Process overview

Our process is illustrated in Fig. 1. It involves a two-stage pretreatment, composed of a mild thermomechanical stage without added acid catalyst, combined with mechanical attrition using a ball-mill. The thermomechanical stage can be carried out in equipment similar to that used commercially for the production of fibre for medium density fibreboard and produces a solid residue largely as individual fibres. While the fibres themselves are not responsive to enzymatic hydrolysis, attrition by wet ball-milling yields a digestible substrate. Treatment of this ball-milled substrate with commercially available enzyme cocktails affords a mainline sugar syrup containing the monomeric sugars, plus a solid residue containing mainly lignin and unhydrolysed carbohydrates. The solid residue can either be used to isolate the lignin for downstream processing, or be burnt as source of process energy.

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

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 [40]. 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 [38]. 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 [41] of 0.57 versus 1.4–5.4 for steam pretreatment of softwoods in the presence of added acid catalysts [42].

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 [43]. 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) [44].

The lignin residue remaining after the enzyme-based process for converting lignocellulosic substrates to sugars is a potential future feedstock for conversion into a wide variety of sustainable products [45, 46]. Under the acidic conditions present during hydrothermal treatment lignin can be both partially depolymerised via cleavage of β-O-4 ether interunit linkages and repolymerised via condensation between C-α and the aromatic ring of adjacent phenylpropane units [47–49]. We therefore investigated the extent to which the lignin was being modified in our process. Analysis of the levels of β-O-4 ether interunit linkages in the pretreated substrates by thioacidolysis [50] showed that the level of β-ethers remained essentially unchanged in the fibre after steaming for 3 min, and decreased by only ~30% after 72-min steaming (Fig. 2). In contrast, the lignin in a reference steam-exploded wood (SEW) prepared from P. radiata wood using the conditions identified for this substrate by Clark and Mackie [29] contained no detectable β-O-4 ethers. Furthermore, analysis of the lignin by nitrobenzene oxidation [51] showed that uncondensed phenylpropane units decreased by ~30% after 72-min steaming, compared to a >80% reduction in the reference SEW substrate. These results show that, relative to the reference steam explosion pretreatment, the lignins from this process have undergone only limited modification during steaming.

Hemicellulose-rich pressate

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 [28], 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.

Hydrolysis of the soluble oligomers in the pressate to the constituent monomers, required for many end-use applications such as fermentation to ethanol, can be accomplished by heating the pressate at 121 °C for 60 min in the presence of 1% w/w sulphuric acid (Fig. 3). More severe hydrolysis conditions could be used to ensure hydrolysis of the 8% remaining oligomers. However, this must be balanced against the greater pentose sugar degradation under these conditions [52] and higher costs due to higher temperatures, longer times and additional chemicals for hydrolysis and neutralisation.

Ball-milling

Wet ball-milling in a vibratory mill at 5% solids content dramatically increased the digestibility of the steamed fibre substrates [38]. Figure 4 shows that both the responsiveness of the fibre to ball-milling and the eventual extent of conversion to glucose after ball-milling for 120 min decreased in the order of 72-min steaming >3-min steaming >no steaming. The greater responsiveness of the more severely pretreated fibres to ball-milling is consistent with earlier results which have found that treatments which remove the hemicelluloses and/or lignin weaken the network structure of the polymer matrix, reducing the energy requirement for mechanical attrition [22, 24, 53–55]. In a closely aligned study, Shikinaka et al. [56] very recently reported that softwoods can be converted into glucose in yield of almost 70% by simultaneous wet bead milling and enzymatic saccharification of milled wood. Additional data, including the process energy requirements, would be required to compare the cost-effectiveness and scalability of this purely mechanical approach against other pretreatments, including the one described here.

