A mild thermomechanical process for the enzymatic conversion of radiata pine into fermentable sugars and lignin
- Ian D. Suckling1Email author,
- Michael W. Jack1, 2,
- John A. Lloyd1,
- Karl D. Murton1,
- Roger H. Newman^1,
- Trevor R. Stuthridge1, 3,
- Kirk M. Torr1 and
- Alankar A. Vaidya1
© The Author(s) 2017
Received: 4 October 2016
Accepted: 1 March 2017
Published: 9 March 2017
Conversion of softwoods into sustainable fuels and chemicals is important for parts of the world where softwoods are the dominant forest species. While they have high theoretical sugar yields, softwoods are amongst the most recalcitrant feedstocks for enzymatic processes, typically requiring both more severe pretreatment conditions and higher enzyme doses than needed for other lignocellulosic feedstocks. Although a number of processes have been proposed for converting softwoods into sugars suitable for fuel and chemical production, there is still a need for a high-yielding, industrially scalable and cost-effective conversion route.
We summarise work leading to the development of an efficient process for the enzymatic conversion of radiata pine (Pinus radiata) into wood sugars. The process involves initial pressurised steaming of wood chips under relatively mild conditions (173 °C for 3–72 min) without added acid catalyst. The steamed chips then pass through a compression screw to squeeze out a pressate rich in solubilised hemicelluloses. The pressed chips are disc-refined and wet ball-milled to produce a substrate which is rapidly saccharified using commercially available enzyme cocktails. Adding 0.1% polyethylene glycol during saccharification was found to be particularly effective with these substrates, reducing enzyme usage to acceptable levels, e.g. 5 FPU/g OD substrate. The pressate is separately hydrolysed using acid, providing additional hemicellulose-derived sugars, for an overall sugar yield of 535 kg/ODT chips (76% of theoretical). The total pretreatment energy input is comparable to other processes, with the additional energy for attrition being balanced by a lower thermal energy requirement. This pretreatment strategy produces substrates with low levels of fermentation inhibitors, so the glucose-rich mainline and pressate syrups can be fermented to ethanol without detoxification. The lignin from the process remains comparatively unmodified, as evident from the level of retained β-ether interunit linkages, providing an opportunity for conversion into saleable co-products.
This process is an efficient route for the enzymatic conversion of radiata pine, and potentially other softwoods, into a sugar syrup suitable for conversion into fuels and chemicals. Furthermore, the process uses standard equipment that is largely proven at commercial scale, de-risking process scale-up.
KeywordsPine Ball-milling Biofuels Softwood Enzymatic conversion Galactoglucomannans Sugar yield Energy
Advanced biofuels derived from lignocellulosic biomass, composed of cellulose, hemicellulose and lignin, are seen as a key to the future growth of biofuels. They are not derived from food crops and promise to be more sustainable, offering greater reductions in greenhouse gas emissions compared to conventional biofuels . Potential lignocellulosic feedstocks include wood and wood residues, agricultural residues such as corn stover or sugarcane bagasse and dedicated energy crops such as miscanthus or energy cane.
One of the most promising approaches to the production of lignocellulosic biofuels involves using enzymes to hydrolyse the carbohydrate polymers in the substrate to monomeric sugars and then fermenting these sugars to ethanol . Critical to high sugar yields during enzymatic hydrolysis is an effective pretreatment to disrupt and/or remove the lignin and hemicelluloses encasing the cellulose microfibrils and make the cellulose more accessible to the enzymes [3–9].
Heating lignocellulosic biomass in water, or directly with steam, is one of the simplest and most effective pretreatments. There are many variants on this basic approach, with most hydrothermal pretreatments involving heating the biomass to temperatures of between 160 and 230 °C, often in the presence of acid catalysts [3–5, 10]. However, hydrothermal pretreatments suffer from a number of disadvantages. Firstly, under the acid conditions, the hemicelluloses may be hydrolysed and degraded to produce furans and acetic acid, which inhibit subsequent fermentation stages , and into pseudo-lignin, which can deposit on cellulose surfaces and retard enzymatic hydrolysis . Furthermore, the lignin in the biomass can be modified in the pretreatment process to produce compounds which inhibit subsequent saccharification or fermentation [11, 13, 14] and lignin can also be relocalised within the cell wall to negatively impact cellulose hydrolysis [15–17].
