Ana Rita C.
Morais
abc,
Jian
Zhang
d,
Hui
Dong
e,
William G.
Otto
f,
Thapelo
Mokomele
g,
David
Hodge
f,
Venkatesh
Balan
eh,
Bruce E.
Dale
ei,
Rafal M.
Lukasik
a and
Leonardo
da Costa Sousa
*e
aUnidade de Bioenergia e Biorrefinerias, Laboratório Nacional de Energia e Geologia, I.P., Estrada do Paco do Lumiar 22, 1649-038 Lisbon, Portugal
bLAQV-REQUIMTE, Department of Chemistry, Faculty of Science and Technology, Universidade NOVA de Lisboa, Lisbon, Portugal
cDepartment of Chemical and Petroleum Engineering, University of Kansas, Lawrence, KS 66045, USA
dState Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China
eBiomass Conversion Research Laboratory, Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing, MI 48824, USA. E-mail: sousaleo.eng@gmail.com
fDepartment of Chemical & Biological Engineering, Montana State University, Bozeman, MT 59717, USA
gDepartment of Process Engineering, Stellenbosch University, Private Bag X1 Matieland, Stellenbosch, South Africa
hDepartment of Engineering Technology, College of Technology, University of Houston-Sugarland campus, TX 77479, USA
iGreat Lakes Bioenergy Research Center, Michigan State University, East Lansing, MI 48824, USA
First published on 21st April 2022
A novel ammonia-based pretreatment for densified lignocellulosic biomass was developed to reduce ammonia usage and integrate with viable biomass logistics scenarios. The COmpacted Biomass with Recycled Ammonia (COBRA) pretreatment performed at 100 °C allows >95% conversion of sugarcane bagasse (SCB) carbohydrates into soluble monomeric and oligomeric sugars (glucose and xylose) using industrially relevant 6% glucan loading (∼21% solids loading) enzymatic hydrolysis conditions at reduced enzyme loadings. Pretreatment via COBRA with simultaneous lignin extraction (COBRA-LE) improved Saccharomyces cerevisiae 424A(LNH-ST) metabolic yield from 89% to 97.5% relative to COBRA without delignification, allowing a process ethanol yield of 71.6%. A technoeconomic analysis on SCB biorefining to ethanol in the state of São Paulo, Brazil, compared COBRA to other mature technologies, such as AFEX and steam-explosion. Amongst all scenarios studied, biorefineries based on COBRA-LE pretreatment offered the lowest average minimum ethanol selling price of US$1.45 per gallon ethanol. COBRA pretreatment was subsequently tested on perennial grasses and hardwoods, and >80% total sugar yields were achieved for all cases.
The biomass fractionation approach requires selective lignin removal from carbohydrates to maximize product yields from sugar and lignin streams, while simplifying product separations downstream. In addition to the value increment that lignin conversion can provide to the biorefinery, maximizing the removal of lignin prior to enzymatic hydrolysis also helps to reduce biomass recalcitrance and increases enzymatic activity on the pretreated carbohydrate-rich stream.11–13 Reducing enzyme loading during enzymatic hydrolysis has been a primary objective in biomass conversion, not only because it positively impacts the final biofuel price, but it also serves to mitigate risk due to uncertain bulk enzyme prices.14 An effective approach to significantly reduce enzyme loading is the use of pretreatment/fractionation technologies that delignify lignocellulosic biomass while manipulating the native cellulose I (CI) crystalline structure to either amorphous or other more digestible crystalline cellulose allomorphs, such as cellulose III (CIII).15–18 Pretreatments with ionic liquids (ILs), ammonium salts (ammonium thiocyanate in liquid ammonia) and liquid ammonia (Extractive Ammonia (EA) pretreatment) are approaches that can both selectively isolate usable lignin from carbohydrates and improve the enzymatic digestibility of cellulose by altering its crystalline structure.3,19–21 However, these methods still need to improve their economic and environmental sustainability. For example, although ionic liquids are highly effective in reducing biomass recalcitrance, they are nonetheless expensive and difficult to recycle.22,23 Recent research efforts to develop low-cost ILs and new IL recycling strategies reduced the effective IL cost to ∼US$5 per kg, which is still a relatively expensive proposition for biomass pretreatment.23
EA has proven to be a very effective ammonia-based pretreatment technology, generating biofuel yields comparable to those obtained for 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]) pretreatment, and significantly higher than those obtained for AFEX‡ and Dilute Acid (DA) pretreatments at relatively low enzyme loadings.3 EA pretreatment uses liquid ammonia-to-biomass at ratios greater than 3:
1 w/w to completely submerge the biomass in liquid ammonia, thereby forming highly digestible CIII and extracting nearly 50% of the lignin in corn stover (CS) without significant carbohydrate losses.3 The lignin extracted during EA pretreatment and recovered after enzymatic hydrolysis of EA-pretreated biomass is relatively intact, maintaining most of the aryl–ether crosslinks and minimal condensation levels. Such lignin materials are viable substrates for conversion to an array of valuable aromatic platform chemicals.3,5,24
Although ammonia is a less expensive chemical (∼US$0.5 per kg) for pretreating lignocellulosic biomass and is easier to recycle than ILs, a recent analysis on EA pretreatment showed that the key factor determining its economic sustainability is the liquid ammonia-to-biomass ratio required to effectively generate CIII and extract lignin.3 Ammonia evaporation and re-condensation during recycling requires considerable energy for EA pretreatment relative to AFEX. For example, EA pretreatment with 3:
1 w/w ammonia-to-biomass ratio requires about 60% of the high heating value (HHV) of the ethanol produced in order to recycle the ammonia, while AFEX only requires about 36%.3 Reducing the energy requirements during ammonia pretreatment, while converting CI to CIII, cleaving LCC linkages and achieving biomass delignification remain as major challenges for ammonia-based pretreatments.
