Saccharification of recalcitrant biomass and integration options for lignocellulosic sugars from Catchlight Energy’s sugar process (CLE Sugar)

Biomass feedstocks

The wood chips were re-chipped with a Bearcat garden chipper with a 1.91 cm screen to obtain smaller size chips. 3-mm round-hole fines were removed to avoid circulation problems in a 28.3-liter pilot pretreatment reactor. The resulting wood chip size distribution for the softwood chips was 24% “pin size” chips (passes a 7 mm round hole screen, retained on a 3 mm round hole screen), and 76% “accept size” chips (retained on a 7 mm round hole screen). All softwood chips were less than 8 mm thickness. The lengths of the re-chipped chips ranged approximately from 20 to 40 mm.

Pretreatment

Enzymatic hydrolysis and total sugar yield

Biomass feedstocks

The softwood feedstock was forest chips of Loblolly pine, so they contained bark. Hardwood chips used in this test were debarked maple chips from Michigan. Both the un-debarked Loblolly pine chips and debarked maple chips were re-chipped with a Bearcat garden chipper with a 1.91 cm screen to obtain smaller chips, and 3-mm round-hole fines were removed. Switchgrass biomass is Alamo variety harvested in winter from a Catchlight Energy field trial in the southeastern United States.

Acidic calcium bisulfite reagent

An acidic calcium bisulfite solution was used as a sulfonation reagent in the pretreatment of biomass. Calcium bisulfite was produced by constantly purging pure sulfur dioxide into a calcium oxide solution. The final calcium bisulfite concentration contained about 2–4% total sulfur dioxide, of which about 1% was free sulfur dioxide. The pH of this calcium bisulfite solution was about 1.4.

The total sulfur in the reagent was determined by a standard ASTM D 1552–90 method of sulfur in petroleum products (high temperature method) in Leco S632 Sulfur Determinator (St. Joseph, Michigan). The sulfur result was used to calculate the total equivalent sulfur dioxide (bound and free). The calcium in the liquor was measured by a standard method equivalent to EPA Method 200.8, revision 5.4, of EPA/600/R-94/111. The calcium in the liquor was analyzed by an inductively coupled argon plasma mass spectrometry (ICPMS) (Thermo Elemental X Series II, Waltham, MA). The bound sulfur dioxide in the calcium bisulfite was determined by the total amount of soluble calcium in the liquor, and the free sulfur dioxide was determined by subtracting the bound sulfur dioxide from the total sulfur dioxide.

Pilot pretreatment digester

A 28.3-liter (one cubic foot) digester with a heat exchange was used in biomass pretreatment with an acidic calcium bisulfite reagent. The digester and the heat exchanger are made of SA-515 Gr 70 alloy and the digester head is made of SA-515 Gr C alloy. The piping for calcium bisulfite reagent circulation during the pretreatment is made up of 304 stainless. During pretreatment, steam indirectly heats up the cooking liquor that circulates back to the digester.

Sulfonation conditions

The re-chipped biomass materials were sulfonated in the pilot pretreatment reactor. In each cook, 3.0 kilograms (oven dried - OD basis) of woody biomass material were used at a biomass-to-liquor ratio of about 4.0, and 2.2 kilograms (oven dried basis) of switchgrass was used at a biomass-to-liquor ratio of about 4.5.

The biomass materials were first steamed to 90°C to remove air in the void space of the biomass. After the condensation was drained, the woody biomass was charged with a cool liquor of calcium bisulfite at 12.5% on wood. The pre-steamed un-debarked Loblolly chips were heated up to 165°C in 15 minutes and held at 165°C for 75 minutes. The pre-steamed debarked maple chips were heated up to 155°C in 15 minutes and held at 155°C for 75 to 120 minutes. Similarly, the pre-steamed switchgrass was heated up to 155°C in 15 minutes and held at 155°C for 75 minutes. The wet cooked biomass and the prehydrolysate amounts were measured for mass balance calculation. After cooking, a prehydrolysate “spent liquor” fraction was drained and the cooked chips were collected after having relieved the pressure in the pretreatment reactor by draining the spent liquor and venting. The cooked biomass solid content was measured by drying a biomass sample of about 70 wet grams in an oven set at 105°C for overnight. This solids content was used to determine the total recovered solid biomass after the pretreatment.