Mechanical milling processes such as ball-milling can also be used to improve the enzymatic hydrolysis of lignocellulosic materials by increasing the surface area of the cellulose. However, the amount of energy required is normally considered to be prohibitively high . Nevertheless, a number of researchers have suggested that refining or ball-milling at moderate energy inputs can be beneficially applied to increase digestibility after hydrothermal and chemical pretreatments [19–25].
Softwoods, such as Pinus radiata, pose particular challenges in enzymatic processes. Firstly, softwoods are amongst the most recalcitrant lignocellulosic substrates in enzymatic processes, typically requiring both more severe pretreatment conditions and higher enzyme doses than hardwoods or agricultural residues [6, 26, 27]. Secondly, galactoglucomannans (GGMs) are the dominant hemicellulose sugars in softwoods, whereas xylans are the main hemicelluloses in hardwoods and agricultural residues . With GGMs making up 15–20% of the wood mass in softwoods, efficient conversion of this polymer to its constituent C6 sugars is critical for good overall yields. While a number of pretreatments have been investigated for softwoods , including processes based on steam explosion [29–31], single- and two-stage acid treatments [32, 33], sulphite treatments [34, 35], organosolv pulping  and alkaline pulping [20, 37], there is still a need for a high-yielding, industrially scalable and cost-effective pretreatment for these substrates.
We describe here a new efficient process for the conversion of softwoods into monomeric sugars in high yields. This process, developed in a programme of work, involves a novel combination of known steps combined and operated in a specific way, affording high yields of fermentable monomeric sugars using reasonable doses of current commercial enzyme cocktails. Specifically here we describe the overall process, including its rationale and overall performance, with recent [38, 39] and future publications providing more detail on the specific steps within the process.
Results and discussion
To increase the overall monomeric sugar yields, a compression screw is incorporated between the steaming and disc refining stages to squeeze out a pressate rich in solubilised hemicelluloses. The resulting pressate is hydrolysed with dilute acid in a subsequent step to afford a pressate syrup containing mainly C6 hemicellulose-derived monomeric sugars. In this way, the GGMs can be converted to the constituent monomeric sugars without requiring an enzyme cocktail containing the enzymes needed to fully degrade these hemicelluloses.
Each of these steps is then discussed in more detail in subsequent sections, illustrated with data from two steaming conditions.
Thermomechanical pretreatment was carried out at a pilot scale in equipment commonly used during the production of wood fibre for newsprint and medium density fibreboard . Fresh P. radiata chips were first softened by atmospheric steaming at 80 °C for 5 min and then passed through a compression screw. This squeezed out a small amount of material, ca. 0.5% on OD chips, which was discarded as waste, as it contains low levels of sugars. It is however rich in wood extractives, which could potentially be isolated as a saleable co-product.
The compressed chips were then steamed at 7.5 bar (173 ± 2 °C) and passed through a second compression screw to afford a concentrated pressate rich in hemicellulose sugars, plus a solid residue. We have recently reported that when the steaming time at 173 °C is increased from 3 to 144 min, a greater proportion of the hemicelluloses are solubilised and removed into the pressate . Notably, these steaming conditions are mild relative to those commonly employed during other dilute acid treatments and steam explosion treatments. For example, steaming at 173 °C for 72 min corresponds to combined severity factor  of 0.57 versus 1.4–5.4 for steam pretreatment of softwoods in the presence of added acid catalysts .