Furthermore, although ammonia pretreatments perform well on herbaceous monocots and generate highly fermentable hydrolysates, they have not yet shown comparable performance on herbaceous dicots, hardwoods and softwoods under low severity processing conditions.25–28 Thus, ammonia pretreatments are still seen as less versatile for pretreating mixed lignocellulosic feedstocks, relative to IL-based, organosolv, steam explosion (StEx), deep eutectic solvents (DES) pretreatments and Reductive Catalytic Fractionation (RCF), among others. The ability to successfully pretreat a wide range of lignocellulosic feedstocks is particularly important if the objective is to create very large scale biorefineries to benefit from economies of scale and reduce the Minimum Biofuel Selling Price (MBSP). The larger the biorefinery capacity, the wider the biomass collection radius is likely to be, which probably also increases the available feedstock diversity, except for those relatively rare geographic locations where monocultures are available in large, contiguous areas of land (e.g., corn in the US Midwest and sugarcane in Sao Paulo, Brazil, among others).
In a field that wishes to sustainably valorize lignocellulosic biomass and compete in a market dominated by petroleum-derived commodities, the scale of biorefineries is a critical topic that has been largely overlooked in the literature. For example, the US has been reducing the number of active petroleum refineries while increasing their production capacity (i.e., increasing the refinery scale), with the intention of reducing operating costs and depreciation.29 In 2014, the US average capacity for crude oil processing per refinery was 128.7 thousand barrels per day, which corresponds to about 17800 Mg crude oil per refinery, per day.29 In contrast, most lignocellulosic biorefinery models in the literature assume capacities of around 2000 Mg dry biomass per day,30,31 which is very far from the scale at which petroleum is refined. There are major challenges associated with the scalability of systems that depend on highly variable, low-density solid substrates such as lignocellulosic biomass. The larger the biorefinery, the greater the biomass collection radius and transportation distances from the field to the biorefinery. As consequence, delivering low-density biomass to those large biorefineries (hereafter called ‘mega-biorefineries’) becomes more complex and expensive, resulting in the need for biomass milling, densification and drying in regional processing depots (RPDs) located closer to the biomass production fields. As such, biomass storage and long-haul shipment to mega-biorefineries can be simplified.32,33 We note also that paying farmers more for biomass induces them to grow much more biomass, leading to shorter supply chains with reduced transportation costs and much larger biorefineries (potentially with capacities greater than 20
000 Mg per day), with the attendant economies of scale and only small effects on biofuel selling price.34 Overall, the issue of biorefinery scale and the associated logistics needs much more study.
Based on these considerations, this work describes a highly efficient pretreatment technology using low chemical and energy inputs that is effective on a wide variety of lignocellulosic feedstocks, extracts usable lignin with preserved chemical functionalities and that fits within a scalable bioeconomy concept. The overall system accounts for feedstock availability and diversity, feedstock logistics and the need for very large scale biorefineries with their greater economic sustainability (Fig. 1A). Here, a new pretreatment technology called ‘COmpacted Biomass pretreatment with Recycled Ammonia’ (COBRA) is studied for the first time. COBRA pretreats low moisture, densified feedstocks at temperatures under 100 °C, thus allowing liquid ammonia-to-biomass ratios below 1:
1 to fully submerge the densified solids and convert CI to CIII, maximizing both carbohydrate conversion and usable lignin recovery (Fig. 1B). The economic and environmental sustainability of the COBRA-based bioeconomy is elaborated. Also, a case study on a COBRA-based bioeconomy is evaluated for sugarcane bagasse in the State of São Paulo, Brazil, relative to other competing technologies that use loose feedstocks, such as AFEX, EA and StEx pretreatments.
Alternatively, if we assume a feedstock logistics model based on densified biomass, as shown in Fig. 1, much larger quantities of densified biomass can be loaded per unit volume of a pretreatment reactor relative to loose or baled biomass. This can result in several potential advantages relative to pretreatment of loose biomass, as the volume of liquid ammonia required to fully submerge densified biomass is considerably lower than that required to submerge loose or baled biomass. Thus, submerging densified biomass in liquid ammonia enables conversion of CI to CIII, the cleavage of LCC linkages and selective extraction of lignin from biomass, as has been described for EA pretreatment (e.g., 3:
1 ammonia to biomass ratio), but also using low ammonia levels as described for AFEX pretreatment (e.g., 1
:
1 ammonia to densified biomass mass ratio). The lower ammonia loading requirement greatly reduces energy costs for ammonia recycling, while potentially producing highly digestible feedstocks. Also, greater biomass density allows for smaller pretreatment reactor volumes for a fixed pretreatment time, which reduces capital costs. Alternatively, it allows longer pretreatment residence times for the same reactor size relative to conventional AFEX, EA and StEx, all of which operate with loose biomass. In this article, the COBRA pretreatment has been developed, optimized, and evaluated for various pretreatment conditions (with and without lignin extraction), enzyme loadings and feedstocks.
Furthermore, the enzyme cocktail for optimal conversion of COBRA-pretreated SCB to fermentable glucose + xylose sugars was determined (71 wt% CTec3: 23 wt% HTec3: 6 wt% Multifect Pectinase) (Fig. S5 and S6, ESI†) and used to fully explore the enzymatic hydrolysis potential of COBRA-pretreated SCB. Fig. 2A and B show that COBRA pretreatment performed under the mildest condition scrutinized here, i.e., 75 °C, 1:
1 NH3
:
BM loading g/g for 4 h of residence time, and 15 mg of enzyme loading converted >88% glucan and >85% xylan, for a combined sugar yield of 63 kg per 100 kg of sugarcane bagasse (Fig. 2B).
