Scholarly article on topic 'Integrated enzyme production lowers the cost of cellulosic ethanol'

Integrated enzyme production lowers the cost of cellulosic ethanol Academic research paper on "Agricultural biotechnology"

0
0
Share paper
OECD Field of science
Keywords
{""}

Academic research paper on topic "Integrated enzyme production lowers the cost of cellulosic ethanol"

Modeling and Analysis flSiOfpr

__Blofuels Bloproducbs & iBIorefkikig

Integrated enzyme production lowers the cost of cellulosic ethanol

Eric Johnson, Atlantic Consulting, Gattikon, Switzerland

Received September 8, 2015; revised November 20, 2015; and accepted December 15, 2015 View online February 18, 2016 at Wiley Online Library (wileyonlinelibrary.com); DOI: 10.1002/bbb.1634; Biofuels. Bioprod. Bioref 10:164-174 (2016)

Abstract: Previous studies of cellulosic-ethanol production have shown that the cost of producing cellulase is surprisingly significant, and that reducing this cost is key to making cellulosic-ethanol economically viable. This study confirms that finding, and compares the costs of the three approaches for producing cellulase: off-site, on-site, and integrated. It finds that the integrated method is the lowest cost, primarily because it substitutes an inexpensive feedstock, biomass, for a relatively expensive one, glucose. This substitution also makes the ethanol a 100% second-generation biofuel, i.e., it uses no 'food for fuel'. This study also compares the activity of cellulase produced by the integrated method versus that produced by the off-site method. Laboratory trials of the two show the 'integrated' cellulase to be better or equal to commercially available 'off-site' cellulase in converting cellulose to sugar. © 2016 The Authors. Biofuels, Bioproducts, Biorefining published by Society of Chemical Industry and John Wiley & Sons, Ltd.

Keywords: cellulosic ethanol; cellulase; bioethanol; cost; integrated cellulase; food for fuel

Introduction

Cellulose and hemi-cellulose are the most abundant types of biomass on Earth. As interest in renewable energy has surged over the past decade, so too has interest in developing a process for converting this biomass to ethanol at a cost that could compete with that of conventional gasoline (for which the ethanol can substitute).

One of the key challenges to cost-competitiveness,1-3 is the cost of the enzyme, the cellulase that converts polymeric cellulose into single molecules of sugar which are then fermented into ethanol. This challenge has been broadly recognized only recently,4 and to most observers it came as a surprise, because most prior studies 'significantly underestimated the contribution of enzyme costs to biofuel production'. Therefore, 'a significant effort is still required to lower the contribution of enzymes to biofuel production costs'.4

Cellulase is produced in an industrial fermentation process. Fungi feed on a stream of incoming nutrients, converting them to outgoing cellulase. This production process is typically characterized by two main variables:

• Location: Traditional cellulase production is off-site to the ethanol plant, i.e., at a central factory that supplies numerous cellulosic ethanol operations. On-site production of cellulase, adjacent to the ethanol plant, has more recently been presented as a potentially lower-cost option, by researchers.4-9

• Feedstock: Both off-site and on-site cellulase production use glucose as the primary feedstock. However, in an on-site configuration, it is possible to substitute glucose with much-cheaper cellulosic biomass - the same material that at the same site is being converted to ethanol. By using the same feedstock and location, it is possible to integrate cellulase and ethanol production - so this approach is called 'integrated'.

Correspondence to: Eric Johnson, Atlantic Consulting Obstgartenstrasse 14 CH-8136 Gattikon, Switzerland E-mail: ejohnson@ecosite.co.uk

This paper examines these three approaches to cellulase production: off-site, on-site, and integrated. First the approaches are described, then the mass balances are reviewed, and from that the costs are estimated and compared. In a final section, the paper examines another question regarding the cost of cellulase production: the activity of the cellulase, i.e., its ability to convert cellulose and hemi-cellulose from polymers into monomolecular sugars. Based on laboratory experiments, this paper compares the activity of cellulases that are produced from glucose and from biomass.

