with water and neutralized with dilute sodium hydroxide to pH 5. Mixture was re-centrifuged and washings were discarded. Pellet obtained was suspended in 25ml sodium acetate pH 5 buffer to achieve solid to liquid loading of 1:10 and cellulase enzyme @ 5% (w/w) on dry substrate basis was added to the mixture. Cellulase enzyme used was Cellulase 5000 from Specialty Biochemicals (Chino, CA). Suspension was shaken for 1.5 hours at 50oC. Enzymatic reaction was terminated by boiling for 10 minutes. After enzymatic treatment suspension was centrifuged and supernatant was analyzed for total reducing sugars using DNS assay. Total sugars were reported in glucose equivalents as milli-moles per liter. Acid treatment was carried out on both particle sizes PS<132μm and PS>132μm respectively.

Extrusion

Part-1

Optimization of extrusion conditions for wheat bran

Wheat bran was hydrated to 25% moisture and kept overnight under refrigeration conditions for proper hydration and equilibration before extrusion. Hydrated bran was processed on a lab-scale twin screw extruder at different screw speeds (RPM) and various temperatures in die zone using a lab-scale twin screw extruder. Following combinations were studied 220 RPM and 110oC; 420 RPM and 110oC; 320 RPM and 130oC; 220 RPM and 150oC; 420 RPM and 150oC. The extruder screws were configured to have a constantly decreasing pitch, and two kneading blocks and a reverse screw element were used to enhance the shear on the material. Following extrusion, wheat bran was dried at 45oC overnight in a forced draft oven to an approximate moisture level of 4.5%. Dried extruded wheat bran was ground to finer particle size. The ground samples were treated with Cellulase 5000 enzyme (Specialty Biochemicals, Chino, CA) @ 5% (w/w) on dry substrate basis in pH 5 water. The incubation time with enzyme was 30 minutes and solid to liquid loading was 1:15. Supernatant from enzymatic hydrolysis was analyzed for total reducing sugars using standard DNS assay. Non-enzymatic samples were also included for comparison.

Comparison of extrusion with acid pre-treatment

Following extrusion optimization for wheat bran, sampled from two better performing conditions were quantified in terms of sugar yield were selected. Here extrusion conditions 220 RPM and 110oC; 420 RPM and 150oC respectively were chosen for comparison with acid hydrolysis. After extrusion at chosen conditions, wheat bran was dried at 45oC overnight in a forced draft oven to an approximate moisture level of 4.5%. Dried extruded wheat bran was ground to finer particle size.

Two and half grams of ground samples were suspended in 25ml sodium acetate pH 5 buffer to achieve solid to liquid loading of 1:10 and cellulase enzyme @ 5% (w/w) on dry substrate basis was added to the mixture. Cellulase enzyme used was Cellulase 5000 from Specialty Biochemicals (Chino, CA). Suspension was shaken for 1.5 hours at 50oC. Enzymatic reaction was terminated by boiling for 10 minutes. After enzymatic treatment suspension was centrifuged and supernatant was analyzed for total reducing sugars using DNS assay. Total sugars were reported in glucose equivalents as milli-moles per liter.

Results

Extrusion improved structural modification of wheat bran. Extruded, ground and enzyme treated wheat bran had higher sugar release than ground and enzyme treated wheat bran (Fig 1). For obvious reasons enzyme addition boosted sugar recovery and total sugars were higher when enzyme was included both in control and extruded wheat bran (Fig 1). Particularly two combinations 220 RPM and 110oC; 420 RPM and 150oC worked well. Therefore, they were included for comparison with acid treatment.

Both extruded and enzyme treated (420 RPM@150oC and 220 RPM@110oC), and acid and enzyme treated wheat bran performed better than two ground and enzyme treated controls PS<132μm C and PS>132μm C (Fig 2). Extrusion was at par with acid treated wheat bran (Fig 2).

Future studies

X-ray diffraction studies for detecting structural changes in cellulose (loss in crystallinity) due to various pre-treatment methods

Further optimization of extrusion pre-treatment.

Optimization of enzymatic treatment to study combinations of enzyme (like xylanase) with cellulase.

Study acid hydrolysis in combination with extrusion pre-treatment.

Simulate the high temperature acid hydrolysis conditions typically used in industry.

Investigate anti-oxidant recovery from wheat bran.

Study the profile of the sugars released by various pre-treatment methods (C5 versus C6).