As expected, combined sugar yields were greater for the highest temperature studied (e.g., 100 °C, 1:
1 NH3
:
BM loading g/g for 3.5 h and 15 mg of enzyme loading), resulting in an increase of 6% glucan and 8% xylan yields relative to COBRA-pretreated SCB at 75 °C, when hydrolyzed with 15 mg g−1 glucan enzyme loading. As observed with EA, COBRA pretreatment temperature does not affect the formation of CIII, as it is formed at both 75 °C and 100 °C using 1
:
1 NH3
:
BM (g/g) (Fig. S4, ESI†), but it does promote a more effective cleavage of ester bonds and greater lignin solubilization, as previously reported by da Costa Sousa et al., making the CIII more accessible to cellulases.3 However, higher temperatures generate higher operating pressures (60 bar at 100 °C). For example, a pressure of 30 bar was observed when COBRA was performed at 75 °C, which is similar to the pressure found for AFEX pretreatment at 120 °C.42 AFEX uses higher moisture levels (approximately 60% of biomass dry weight)43 than does COBRA, reducing the pressure for a given temperature relative to using solely anhydrous ammonia to pretreat dried biomass. However, those moisture levels suitable for AFEX prevent CIII formation during ammonia pretreatment, thus reducing pretreatment effectiveness.3,16,17 COBRA pretreatment is performed on storage-grade, densified biomass with about 10 wt% moisture to prevent microbial decomposition of the biomass. Thus, when the temperature is raised to 100 °C, the pressure increases to about 60 bar, approaching the pressures observed for EA pretreatment. Note that EA pretreatment is also performed using anhydrous ammonia, but on dried loose biomass instead of densified biomass.3 Reducing ammonia loading from 1
:
1 to 0.75
:
1 NH3
:
BM (g/g) during COBRA pretreatment at 75 °C did not significantly impact the glucan and xylan conversion.
Detailed knowledge on how the biomass bulk density changes during COBRA pretreatment and related thermodynamic property measurements are required to predict the lowest possible ammonia loading for a given operational condition, and to maintain high process sugar yields. Nonetheless, based on the ammonia density at 75 °C, and determining experimentally the water adsorption capacity of SCB pellets as a surrogate for ammonia, while accounting for the respective pellet bulk volume expansion at saturation conditions, we estimate that 0.75:
1 NH3
:
BM (g/g) is close to the lower limit required to fully submerge the densified biomass in anhydrous ammonia. Also, preliminary studies confirmed that NH3 loadings below 0.75
:
1 NH3
:
BM g/g significantly reduced sugar yields for COBRA pretreated CS (data not shown).
Comparing the three COBRA pretreatment conditions tested in Fig. 2, it is evident that pretreatment performance did not vary significantly for the highest enzyme loading (15 mg g−1 glucan). However, enzyme loading significantly impacts the overall sugar release for all pretreatment conditions, and more so for the lower temperature COBRA pretreatment conditions. Thus, enzyme levels might be reduced for COBRA pretreatment, but might in turn require higher pretreatment temperatures (and pressures). Also, high levels of soluble gluco- and xylo-oligosaccharides were released during high solid loading enzymatic hydrolysis, accounting for 10–15% of the total soluble sugar under some conditions (Fig. 2). The specific properties of these oligosaccharides (including linkage analysis, composition, chemical structure) should be studied so that specific enzymes can be added to the enzyme cocktails to improve the conversion of those soluble carbohydrates to fermentable sugars.
The impact of lignin extraction during COBRA pretreatment was evaluated by removing the liquid ammonia-soluble lignin from the bottom of the reactor, passing it through a sintered filter under pressure (COBRA-LE), as described in the Experimental section. Hereafter the COBRA process with lignin extraction is identified as COBRA-LE. COBRA-LE pretreatment performed at 100 °C extracted about 26% of the original lignin from SCB, resulting in 4% point improvement in glucan conversion (97% overall glucan conversion) over that observed for COBRA pretreatment performed at similar operational conditions without lignin extraction.
Biomass delignification during EA pretreatment has shown a similar effect on enzymatic saccharification to that observed for COBRA-LE. da Costa Sousa et al. reported a glucan conversion improvement of 6% points after removing lignin from CS during EA pretreatment at 120 °C and 6:
1 NH3
:
BM g/g loading, yielding 89% overall glucan conversion.3 However, no significant improvement in total glucan conversion was found for COBRA-LE performed at 75 °C relative to COBRA performed using the same conditions, likely due to low delignification yield (∼19%). Although carbohydrate conversion from COBRA-LE pretreated biomass improved somewhat relative to COBRA, the total sugar yields from pretreated biomass did not improve upon those obtained with COBRA pretreatment. For example, the combined sugar yield at the most severe COBRA-LE pretreatment condition (100 °C, 1
:
1 NH3
:
BM g/g loading, 3.5 h reaction time) was 65.7 kg sugar per 100 kg untreated SCB, while COBRA achieved 67.4 kg sugar per 100 kg untreated SCB. This is due to the fact that a small fraction of carbohydrates, notably xylan, was extracted with lignin during COBRA-LE pretreatment and was never converted during enzymatic hydrolysis. However, both COBRA and COBRA-LE pretreatments enabled soluble sugar yields, including oligosaccharides, close to the theoretical maximum for the SCB used in this work, i.e., 72.5 kg sugar per 100 kg untreated SCB, using relatively mild operating conditions and low enzyme loadings during high solid loading enzymatic hydrolysis (Fig. 2B).