Process descriptions of cellulase production approaches

Based on a literature review and discussions with researchers and operators, descriptions have been compiled for the three approaches to cellulase production: offsite, on-site, and integrated (Fig. 1).

In all cases, the basic process is as follows. Feedstocks are fed to micro-organisms that produce an enzyme complex, cellulase. The cellulase is used to catalyze the breakdown of cellulose and hemi-cellulose into monosaccharides, which are then fermented into ethanol.

Off-site (glucose-fed)

In this approach, cellulase is produced off-site, at a central plant, from which it is shipped out to ethanol plants. The primary feedstock is glucose. Secondary feedstocks are a cocktail containing mainly nitrogen, phosphate and potassium compounds. Corn steep liquor can also be added as a source of protein.8

Figure 1. The three approaches to cellulase production.

The protein coming out of the fermenter contains a mixture of cellulase and other proteins. This is then formulated, i.e., purified to cellulase. Formulation is required in order to stabilize the cellulase for shipment, i.e., to keep it viable and active during transport. Post-shipment, the formulated cellulase is then fed to the ethanol plant.

On-site (glucose-fed)

This approach is essentially the same as that of off-site production, except that the cellulase plant is located on-site, adjacent to the ethanol plant. Both plants are stand-alone. Being 'next door' eliminates the need for formulation; the protein mixture is fed directly to the ethanol plant without purification and stabilization. The scale of cellulase production is larger in an off-site than in an on-site plant, which is sized to accommodate one ethanol plant rather than many.

Integrated (cellulose-fed)

This approach is on-site, but not stand-alone. Instead, the cellulase production is:

• Integrated, by both process and energy flows, directly into the ethanol plant.

• Fed by cellulose, not glucose. The same, pre-treated cellulosic feedstock is used for both cellulase and ethanol. A fraction of the cellulose going into the ethanol plant is diverted to cellulase production; the cellulose substitutes glucose as the primary feedstock for cellulase.

The volume of cellulase production is similar to that of an on-site, glucose-fed plant, and is sized to fit the production capacity of ethanol. Yields are assumed to be similar, because of the ability of process designers and operators to optimize the interaction of microorganism, substrate and product. This can be done with native or recombinant strains,10 and is a focus of ongoing research.11-14

With integrated enzyme production, it is possible to use complex substrates such as straw to produce enzymes with high activity, compared to those grown on glucose or sucrose. Enzyme production from a complex substrate is comparable to that of the simple substrate like sugars, if the process is engineered specifically for the chosen microorganism. Delabona et al.15 have shown that with selected micro-organisms, steam pre-treated bagasse provides higher FPase, xylanase, ^-glucosidase than do sucrose, glycerol and lactose. Pullan et al.16 have shown that the inducing substrate used for the production of enzymes has marked effects on the enzyme cocktail achieved. These findings highlight the limitations of using simple inducing

substrates to generate complex mixtures of enzymes. Using simple substrates with de-repressed fungal strains denies the benefits of fungi's enzymatic capability for complex substrates.

Neither human nor animal food is used as a feedstock, making this process the only 100% second-generation process for bioethanol.

Mass balances of cellulase and cellulosic ethanol production

Published cost estimates of cellulase production are almost always expressed with respect to ethanol output, i.e., US$ per unit of ethanol. Moreover, feedstock inputs and product yields are critical factors in determining cellulase costs. So to analyze the costs transparently, it is necessary to clarify the mass balances involved. Of these, there are three primary ones:

• Biomass:ethanol - the amount of biomass feedstock coming into the ethanol plant relative to the amount of ethanol going out.

• Feedstocks:cellulase - the amount of sugar or biomass into the enzyme plant relative to the cellulase product.

• Cellulase:biomass - the 'enzyme loading' of the ethanol plant, the amount of cellulase charged to the ethanol plant, relative to the amount of biomass feedstock charged to it.