Analysis of the ball-milled fibres by field emission scanning electron microscopy revealed extensive disruption of the fibres after ball-milling to produce much finer cell wall fragments (Fig. 5). The surfaces of these fine fragments also show extensive delamination of the fibre wall with a loosened fibrillar structure. This indicates that ball-milling delaminates the fibre wall, loosens the ultrastructure of the wall fragments and possibly removes some of the matrix material from the fibre surfaces, resulting in greatly increased surface area [39]. This increased surface area on both the outside and inside of the wall fragments explains the enhanced digestibility from ball-milling, as the accessibility of the cellulose to the enzymes is a well-known determinant of digestibility, e.g. Ref. [57].

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 [23] 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.

Saccharification

The extensive delamination and opening up of the fibre wall occurring on ball-milling means the initial rate of hydrolysis of the ball-milled substrate is high, with ca. 90% of the final hydrolysis of glucosyl residues occurring within 5 h (Fig. 6). This is much more rapid than for the reference steam-exploded wood prepared from the same P. radiata wood.

Both the main hemicelluloses in the substrate, i.e. the GGM and xylan, were rapidly solubilised during enzymatic saccharification. Figure 7 shows that both the mannosyl and xylosyl structural units in the ball-milled substrate were rapidly solubilised during enzymatic saccharification at rates indistinguishable from that of the glucosyl units.

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 [63].

Impact of new cocktails and PEG

With the ball-milled softwood substrates, replacing the Celluclast/Novozyme 188 cocktail with an equivalent dose of the newer Cellic CTec2 cocktail had no major effect on the final extent of conversion (Fig. 8). However, addition of 0.1% w/w PEG (polyethylene glycol 4000) during saccharification greatly improved the effectiveness of saccharification of our substrate using this newer cocktail, particularly at lower enzyme doses. PEG and other additives are believed to be effective because the PEG interacts with the lignin to reduce non-productive binding of enzymes with the lignocellulosic substrates [64–66].

Overall sugar yields

Increasing the time of steaming in the pilot plant increases the overall yield of sugars per tonne of input wood (Fig. 9). This can be attributed to two factors. Firstly, the digestibility of the pulps after a standard 60-min ball-milling increases as the steaming time is increased (c.f. Fig. 4), more than compensating for the lower pulp yield following steaming. Secondly, hydrolysis of the GGMs increases with increasing steaming time, meaning they are more soluble in the aqueous phase and are separated into the pressate where they can be hydrolysed by acid (c.f. Table 1). In this way, the GGMs are then more efficiently converted to monomeric sugars than would be the case if they remained in the fibre and were only incompletely hydrolysed in the enzymatic saccharification stage.

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 [3] and recent yields of up to 86% from loblolly pine by bisulfite pulping [34] and 84% from lodgepole pine by the SPORL process [67]. 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

Tests undertaken at National Renewable Energy Laboratory showed that freeze-dried samples of both the mainline sugars and hydrolysed pressate sugar syrup produced from chips steamed for 72 min could be readily fermented to ethanol. Fermentation of the mainline glucose-rich syrup at a concentration of 150 g/L sugars using Saccharomyces cerevisiae strain D5A (which metabolises both glucose and mannose) and the glucose–xylose co-fermenting bacterium Zymomonas mobilis strain 8b gave high ethanol yields, >90% based on fermentable sugars (Fig. 10a). Glucose was completely consumed in both cases. One advantage of our process is that detoxification of this mainline sugar syrup is not required prior to fermentation, consistent with only low levels of fermentation inhibitors produced in the mild thermomechanical pretreatment.

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.

Thermomechanical pretreatment

The fibre and pressate samples prepared via trials were performed in the Scion fibre processing pilot plant [40]. 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 [29] 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.

Ball-milling

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.

Pressate hydrolysis

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

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.

Analyses

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 [69] 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. [70].

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 [50].

Uncondensed phenylpropane lignins in the in situ fibre lignins were determined in duplicate by nitrobenzene oxidation following the method of Chen [51].

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

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 OD_600nm 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 OD_600nm 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 [70]. Because mannose and arabinose co-elute using this column, arabinose concentrations were determined by ion chromatography using a Dionex PA1 column [69] and used to calculate the mannose concentrations.