The solid residue was disc-refined under pressure to produce a pulp containing largely individual fibres. Under these conditions, the fibres separate at the lignin-rich middle lamella layer, as the temperature exceeds the glass transition temperature of lignin . The nominal refining energy here is 300 kWh/ODT. However, refining energies during similar commercial processes, e.g. medium density fibreboard production, are considerably lower than required in our pilot plant due to the larger scale and optimised plate design, typically ~120 kWh/ODT (0.43 GJ/ODT) .
Mass and component balances for trials using 3- and 72-min steaming
Combined severity factor
Wood or fibre, kg/ODT wood
Mass, kg/ODT wood
Total carbohydrates, kg/ODT wood
Ball-milled fibre digestibilityd, % of glucosyl residues
Monomeric sugars, kg/ODT initial woode
The pressate from steaming for 72 min contained high concentrations, averaging 127 g/L, of hemicellulose sugars, making it particularly suitable for subsequent processing (Table 1). This is because steaming is carried out at high solids loading using direct steam heating, so only a low amount of pressate is produced, ca. 1.3 kg/kg OD chips entering the process.
The hemicellulose sugars are largely present in the pressate as soluble oligomers, with the concentrations increasing as the steaming time is increased (Table 1). In the pressate after 72-min steaming, the galactan:glucan:mannan ratio was 0.7:1:3.0, consistent with removal of GGMs from the wood and little cellulose dissolution. Softwoods are believed to contain two different GGMs, the galactan-rich having a galactan:glucan:mannan ratio of 1:1:3 and the other having a ratio of 0.1–0.2:1:3–4 , so our results suggest preferential dissolution of the galactan-rich GGM. Smaller amounts of arabinoxylans were also removed, but in this case largely as the monomeric sugars.
For pressates produced from 72-min steaming, levels of the fermentation inhibitors acetic acid (2.5–4.4 g/L) and furans (1.5–3.3 g/L furfural + hydroxymethylfurfural) are sufficiently low that detoxification is not required prior to fermentation (see below). By comparison, the liquid from the reference SEW treatment contained 7.1 g/L of acetic acid and 4.0 g/L of furfural plus hydroxymethylfurfural.
The benefits of wet ball-milling have been observed using a number of different types of ball-mill, including both steel and ceramic vibratory mills, a 105 L ceramic tumbling ball-mill, a vertical stirred ball-mill and a vibratory rod mill (data not shown). While low or high consistency refining or treatment in a SupermassColloider  did enhance the digestibility of the fibre, all were considerably less effective than wet ball-milling when compared at the same energy level (data not shown).
Wet ball-milling using ceramic mills affords a substrate containing some ash (≤6%), as a result of a loss of material from the ceramic balls during milling. The ash content varies depending on the substrate, ceramic ball-milling device used and milling time. All subsequent saccharification results have been corrected for ash in the ball-milled material, as ceramic mills are unlikely to be used on a commercial scale.
Calculated energy inputs during pretreatment of softwoods
While the solubilised glucosyl and xylosyl units were completely converted to monomers after 24 h enzyme treatment, conversion of the solubilised mannosyl units to mannose (and galactosyl units to galactose, data not shown) was <30% complete (Fig. 7). Our results suggest that the Celluclast/Novo 188 cocktail contains some β-mannanase activity needed to convert the GGM to soluble oligosaccharides, but lacks sufficient β-mannosidase or α-galactosidase activity to completely hydrolyse these oligomers to mannose and galactose. It has been reported that some non-specific cellulases, particularly endoglucanases, have significant β-mannanase side activities towards mannans .
Impact of new cocktails and PEG
Overall sugar yields
Total monomeric sugar yields of up to 76% of theoretical are obtained in the process, rising to 83% if the soluble oligomers are included. The latter are mainly as mannosyl residues remaining in the sugar syrup after enzymatic saccharification. This yield is comparable to reported total sugar yields from a range of other acid-catalysed processes for softwoods  and recent yields of up to 86% from loblolly pine by bisulfite pulping  and 84% from lodgepole pine by the SPORL process . There is also an opportunity to further increase the overall yield by applying higher enzyme doses (c.f. Fig. 8), but this would need to be balanced against the additional cost of the enzyme.