![]() | ||
Fig. 3 Comparison of COBRA, COBRA-LE, EA, AFEX and steam explosion (StEx) pretreatments in terms of sugar and ethanol (EtOH) yields. Total sugar yields were calculated considering glucose, gluco-oligomers, xylose and xylo-oligomers. Ethanol yields were calculated on the basis of 100 kg of untreated sugarcane bagasse input after 120 min of fermentation time. The theoretical maximum for sugar and ethanol yields was calculated based on the initial glucan and xylan contents in untreated sugarcane bagasse. # The sugar and ethanol yields from “AFEX – bagasse” and “StEx – bagasse – whole slurry” were obtained by Mokomele et al.40¤The potential ethanol yield from oligomers was estimated based on the metabolic yields and sugar consumption obtained in each operational condition (Table S1 in ESI†). ¥The potential ethanol yield from soluble sugars was estimated considering the complete conversion of soluble sugars into ethanol with the highest metabolic yield obtained (97.5%). All the enzymatic hydrolysis liquors were produced at 6% glucan loading (w/w, glucan) for 96 h of hydrolysis time. COBRA, COBRA-LE and EA enzymatic hydrolysis were performed with 15 mg protein per g glucan, while AFEX and StEx were carried out with 25 mg protein per g glucan. |
For COBRA-pretreated SCB, the total sugar yield decreases with reducing pretreatment severity, generating lower levels of soluble sugars. However, most of that difference is due to increased oligomeric carbohydrates in the hydrolysate at higher pretreatment severities. There is no substantial difference in monomeric sugar yields between the various COBRA pretreatment conditions: the fermentable sugar yields for the most severe and the least severe COBRA pretreatment conditions were, respectively, 56.0 ± 2.6 and 52.8 ± 4.9 kg per 100 kg SCB. However, if the potential conversion of oligosaccharides to ethanol is also considered, larger differences in ethanol yield can be observed (Fig. 3) between these two conditions. In addition, if we assume that all soluble carbohydrates are converted to ethanol by S. cerevisiae 424A (LNH-ST) with a metabolic yield of 97.5%, the highest metabolic yield observed for all conditions tested herein, 90% of the theoretical ethanol yield from SCB, i.e., 36.9 kg ethanol per 100 kg SCB, could be obtained for the most severe COBRA pretreatment condition.
Significant improvements in ethanol yields were observed when lignin was extracted during COBRA pretreatment (COBRA-LE). Although fermentable sugar yields were slightly lower than those observed for the highest COBRA severity, the fermentation performance on COBRA-LE hydrolysates was much greater than that observed for COBRA hydrolysates, achieving 97.5% metabolic yield (ESI Table S1†). This result is expected, as compounds that inhibited the yeast strain were likely removed during the lignin extraction process.3,48 As previously discussed, a total of 65.7 ± 1.8 kg sugar per 100 kg SCB was solubilized during 6% glucan loading enzymatic hydrolysis for the highest COBRA-LE pretreatment severity. This result includes monomeric and oligomeric sugars, representing about 90% of the theoretical maximum sugar yield from SCB (72.5 kg sugar per 100 kg SCB), of which 76.5% are fermentable monomeric sugars (55.4 kg sugar per 100 kg SCB). The experimental ethanol yield for COBRA-LE pretreatment performed at the highest severity was 26.5 kg ethanol per 100 kg SCB, which is 71.6% of the theoretical. In contrast, the same COBRA pretreatment condition only achieved 69.4% of the theoretical maximum yield, despite generating higher sugar yields. If all soluble sugars, including oligosaccharides, were consumed and converted to ethanol with 97.5% metabolic yield, about 32.7 kg ethanol per 100 kg biomass would be produced, or about 88.4% of the theoretical maximum.
As previously mentioned, these results obtained for COBRA and COBRA-LE pretreatments further highlight the importance of understanding the recalcitrant nature of soluble oligosaccharides present in COBRA-derived hydrolysates. In addition, more robust microorganisms than that used in this study should be developed to maximize ethanol yields from the available sugar present in COBRA-derived hydrolysates. Lower overall xylose consumption (range 84.5 ± 3.7% to 91.1 ± 0.1%) was the main factor responsible for the lower ethanol yields. Improved xylose consumption can be achieved by extracting lignin from SCB or by adding nutrients to the hydrolysate,49 however, both require additional processing costs. For instance, COBRA-LE performed at 100 °C resulted in a delignification yield of 26%, improving the xylose consumption by approximately 8% points relative to COBRA at 100 °C. In addition to enabling a higher ethanol yield, COBRA-LE pretreatment generates a lignin stream that can be further processed and purified, and potentially becomes a revenue source for the biorefinery (see technoeconomic evaluation section below).5,24 Previous studies have demonstrated that the lignin derived from ammonia pretreated biomass maintains the β-aryl–ether linkages from lignin intact, which are critical functionalities to perform controlled lignin depolymerization and avoid C–C condensation reactions.50
Also, EA was performed using 6:
1 ammonia to biomass ratio, while the most severe COBRA-LE condition only used a 1
:
1 ratio to produce a greater yield of fermentable sugars and overall soluble carbohydrates (monomers + oligomers). Although EA pretreatment did not promote higher sugar yields relative to COBRA-LE, it did show better fermentation performance. It may be that the higher lignin extraction efficiency during EA pretreatment relative to COBRA-LE resulted in lower levels of inhibitory compounds for fermentation. However, more in-depth studies are required to determine the inhibitory levels of the extracted lignin to S. cerevisiae 424A (LNH-ST), or even the impact of densification conditions on the potential formation of inhibitory compounds for fermentation.