Biomass:ethanol

Biomass in, ethanol out - this is the basic transformation behind cellulosic ethanol. A schematic is presented in Fig. 1, and a more-detailed flowsheet6 of the on-site process with ethanol production is also presented (Fig. 2). Based on an examination of the literature, particularly 6,8,17-19 and some discussions with operators, we derived a base-case mass balance of 1000 kg of dry biomass to 225 kg of pure ethanol (Table 1). This yield of 22.5% is representative of the reported range, lower than some of the studies,6,8 but higher than others20 as well as industry

estimates.18

In the literature, two of these bases are somewhat unclear:

Figure 2. Simplified flow sheet of the entire process (adapted from Humbird et al.6)

Table 1. Cellulose to ethanol, mass balance, base case.

Line item Amount Unit Comment

Biomass, dry 1000 kg Straw

Biomass, dry, carbohydrate content 65% weight (21) adjusted down slightly

Carbohydrate, dry 650 kg

Sugar/carbohydrate yield 80% weight

Sugar to fermentation, of which 520 kg

Glucose 60% weight

Xylose & Arabinose 40% weight

EtOH/sugar yield, theoretical 51% weight

EtOH/sugar yield, % of theoretical 85% weight

EtOH, mass 225 kg

EtOH density 0.789 kg/litre

EtOH, volume 286 litres

EtOH/Biomass, dry: calculated 23% weight

Table 2. Feedstocks to cellulase, mass balance, base case (weight).

Cellulase production approach

Off-site On-site Integrated

Biomass, dry 1000

Carbohydrate content in biomass 650

Glucose/Dextrose 703.2 703.2

Glycerol/sucrose 220.1§

Ammonium sulphate 12.63 12.63 11.67

Monopotassium phosphate 18.05 18.05 16.68

Magnesium sulphate 2.71 2.71 2.50

Calcium chloride 3.61 3.61 3.34

Sodium chloride 27.5 27.5 25.42

Ammonia 32.83 32.83 30.35

Antifoam 54.27 54.27 50.16

SO2 3.73 3.73 3.45

Corn steep liquor 99.62 99.62 92.08

Output

Cellulase, protein 110 128.6 118.871

Protein/glucose or Carbohydrate 15.6% 18.3% 18.3%

Protein/biomass 11.9%

§As a stabilizer in the formulation.

• Dry or wet biomass - Most papers refer to biomass input on a dry basis, but some are not explicit about it. If biomass is measured as wet, usually the assumption appears to be that moisture is 20% of the incoming mass.

• Biomass in its entirety or only as its carbohydrate content - Some papers are unclear as to their basis. We have assumed a carbohydrate content of 65%. An often-cited source21 suggests 67%, but review and discussions with producers suggest that actual carbohydrate content tends to be lower than this, so we adjusted it down slightly.

Feedstocks:cellulase

The most detailed mass balance for this in the literature is presented by Hong et al. (Supporting Information, Table S3), which was derived in part Humbird et al.6 Some detail is also presented by Dunn et al .22

Protein yields on glucose or carbohydrate are higher for the on-site and integrated approaches than for the offsite approach (Table 2), because there are 'protein losses during the purification process, which is only required for offsite production, whereas on-site production leads to dosing a whole fermentation broth. Thus, more protein/cellulase is available from an on-site [or integrated] production strategy.'23 The protein/biomass yield is lower than the

yield from glucose or carbohydrate, because the biomass itself is only 65% carbohydrate.

Other authors,20,24,25 support this general picture, but they do not provide quantitative mass balances. Both Hong et al.8 and Nielsen et al.25 report the use of stabilizer in off-site production. The former quotes the stabilizer as glycerol, the latter as sucrose.

Cellulase:biomass

This mass balance is often referred to as 'enzyme loading', i.e., the amount of enzyme that needs to be charged to the ethanol plant, relative to the amount of biomass being charged to the same plant. Based on Hong et al.,8 MacLean and Spatari,20 and Dunn et al.,22 we specified a base case loading of 1% weight cellulase to substrate, i.e., 10 g enzyme protein to 1 kg of biomass input to the ethanol plant.