Fermentation to ethanol
Fermentation of the mannose-rich pressate syrup at a concentration of 58 g/L fermentable sugars using S. cerevisiae D5A gave ethanol in a yield of 76% based on the level of fermentable sugars in the pressate (Fig. 10b). Mannose was 87% utilised. The lower sugar utilisation and yields can likely be attributed to partial inhibition by the higher level of acetate in this pressate syrup (see above). In a parallel fermentation using pure mannose as a substrate, the mannose was completely consumed.
Our process provides an efficient process for the enzymatic conversion of radiata pine, and potentially other softwoods, into a sugar syrup suitable for conversion into fuels and chemicals. The mild thermal pretreatment coupled with wet ball-milling produces only low levels of fermentation inhibitors, meaning that the resulting sugars can be easily fermented to ethanol. In addition, the lignin from the process remains comparatively unmodified, providing an opportunity for conversion into saleable co-products. Furthermore, the process uses standard equipment that is largely proven at commercial scale, reducing risks during commercialisation.
Enzymes were obtained from Novozymes A/S (Bagsvaerd, Denmark). Filter paper activity units (FPU) were determined according to the IUPAC method and the β-glucosidase activity using p-nitrophenyl-β-glucopyranoside as a substrate . Fresh radiata pine (P. radiata) wood chips, as produced for use in pulp mills, were obtained from a local sawmill. All other chemicals, including PEG-4000 (MW 4000), were purchased from Sigma-Aldrich (Milwaukee, USA) and used as received.
The fibre and pressate samples prepared via trials were performed in the Scion fibre processing pilot plant . This plant operates in a fully continuous mode, with each trial processing approximately 2 m3 of chips (~350 OD kg) producing fibre at a flow rate of approximately 17 OD kg/min. Thus, fresh chips were steamed at 80 °C for 5 min in a 2.5 m3 steaming vessel and then transferred via a chip compression screw into the 3 m3 pressurised steaming vessel maintained at 173 °C (750 kPa) by direct steam injection for 3 or 72 min. The steamed chips were then fed via another chip compression screw (3:1 compression ratio) into the single disc pressurised (650 kPa) refiner (Jylhavaara SD 52/36, 900 mm, 1250 kW) and refined using approximately 300 kWh/ODT energy. The oven dry content of the refined substrates was 50–60%.
The pressates from the two chip compression screw were collected as a single bulked sample for each run, weighed and their solids contents were determined. Mass balances for each trial were calculated on an OD basis from the mass of pulp and pressate solids collected and assuming a pressate density of 1 kg/L and that the mass of input wood chips equals the mass of outputs collected, i.e. no losses occur or volatile compounds are produced through the process.
To produce the “no steaming” fibre, the chips were passed straight through the plant without any steam being applied and were then refined at 0.5 bar inlet pressure and a nominal energy input of 300 kWh/ODT fibre.
Preparation of steam-exploded wood
Pinus radiata wood chips were steam-exploded following the procedure of Clark and Mackie  using the optimum conditions they identified for this species. Briefly, a sample of fresh chips (0.75 OD kg) was impregnated with SO2 (3% w/w) and heated with steam in a 3 L steam gun at 215 °C for 3 min before being rapidly discharged into a cyclone. The resulting solid was washed four times with deionised water to obtain a 54% yield of water-insoluble substrate.
Vibratory ball-milling was carried out for the required time in 1-L porcelain pots on a Schwingmühle VIBRATOM vibratory ball-mill loaded with two hundred 15-mm-diameter alumina balls (ca 1350 g), never-dried pulp (6 OD g) and sufficient 0.01% w/v aqueous solution of sodium azide to give solids content of 4.8%. The slurry of milled solids was transferred from the pots with the aid of additional water and stored at 4 °C.