As AFEX and StEx pretreatments do not modify cellulose crystallinity, nor remove lignin from biomass, an enzyme loading of 25 mg per glucan was required to maximize sugar yields, as shown by Mokomele et al.40 A total sugar yield of 60.3 ± 1.1 kg per 100 kg SCB was obtained under optimal AFEX conditions (140 °C, 1:
1 NH3
:
BM ratio (g/g) for 1 h), which is comparable to that found for COBRA-LE with the lowest severity tested herein and using only 60% of the enzyme loading added to AFEX-pretreated SCB. Although COBRA-LE is conducted for 4 h, and AFEX for 1 h, COBRA-LE uses densified biomass which occupies significantly less reactor volume per unit of biomass treated. Our findings show that the pretreatment productivity is practically the same for COBRA-LE at 75 °C, with 0.75
:
1 NH3
:
BM ratio g/g, for 4 h residence time, and for AFEX performed at 140 °C, with 1
:
1 NH3
:
BM ratio (g/g) for 1 h residence time. Thus, in addition to the logistic advantage of using densified biomass for transportation and enabling mega biorefineries, with better economies of scale, COBRA-LE saves both operation and capital costs due to lower pressures, temperatures and ammonia loadings. The cost of biomass densification may not be offset by reduced transportation costs for scenarios with high biomass availability in a relatively small land area but it is still likely to be less expensive to feed densified biomass into the COBRA pretreatment reactor than to feed undensified, loose biomass.
StEX pretreatment led to modest total sugar yields (47.6 ± 0.9 kg per 100 kg SCB), even with 25 mg protein per g glucan enzyme loading. This low sugar yield is mainly due to low carbohydrate recovery after the pretreatment step. StEx pretreatment requires high reaction severities to improve cellulose digestibility, leading to degradation of sugars into e.g., furans, hampering the production of fermentable sugars at high yields. Among the processes studied herein, StEx showed the lowest sugar recovery and lowest product yield, however, techno-economic analysis, as discussed later in this manuscript, clarifies the economic potential of StEx relative to the other pretreatment-based biorefinery models.
The identification and development of a “feedstock-agnostic” pretreatment that can simultaneously extract lignin from biomass, promote biomass solubilization, and achieve high fermentable sugar yields with minimal use of enzymes and chemicals has been a subject of great interest. For instance, ionic liquid-based pretreatments are claimed to be one of the few “feedstock agnostic” technologies capable of efficiently handling hardwoods, softwoods, agricultural residues, herbaceous dicots and monocots, both as a single and as a blend of various feedstocks.51,52 According to Li et al., [C2mim][OAc] is one of the most effective and versatile ionic liquids reported for biomass pretreatment, as it is able to effectively liberate more than 90% of sugars from eucalyptus and switchgrass during enzymatic saccharification in 24 h.51 However, similar results were not observed for other woody biomasses under the same IL pretreatment (160 °C for 3 h) and enzymatic hydrolysis conditions, as the authors reported only 62% enzymatic digestibility for pine wood, for example. Also, it is important to note that the overall monomeric sugar recovery from pine in that study was only 49.7% after 72 h enzymatic hydrolysis, as a significant portion of the carbohydrates were left in the liquor as oligomers.51 It is worth to mention that a verification experiment on COBRA pretreated pine at 100 °C for 6 h, using with 1:
1 NH3
:
BM loading, followed by 72 h of enzymatic hydrolysis with a non-optimal enzyme cocktail shows nearly 60% monomeric sugar recovery, combined.
Thus, as reported for other pretreatments (e.g., DA, AFEX and many others), ionic liquids pretreatment performance also varies with the feedstock due their inherent compositional and structural differences. Sun et al. reported that the type of wood affects the dissolution yields and rates of the feedstocks in [C2mim][OAc].53 For example, red oak dissolves much faster than southern yellow pine. In addition, the performance of a specific pretreatment technology is not only dependent on the type of feedstock, but also on other factors such as enzyme and solids loading, enzyme cocktail, and particle size. As shown in Fig. 4, the total sugar yields for each individual COBRA-pretreated feedstock, except for corn stover, increased significantly with enzyme loading. For instance, an improvement of 29% points in combined sugar yield was found for prairie grass with increasing enzyme loading from 7.5 to 30 mg protein per g glucan. Interestingly, changing enzyme loadings only had a slight effect on total sugar yields achieved from COBRA-pretreated CS.
To better understand the effect of different enzyme combinations on glucan and xylan conversion, we studied the influence of enzyme combinations on poplar, which was pretreated at 100 °C for 6 h with 1:
1 NH3
:
BM loading g/g, followed by 96 h enzymatic hydrolysis with an enzyme loading of 30 mg protein per g glucan. As shown in Fig. 4B, there are significant differences in combined fermentable sugar yields for the various enzyme combinations tested for poplar (see data for Poplar, Poplar Enz. 2 and Poplar Enz. 3). An increase of 7% combined sugar yield was obtained using enzyme combination 3 (Enz. 3 in Fig. 4B) relative to the standard enzyme combination previously optimized for SCB. Thus, COBRA pretreatment has significantly improved enzyme access to their substrates, but differences in substrate composition require different ratios of the various enzymes in order to maximize carbohydrate conversion. Unlike acid-based pretreatments which require a washing step, COBRA is basically a dry-to dry pretreatment that preserves polysaccharides with little-to-no degradation of sugars. However, the presence of hemicellulose and pectin requires more robust and complex enzymatic cocktails relative to acidic pretreatments, which typically hydrolyze the non-cellulosic fraction of the biomass. Therefore, ammonia-pretreated mixed feedstocks require non-limiting levels of optimized ratios of cellulases, hemicellulases, pectinases, and other accessory enzymes with synergistic key activities to maximize overall sugar yields.54 This is particularly important not only for COBRA pretreatment, but also for other technologies (e.g., AFEX and EA) that do not hydrolyze hemicellulose linkages.3 Enzymatic deconstruction of hemicellulose is highly dependent on a complex range of enzyme activities that must be understood and fine-tuned to become effective on a wider range of substrates.55
In fact, AFEX pretreatment can be performed at the depot on loose biomass, and the AFEX-pretreated material can be densified, stored, and transported to a mega-biorefinery for further conversion to biofuels and chemicals.56,57 Since AFEX-treated biomass is an improved animal feed, the AFEX-based depot can provide both animal feed and biorefinery feedstock, thereby helping to “jump-start” a biorefining industry in the same way that the pre-existing use of corn as an animal feed helped jump-start the corn ethanol industry. Furthermore, unpublished work from our group shows that if biomass is AFEX-treated and then pelletized, milder COBRA pretreatment conditions can be used to achieve comparable sugar yields than those obtained using the most severe COBRA conditions reported in this manuscript. However, this scenario (AFEX and pelletization at depots followed by COBRA on AFEX-treated SCB) was not considered in this specific study and will be a topic of a separate manuscript.