In the literature, there are two aspects of this mass balance that can lead to miscalculations or inaccurate comparisons:

Table 3. Existing, commercial-scale cellulosic-ethanol plants.**

Annual capacity

Operator Location Volume Weight

Abengoa Hugoton, Kansas, USA 25 million1"1" gallons 74,677 tonnes

DuPont Nevada, Iowa, USA 30 million gallons 89,601 tonnes

POET-DSM Emmetsburg, Iowa, USA 25 million gallons 74,677 tonnes

Ineos Vero Beach, Florida, UA 8 million gallons 23,894 tonnes

GranBio Alagoas, Brazil 21 million gallons 62,721 tonnes

Mossi & Ghisolfi Crescentino, Italy 75 million litres 59,175 tonnes

**Sourced from company and press reports. ttlQ6

• Biomass in its entirety, or only as its carbohydrate content - This is the same issue mentioned above. Some papers express enzyme loading per unit of substrate (the entire dry biomass), while others express it per unit of carbohydrate in the biomass.

• Units of enzyme - Most studies refer to mass of 'protein', which, as Humbird et al. 6 point out, 'refers to the total amount of high molecular weight protein in the enzyme broth as determined by assay; not all of this protein is active cellulase'. This can be complicated,

in that different assay methods give different protein weights from the same sample. Some studies refer to filter paper units (FPUs) as a measure of enzyme loading. When off-site cellulase is used, it is not always clear if the enzyme loading refers to the protein alone or to the entire cocktail of protein plus stabilizer.

Overall, annualized mass balance (base case)

With the exception of a plant operated by Ineos, commercial-scale cellulosic ethanol plants range around the size of 70 000-tonne per year capacity (Table 3). So, this was chosen as the size for the base-case mass balance.*

Using that capacity, and then combining the mass balances presented above, an overall mass balance, from biomass to ethanol, was calculated for the three approaches to cellulase production (Table 4). The off-site approach requires less input biomass than the on-site one, because the glycerol/sucrose stabilizer is converted to ethanol, reducing the need for input biomass (and for input cel-lulase). The integrated approach obviously requires more biomass, because some of it is converted to cellulase.

*One reviewer of this paper noted that such plants might be limited by feedstock availability. This is a valid point. For comparitive purposes, nonetheless, the subsequent cost analysis is done on an equivalent-capacity basis, as is common in practice and in the literature.

Table 4. Overall mass balance - biomass and cellulase to ethanol, base case, tonnes per annum.

Cellulase production approach

Off-site On-site Integrated

Biomass, dry, for EtOH 299,042** 310,531 310,531

Biomass, dry, for cellulase 26,123

Glucose/dextrose, for cellulase 19,117 16,980

Glycerol/sucrose§§, for EtOH 5,984

Cellulase, protein 2,990 3,105 3,105

Output

Ethanol 70,000 70,000 70,000

^Adjusted for the glycerol/sucrose feedstock. §§Added as stabilizer to the cellulase

Cost estimates

Costs of the three production methods have been estimated. The bases of this are presented in this section. In the next section, the costs are compiled and compared.

Operating costs

As a base case, the mass balances presented in the previous section (Tables 1, 2, and 4) were applied and normalized to an ethanol output of 70 000 tonnes per year, equivalent to 88.720 million liters or 23.437 million gallons.

Prices for variable inputs (Table 5) were taken mainly from Humbird et al.,6 except for electricity and steam, which were sourced from the SRI Consulting Process Economics Program (SRI PEP), and cellulase transport, which was sourced from an industry estimate. For the cellulase plant, the same utility prices were used, while

Table 5. Prices of variable inputs to the cellulase and ethanol plants.