Tumble ball-milling was performed in a porcelain-lined 105 L tumbling ball-mill equipped with 20-mm-diameter alumina balls. This was loaded with never-dried pulp (1 OD kg) plus water to bring the solids content to 6% and then sealed and rotated for the required length of time. The slurry of milled solids was removed from the mill and stored at 4 °C after the addition of 0.01% w/v sodium azide (unless required for fermentation trials). For a milling time of 180 min, the energy consumption was 1.96 Wh per OD kg fibre as determined by a Metec DVH3113 energy transducer.
Sulphuric acid (24% w/w, 21 mL) was added to the pressate (479 mL, centrifuged and filtered through a 0.45-µm cellulose acetate filter) and heated at 121°C for 1 h in a large laboratory autoclave. The resulting solution was cooled and adjusted to pH 3 by addition of 33% w/w aqueous ammonia (11.3 mL) to give a hydrolysed pressate (456 mL), which was then frozen, freeze-dried or stabilised with 0.01% sodium azide prior to further analysis.
Enzymatic hydrolysis was performed in duplicate on a 5-mL scale at 50 °C using 0.05 M sodium citrate buffer (pH 4.8) containing 0.01% sodium azide at a substrate concentration of 1.5% on a dry basis in 20 mL screw-capped glass tubes. The enzyme cocktails that used either a mixture of Celluclast 1.5L supplemented with β-glucosidase (Novozyme 188 at a ratio of 1 FPU: 1.25 IU β-glucosidase) or Cellic CTec2 were added and the tubes agitated at 180 rpm for the required time in an inclined vibratory shaker. If required, PEG-4000 (0.1% w/v) was also added. The reaction was stopped by plunging the tubes into a boiling water bath for 5 min and then cooling to room temperature. The mixture was then centrifuged at 4000 rpm for 10 min at 25 °C and the concentration of glucose in the supernatant was determined using an YSI-2700 glucose analyser (YSI Incorporated). All results are expressed as anhydroglucose units and are corrected for glucose present in a control treatment carried out as described above, but using denatured enzymes.
The total lignin content was determined on extracted samples as the sum of Klason plus acid-soluble lignins following standard methods (Tappi standard T222 om-88 1988; Tappi standard UM-250 1991) scaled down to analyse 0.25 g wood. Extractives were removed by extraction of the ground samples with dichloromethane in a Soxtec apparatus (Tecator Soxtec System Model HT1043) using a boiling time of 1 h and a rinsing time of 1 h. Monomeric sugars in the filtrates from Klason lignin determinations were analysed by ion chromatography  and the results were expressed as the corresponding anhydrosugar units (glucosyl, xylosyl etc.). Carbohydrates in pressates were similarly analysed in duplicate before (monomeric sugars) and after (total sugars) hydrolysis in 4% sulphuric acid at 121 °C in an autoclave for 60 min. All biomass and liquor analyses were performed in duplicate, with either the replicates shown, or the mean and deviation from mean reported.
Ash in solid samples was determined by ashing at 525 °C following Tappi standard T211 om-93.
Furans and volatile fatty acids in the filtered pressates were determined in duplicate by ion chromatography using an Aminex HPX-87H column following the method of Sluiter et al. .
Releasable β-ethers in in situ lignin were determined in duplicate by thioacidolysis and analysis of silylated monomeric thioacidolysis products by gas chromatography/mass spectroscopy following the method of Pasco and Suckling .
Uncondensed phenylpropane lignins in the in situ fibre lignins were determined in duplicate by nitrobenzene oxidation following the method of Chen .
Samples for field emission scanning electron microscopy were washed, centrifuged and freeze-dried. The freeze-dried pellets were split open to reveal an internal surface and a portion of this surface was mounted on a carbon-adhesive tab on an aluminium sample holder and sputter-coated with chromium. Samples were examined at an accelerating voltage of 3–5 kV on a JEOL 6700F instrument.