Given the many logistics scenarios which will impact feedstock price at the gate of the refinery, this work focuses on comparing centralized lignocellulosic biorefining systems based on various pretreatment technologies applied to SCB processing in the State of São Paulo, Brazil. The geographical region of São Paulo offers high density and availability of SCB where feedstock logistics systems may be easier to implement. Also, considering a region with an unusually high concentration of feedstock does not introduce bias to benefit pretreatment technologies that require densified biomass. On the contrary, it minimizes the economic benefit that densified biomass could have on the overall delivered price of feedstock (ESI Table S2†). As we noted previously, however, transporting, storing, and feeding densified biomass to bioreactors is likely to be considerably more feasible than feeding bulky, loose biomass.
Fig. 5 shows the geolocation of all the active first-generation sugarcane ethanol plants (1G) in the State of São Paulo, Brazil, where a combined excess of 117679 Mg per day of dry sugarcane bagasse is potentially available for second generation biofuels.58 Here, each 1G ethanol plant was considered as a Processing Depot (PD), where the sugarcane bagasse produced was dried, pelletized, and stored before being transported to a designated lignocellulosic mega-biorefinery. A conditional K-means algorithm was implemented to determine the number of mega-biorefineries, their optimal location, and the PDs that supply each mega-biorefinery with their SCB, with the objective of minimizing the average biomass transportation distance per Mg of biomass and maximizing the size of the mega-biorefineries within the following constraints: (1) the location of each mega-biorefinery was picked amongst the existing PDs in the State of São Paulo; (2) a maximum mega-biorefinery capacity of 20
000 Mg of biomass per day was considered, since the average processing capacity for oil refineries is in that order of magnitude,59 roughly ten times the usually assumed biorefinery size60 and (3) all the available excess sugarcane bagasse could be used by the mega-biorefineries to produce ethanol, electricity and/or lignin.
As depicted in Fig. 5, the clustering algorithm has calculated 6 mega-biorefineries (triangles) and their respective PDs (circles), which are distinguished by the different colors on the map. The capacity and average transportation radius per Mg of biomass for every cluster are described in ESI Table S2.† In summary, the results show that the biomass processing capacity of the 6 mega-biorefineries ranged from 19240 (cluster 2) to 20
000 (cluster 0) dry Mg per day, and the average transportation radius per Mg of dry biomass ranged from 64.2 (cluster 2) to 121.8 (cluster 3) km Mg−1 SCB. The average delivered price of biomass transported as bales also varied significantly from the biomass transported as pellets. The delivered price of sugarcane bagasse bales varied from $61.5 to $71.3 per dry Mg SCB, whereas the delivered price of pellets varied from $77.6 to $82.1 per dry Mg SCB, corresponding to clusters 2 and 3, respectively.
The minimum ethanol selling price (MESP) at the mega-biorefinery gate for the clusters shown in Fig. 5 was determined, considering biorefining processes based on different pretreatment technologies. In the present study, the biorefineries based on COBRA and COBRA-LE pretreatment technologies used biomass delivered as pellets, while the remaining biorefineries considered biomass delivered as bales.
Also, three scenarios were examined based on the experimental results and assumptions summarized in Fig. 3. Scenario 1 used the best experimental ethanol yields obtained in this work under the process conditions described in Fig. 3 (highest severity COBRA and COBRA-LE). Scenario 2 assumed that all fermentable sugars were consumed and converted to ethanol with 97.5% metabolic yield (the maximum we have observed). Scenario 3 assumed that all soluble sugars, including oligosaccharides, were consumed and converted to ethanol with 97.5% metabolic yield.
Fig. 6 shows the predicted average MESP for the various scenarios and pretreatment technologies in this study. For Scenario 1, which represents the combined potential of all the technologies used in this work, from pretreatment to fermentation, COBRA-LE and AFEX showed the lowest and very similar MESPs with $1.45 and $1.46 per gallon ethanol, respectively. It is remarkable that COBRA-LE-based biorefineries could still show a slightly lower average MESP than AFEX-based biorefineries, even though the delivered price of densified biomass was significantly higher than baled. This occurs because COBRA-LE achieves higher ethanol yields than any other pretreatment technology tested herein, while requiring 40% less enzyme loading and using the same ammonia loading as AFEX pretreatment.