Raw materials & consumables

Biomass, dry 58.5 $/t

Ammonia 439.38 $/t

Ammonium sulfate 152.00 $/t

Antifoam 800.00 $/t

Biomass, dry 58.50 $/t

Calcium chloride 125.00 $/t

Caustic 149.47 $/t

Cellulase calculated in this work

Cellulase transport*** 52 $/t

Corn steep liquor 55.56 $/t

DAP 966.73 $/t

Glucose/Dextrose 568.57 $/t

Glycerol/sucrose 440.92 $/t

Lime 199.30 $/t

Magnesium sulfate 150.00 $/t

Monopotassium phosphate 800.00 $/t

SO2 303.80 $/t

Sodium chloride 100.00 $/t

Sorbitol 1103.41 $/t

Sulphuric acid 87.96 $/t

Water 0.22 $/t

Utilities

Electricity 0.05 $/kWh

Steam 1.5 $/t

Waste 28.86 $/t

***Applies only to the off-site production approach.

raw material prices were taken from a variety of sources, including Humbird et al.6 for the glucose/dextrose, Index Mundi+ for glycerol/sucrose and the rest from SRI PEP.

Fixed costs for labor, overheads and insurance were taken from Humbird et al.6 For the on-site and integrated approaches, it was assumed that the ethanol and the cel-lulase plants are operated as one unit, i.e., the fixed costs are spread across both plants. For the off-site approach, these were split among the ethanol and cellulase plants. However, total labor, overhead, and insurance costs are the same for all three approaches. We have not assumed that the off-site plant is run any less efficiently; presumably it is part of a larger chemical-processing complex where costs are integrated and optimized.

thttp://www.indexmundi.com/commodities/?commodity=sugar& months=60

Capital costs

A base capital cost for each production approach was estimated according to Humbird et al.,6 then this was adjusted for scale (at a 0.65 exponent). Scales for each approach are slightly different (Table 4), because the biomass input required per unit of ethanol output varies according to the production approach. Off-site and on-site use sugar as a feedstock for cellulase; off-site uses sugar to stabilize cel-lulase. Integrated requires no sugar as feedstock - this is substituted by biomass.

In addition to the 0.65-exponent scaling, other adjustments were made:

• The biomass-cellulase process is sized larger than the sugar-cellulase alternatives, to accommodate the bulkier feedstock.

• A cellulase formulation plant is included in the off-site approach, whereas cellulase formulation is not needed in the other two approaches.

There are no significant economy-of-scale differences between the approaches. Cellulase production in all three cases is at maximum size reported for industrial cellulase production.

This was then added to the capital cost of the ethanol plant, estimated from Humbird et al.,6 to come up with a full plant installed cost (Table 6). Straight-line depreciation was assumed, and this is taken as an annual charge - that does not include finance charges for debt or debt service.

Cost comparison

First the costs of cellulase production are presented, then those of the entire chain through to ethanol. Finally, these are compared to estimates from previous studies.

Production of cellulase

The biggest cost differences between the three production approaches are in the raw materials and consumables (Table 7). Primarily this comes down to the huge gap between the cost of biomass in the integrated plant and the cost of sugars in the off-site and on-site ones. The varying yields of cellulase (Table 2) also play a role, but far more modestly.

All of this rolls through to the cash cost of cellulase (Table 9), which is the cost used to calculate the overall cost of ethanol production (presented in the next subsection).

Production of ethanol

The cash cost of cellulase (Table 8) rolls through to the cash cost of ethanol production (Table 9), where it is the

Table 6. Capital costs, cellulase plant, and full EtOH/cellulase plant, US$.