The cooked chips were very mushy. The cooked wood chips were passed once through an Alpine grinder, without adding any water, to form a pulp-like material. The cooked switchgrass was not refined due to its pulpy status upon removal from the pretreatment reactor.

Biomass Analysis Methods

The carbohydrate analysis in biomass feedstocks and in pretreated biomass samples was determined by hydrolyzing 100 mg of refined materials with 72% sulfuric acid at 127°C for 60 minutes for a complete carbohydrate hydrolysis. The monomeric sugars from completely acid-hydrolyzed biomass were analyzed quantitatively in the dilute sample for glucose, xylose, mannose, arabinose and galactose in a Dionex ion exchange chromatography. The Dionex chromatography is equipped with a CarboPac PA1 (Dionex P/N 035391) 4 mm × 250 mm ion-exchange column and a Dionex ED 40 pulsed amperometric detector with gold working electrode and solid state reference electrode. The monomeric sugar results were used for the carbohydrate composition calculation in a biomass sample or a pretreated biomass sample.

All biomass carbohydrates were completed in single analysis with duplicate tests of a known composition control (e.g. debarked and clean Southern Loblolly pine chip sample) to assure the accuracy of the analysis in Weyerhaeuser’s analytical group.

In this study, lignin, uronic acids, acetyl groups, extractives and ash were not quantitatively studied.

Enzymatic hydrolysis

For enzymatic hydrolysis, a pre-mixed enzyme cocktail was formulated with a cellulase product (Sigma Cat. No. C2730) at 99.5 mg protein/mL, a beta-glucosidase product (Sigma Cat. No. C6105) at 42.5 mg protein/mL, and a xylanase (Sigma Cat. No. X2753) at 3.4 mg protein/mL. The total mixed Sigma enzyme protein titer was 145.5 mg/mL. The Sigma enzyme cocktail has a density of 1.1 g/ml. Cellic® CTec2 enzyme product was provided by Novozymes and was used in the high consistency pretreated biomass hydrolysis. The CTec2 enzyme product has a density of 1.2 g/ml. In the enzymatic hydrolysis, the enzyme dosage is defined as gram of enzyme product per oven dry (OD) gram of pretreated biomass. Unwashed pretreated biomass was used in all the enzymatic hydrolysis tests. The enzymatic hydrolysis was conducted in screw-capped 50-mL volume in 125 mL Erlenmeyer flasks in an orbital shaking incubator, controlled at 50°C and 200 r.p.m. The hydrolysis pH was controlled at pH 4.8 by a 50 mmol sodium citrate buffer.

Sugar analysis

One milliliter hydrolysis sample was weighed and diluted into a total volume of 10 mL in deionized water. The sample was then centrifuged and the supernatant was used for sugar analysis in a HPLC. A Shimadzu HPLC equipped with a 300 × 7.8 mm Bio-Rad Aminex HPX-87P Column (Cat. No. 125–0098) was used to analyze glucose, xylose, mannose, arabinose and galactose in the hydrolysate from an enzymatic hydrolysis and in the prehydrolysate from a cook. The 87P column was run with water as an eluent at 0.6 ml/min at 85°C. The acetic acid in the prehydrolysate was analyzed in a 300 × 7.8 mm Bio-Rad Aminex HPX-87H Column (Cat. No. 125–0140) with 0.005M sulfuric acid as an eluent at 0.6 ml/min at 65°C. Furfural and hydroxymethylfurfural were analyzed in either the HPX-87P or the HPX-87H column.