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Fig. 6 Average MESP ($ per gallon ethanol) calculated for mega-biorefinery systems based on various pretreatment technologies, implemented in the State of Sao Paulo, Brazil. COBRA and COBRA-LE pretreatment conditions considered for this study were temperature of 100 °C, residence time of 3.5 h, NH3![]() ![]() ![]() ![]() |
The lignin selling price in this base case for COBRA-LE and EA pretreatment-based biorefineries was similar to the price of densified biomass at $75 per dry Mg lignin, and consequently did not result in any additional value, nor loss, to the biorefinery. The average MESP calculated for StEX-based mega-biorefineries was significantly higher than for any other process studied herein ($1.98 per gallon ethanol), because the experimental ethanol yield obtained was just 16.2 g per 100 kg untreated SCB due to poor xylose yields and conversion by the recombinant S. cerevisiae 424A(LNH-ST) strain used in this work (Fig. 3). If we assume that all fermentable sugars were consumed during fermentation with a metabolic yield of 95.7%, the average MESP for StEx-based mega-biorefineries drops to $1.46 per gallon ethanol. This result shows the importance of improving fermentation strains that can tolerate the presence of inhibitory components in hydrolyzates derived from StEX-pretreated biomass. To a lesser extent, the MESP was also significantly reduced for the other pretreatment technologies in Scenario 2 relative to Scenario 1. In Scenario 2, AFEX pretreatment is predicted to have the lowest MESP at $1.38 per gallon ethanol, followed by COBRA pretreatment at $1.39 per gallon ethanol. Though the ethanol yield is significantly higher for COBRA and COBRA-LE at lower enzyme loadings relative to AFEX pretreatment under Scenario 2, that difference was not sufficient to offset the higher delivered feedstock price of biomass pellets relative to bales. However, MESP differences are not very significant between these three pretreatment technologies, while COBRA technologies still benefit from the better logistics platform offered using densified biomass and the 40% lower enzyme loading relative to AFEX pretreatment. The largest impact of COBRA and COBRA-LE pretreatment technologies on reducing the MESP is showcased in Scenario 3, in which all soluble carbohydrates (monomeric and oligomeric sugars) available after enzymatic hydrolysis are converted to ethanol with a metabolic yield of 97.5%. As shown in Fig. 6, the average MESP of COBRA-LE and COBRA-based biorefineries under Scenario 3 could decrease to $1.18 and $1.15 per gallon ethanol, respectively, whereas the remaining pretreatment technologies would not enable average MESPs below $1.25 per gallon ethanol. This occurs primarily because COBRA pretreatments enable over 90% carbohydrate conversion, including sugar oligomers, which are currently not used by the recombinant S. cerevisiae 424A (LNH-ST) strain.
However, this analysis shows a clear path for improving the economic viability of liquid biofuels, which also applies to other fermentation-based biochemicals. It is critical to understand the fundamental reasons why soluble oligosaccharides accumulate during hydrolysis of COBRA and COBRA-LE pretreated biomass. Enzyme technology could improve, notably better hemicellulase cocktails, to facilitate lignocellulosic biomass deconstruction to fermentable sugars. Alternatively, microorganisms could be developed to effectively address the oligosaccharide conversion problem, in addition to improved sugar consumption and biofuel metabolic yield.
The comparative TEA performed here demonstrates that EA pretreatment, although effectively converting carbohydrates to fermentable sugars, did not show economic advantages relative to COBRA pretreatment technologies, nor relative to AFEX. This was mainly because EA uses a 6:
1 ammonia-to-biomass ratio, and extracted of a small fraction of the carbohydrates into the lignin stream, thereby giving slightly lower soluble carbohydrate yields relative to COBRA.
A sensitivity analysis was done to determine how the assumed extracted lignin price affects the average MESP for both types of biorefineries, as shown in Fig. 7. Here, the price of the extracted lignin varied from $50 to $125 per Mg lignin for the 3 scenarios discussed above. Based on Fig. 7, the average MESP is not very sensitive to lignin price, as only 25–30% of the lignin present in SCB was extracted. As such, the average MESP for EA pretreatment-based biorefineries in every scenario calculated in this study was never lower than that shown for AFEX-based biorefineries, even when the price of lignin was assumed to be $125 per Mg lignin.
Anhydrous liquid ammonia cylinders equipped with a dip tube were procured from Airgas (Radnor, PA, USA) for ammonia pretreatment. Solvents, sugar standards, acids and bases were purchased from Sigma Aldrich (St Louis, MO, USA).
Cellic® CTec3 (batch number VDNI0002) and Cellic® HTec3 (batch number VIN00001) enzymes were kindly donated by Novozymes North America, Inc. (Franklinton, NC, USA) and Multifect Pectinase (batch number 4861295753) enzyme was kindly donated by DuPont Industrial Biosciences (Palo Alto, CA, USA). The protein concentration in enzyme solutions was determined using Kjeldahl nitrogen analysis method (AOAC Method 2001.11, Dairy One Cooperative Inc., Ithaca, NY, USA).1,2
COBRA pretreatment for high-solid-loading enzymatic hydrolysis was carried out using an in-house built reactor of 700 mL with a similar design as the one of 33 mL. In the case of these reactors, a desired amount of sugarcane bagasse (dry weight basis) was added into the reactor and the ammonia was loaded gravimetrically by weighing the ammonia transferred from a pre-weighed vessel to the reactors. Immediately after filling the system with ammonia, the reactors were heated up and kept at the desired temperature for defined reaction time. All the subsequent steps were identical to those described for the small-scale reactors.