Fermentation experiments were performed in duplicate at the National Renewable Energy Laboratory, using both Zymomonas mobilis strain 8b (a glucose + xylose co-metabolising strain) and Saccharomyces cerevisiae D5A (preferentially glucose metabolising strain). The sugar syrups used were produced from chips steamed for 72 min and large-scale pressate hydrolysis or saccharification of pulp tumble ball-milled for 300 min using Cellic CTec2 (20 FPU/OD g substrate) for 48 h at 50 °C and 5% solids loading. The freeze-dried syrup powders were rehydrated into the appropriate concentrations: 150 g/L fermentable sugars (glucose + xylose for Z. mobilis 8b and glucose + mannose for S. cerevisiae D5A) for the mainline sugar syrup and 58.4 g/L fermentable sugars for the pressate syrup. Fermentations of the sugar syrup by Z. mobilis 8b and the hydrolysed pressate using S. cerevisiae D5A were accompanied by pure sugar fermentations as controls with matching sugar concentrations and nutrients (data not shown).
The inoculum was prepared by adding 1.0 mL of thawed cell suspension from a cryovial into 9 mL of fermentation medium. The fermentation medium for Z. mobilis 8b was 10 g/L yeast extract and 2 g/L KH2PO4 supplemented with 100 g/L glucose and 20 g/L xylose. For S. cerevisiae D5A, an identical medium was used, except KH2PO4 was replaced with 20 g/L peptone and xylose was replaced with an equal concentration of mannose. The inoculum was incubated at 33 °C (Z. mobilis 8b) or 37 °C (S. cerevisiae D5A) for 8 h and then the optical density was measured at 600 nm.
Once the optical density of the inoculum reached 0.01 units, it was transferred to the Biostat Q-Plus fermenter containing 300 mL of corresponding fermentation medium (in the case of Z. mobilis 8b, the xylose concentration in the medium was increased to 20 g/L) supplemented with 1 g/L sorbitol as an internal standard. For the Z. mobilis 8b, fermentation was performed at 33 °C and 300 rpm with the pH controlled at 5.75 using 4 M KOH. The fermentation continued until the OD600nm reached approximately 10 units (about 17 h). The S. cerevisiae D5A fermentation was performed at 37 °C and 300 rpm and pH was controlled at 5.10 using 4 M NaOH. The fermentation continued until the OD600nm reached approximately 15 units (about 18 h). Then cultures were transferred to the sugar syrup or pure sugar solutions at a volume ratio of 1:9 and fermentation continued for 30 h. Both fermentations were performed in duplicate.
Ethanol concentrations were monitored by HPLC using a BioRad HPX-87H organic acid column and sugar concentrations were measured by HPLC using a Shodex SP0810 carbohydrate column . Because mannose and arabinose co-elute using this column, arabinose concentrations were determined by ion chromatography using a Dionex PA1 column  and used to calculate the mannose concentrations.
cellobiase activity units
filter paper activity units
oven dry metric tonnes
- OD600nm :
optical density at 600 nm
polyethylene glycol 4000
- SO2 :
Steam exploded wood
sulphite pretreatment to overcome recalcitrance of lignocellulose
All authors (MJ, JL, KM, RN, TS, IS, KT and AV) contributed via a project lead team to the project conception, experimental design, interpretation of the results and the preparation of this manuscript. IS led and coordinated the overall project, and drafting of this manuscript. JL also had specific responsibility for the ball-milling, KM for the thermomechanical pulping, KT for the lignin analysis and AV for the enzymatic saccharification. All authors except RN (deceased) have read and approved the final manuscript.
We want to thank Andrew Lowell, Robert Nelson and Nancy Farmer from the National Renewable Energy Laboratory in Golden, CO for the fermentation study and analyses, Lloyd Donaldson (Scion) for the SEM analyses, and the expert technical assistance from Claire Armstrong, Sylke Campion, Sara Carey, Gavin Durbin, Pat Gray, Sunita Jeram, Karen Love, Katrina Martin, Bernadette Nanayakkara, Maxine Smith and Garth Weinberg (all Scion).
The authors declare that they have no competing interests.
Availability of data and materials
No supporting information has been deposited in publicly available repositories.
This research was supported by the New Zealand Ministry of Business Innovation and Employment via Scion Core funding.
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