To assess the influence of lignin removal on enzymatic hydrolysis yields, COBRA pretreatment was performed with lignin extraction, hereinafter referred to as COBRA–LE. COBRA–LE was carried out at the same operational conditions as regular COBRA pretreatment. In COBRA–LE pretreatment, the bottom of the reactor was connected to a high-pressure lignin collection vessel, whilst the top of the reactor was connected to a nitrogen line. After reaching the required reaction time, the ammonia was drained along with the dissolved lignin from the reactor to the lignin collection vessel. The exhaust valve from the lignin collector was slowly opened to remove ammonia from the system. Right after, the nitrogen was introduced through the top of the reactor to keep the pressure in the system approximately at 21 bar. This procedure allowed the nitrogen flowing through the system, and helped to flow the liquid ammonia with the dissolved lignin down to the lignin collector, passing through a sintered filter installed in the bottom of the reactor.4 After lignin extraction, the nitrogen flow was cut off to allow the system releasing the pressure slowly. For mass balance purposes, the pretreated sugarcane bagasse was transferred from the reactor to a pre-weighted tray, which was placed under the fume hood for 48 h to remove any potential traces of ammonia. The pretreated sugarcane bagasse was weighted, and its respective moisture content was measured as described above.
A conditional K-means clustering algorithm was coded in Python,9 to determine where the lignocellulosic biorefineries (mega-biorefineries) should be located and the group of BPDs that should supply each of those mega-biorefineries with SCB feedstock. The objective of the clustering algorithm was to minimize the average SCB transportation distance per dry ton of biomass from the BPDs to the mega-biorefineries included in the entire system and maximize their individual processing capacity up to a limit of 20000 Mg of dry SCB per day. As a set condition in the algorithm, all the available SCB was forced to be included for processing in a mega-biorefinery, and each BPD was set to deliver their entire SCB production to a single mega-biorefinery. The average transportation distance per ton of dry SCB was determined for each of the optimal clusters composed by a mega-biorefinery and respective biomass supplying depots. The mapping of the optimal clusters was performed using the ggmap package in R (https://www.r-project.org) and the georeferenced data labeled with the optimal cluster number assigned by the K-means algorithm.
The minimum ethanol selling price (MESP) at the gate of each of the mega-biorefineries was evaluated considering a 10% internal rate of return (IRR), using a modified version of the Excel-based model developed for prior work for techno-economic modeling of AFEX pretreatment12 and was, in turn, based on the 2012 NREL technoeconomic model.13 AFEX, COBRA, COBRA-LE, StEx and EA pretreatments were simulated.
The biorefinery models assumed the implementation of 6 centralized mega-biorefineries with delivered feedstock prices as described in Table S2.† The installed cost of the equipment used in the pretreatment area was calculated through equipment sizing and equations reported in the literature.14 The installed capital costs used in the model for feedstock handling (area 100) 2000 Mg per day biorefinery were estimated as $24.2 M for AFEX, StEX, and EA and only $4.5 M for COBRA and COBRA-LE based on a biorefinery handling pellets rather than loose biomass.12 The total installed capital costs assumed for pretreatment (area 200) for a 2000 Mg per day biorefinery in the model were estimated as $19.5 M for AFEX, $20.6 M for EA, $6.7 M for StEx, $17.1 M for COBRA, and $17.1 M for COBRA-LE. The remaining process areas in the NREL model (ethanol recovery, wastewater treatment, storage, boiler and utilities) were sized using the six tenths rule and the mass balances obtained from the Excel model. The installed costs of the equipment required for enzyme production were eliminated and it was assumed that the enzymes were purchased from a commercial source. The heat and power required to support the production of ethanol were produced through the combustion of unhydrolyzed solids. The excess electricity produced was assumed to be sold to the grid. The following chemical, biomass, and enzyme costs, and electricity selling price were used in the model calculations (in 2012 dollars): NH3 at $530 per Mg, H2SO4 at $87 per Mg, lime at $107 per Mg, biomass as described in Table S2,† cellulase at $3600 per Mg, hemicellulose at $4500 per Mg, and electricity at $0.0572 per kW h. All monetary values reported in this work are in 2012 US dollars. Ammonia recovery for COBRA, COBRA-LE, EA and AFEX was assumed to be 98% based on nitrogen balances between the untreated and pretreated biomass.
The technoeconomic analysis for the various pretreatment technologies scrutinized in this work were performed using three different scenarios. In Scenario 1, the enzymatic hydrolysis and fermentation performances assumed were those obtained experimentally by the optimal conditions found for each of the pretreatments evaluated. In Scenario 2, the fermentable sugar yields obtained experimentally were considered, however, it was assumed that all sugars were consumed and converted to ethanol with metabolic yield of 97.5%. Finally, Scenario 3 assumed that all soluble sugars generated experimentally during enzymatic hydrolysis, including glucose, xylose and respective oligomers, were converted to ethanol with 97.5% metabolic yield during fermentation.
COBRA pretreatment has proven to be flexible in terms of the feedstocks that it can handle effectively, from hardwoods to herbaceous monocots and dicots. It is possible that, in the future, it can be effective in softwoods provided that appropriate hemicellulase cocktails are developed to effectively hydrolyze galactoglucomannans. This feature is likely important for implementing mega-biorefineries around the globe. Therefore, COBRA-based pretreatments seem to have most of the traits that one should find in the ideal pretreatment, (1) the ability to treat a variety of densified feedstocks under relatively mild conditions, (2) carbohydrate conversions greater than 95% during high solid loading enzymatic hydrolysis, (3) highly fermentable hydrolysates and (4) a lignin stream with most of the native lignin functionalities for further processing to yield aromatic precursors. Further work should target the hydrolysis of incompletely hydrolyzed oligosaccharides and carbohydrate hydrolase cocktails dedicated to target softwood hemicellulose more effectively.
Footnotes |
† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d2gc00488g |
‡ ™AFEX is a trademark of MBI International. |
This journal is © The Royal Society of Chemistry 2022 |