Cellulase production approach

Off-site On-site Integrated

Cellulase capacity cost 9,319,261 8,166,476 8,166,476

EtOH full plant installed cost 214,319,261 213,166,476 216,203,445

Plant life, years 15 15 15

Capital charge, annual 14,287,951 14,211,098 14,413,563

Table 8. Cellulase annual cash costs, US$.***

Cellulase production approach

Cost item Off-site On-site Integrated

Raw materials & consumables 15,804,194 11,694,344 3,568,072

Utilities 1,794,251 1,863,189 1,863,189

Fixed costs

Labor 385,221 included in ethanol included in ethanol

Overheads & insurance 256,322 included in ethanol included in ethanol

SUM 18,239,988 13,557,533 5,431,261

***For 70 000-tonne/year ethanol production

Table 7. Cellulase raw materials

and consumables annual cost, US$.m

Cellulase production approach

Off-site On-site Integrated

Raw materials & consumables

Biomass, dry 0 0 1,528,220

Glucose/Dextrose 10,869,361 9,654,492 0

Glycerol/sucrose 2,638,298 0 0

Chemicals and 2,296,535 2,039,852 2,039,852

process aids

SUM 15,804,194 11,694,344 3,568,072

tt1"For 70 000-tonne/year ethanol production

Table 9. Cellulosic ethanol annual cash costs, USS.888

Cellulase production approach

Cost item Off-site On-site Integrated

Raw materials & consumables

Biomass, dry 17,493,945 18,166,090 19,694,310

Sulphuric acid 625,322 649,348 703,974

Ammonia 1,657,129 1,720,799 1,865,561

Corn steep liquor 230,864 239,734 259,901

DAP 492,611 511,538 554,571

Sorbitol 174,222 180,916 196,135

Cellulase 18,239,988 13,557,533 5,431,261

Cellulase transport 466,647

Caustic 1,207,937 1,254,348 1,359,870

Lime 640,085 664,678 720,594

Water 116,406 120,879 131,048

Utilities

Electricity 287,000 287,000 287,000

Steam 451,500 451,500 451,500

Waste 592,903 615,683 667,477

Fixed costs

Labor 2,114,779 2,500,000 2,500,000

Overheads & insurance 6,000,000 6,000,000 6,000,000

SUM 50,791,338 46,920,046 40,823,204

§§§For 70 000-tonne/year ethanol production

major cost difference between the three approaches. The bases of raw materials and consumables costs are presented in the previous two sections of this paper. Utilities consumptions are taken from Humbird et al.6 and prices from (Table 5). Fixed costs are taken from Humbird et al.6 with the off-site labor costs adjusted for the labor that occurs off-site (Table 8).

The cash costs (Table 9) and the capital costs (Table 6) of course roll through to full costs for cellulosic ethanol production (Table 10).

When full costs are itemized and normalized on the basis of per-gallon ethanol output (Table 11), it becomes clear that there are four items that when compiled account

for a bit more than 80% of full costs: overheads & insurance, capital/finance charges, biomass feedstock and cellulase production. The cost of cellulase is by far the most variable by production approach. With off-site, it accounts of 28% of full costs, with on-site it reduces to 22% and with integrated it falls to 10%.

Cellulase costs are significant, and variable by production approach

Following the findings of Cherry and Fidantsef,1 Fang et al.,2 and Brijwani et al.,3 Klein-Marcushamer et al.4

Table 10. Cellulosic ethanol annual full costs, US$.****

Cellulase production approach

Cost Off-site Off-site Off-site

Cash costs, $/year 50,791,338 46,920,046 40,823,204

Capital/finance costs, $/year 14,287,951 14,211,098 14,413,563

Sum, $/year 65,079,289 61,131,144 55,236,767

Sum, $/tonne ethanol 930 873 789

Sum, $/litre ethanol 0.73 0.69 0.62

Sum, $/gallon ethanol 2.78 2.61 2.36

****For 70 000-tonne/year ethanol production

Table 11. Cellulosic ethanol annual full costs, itemized per gallon, US$.tm

Cellulase production approach

Cost Off-site Off-site Off-site

Raw materials & consumables

Biomass, dry 0.75 0.78 0.84

Sulphuric acid 0.03 0.03 0.03

Ammonia 0.07 0.07 0.08

Corn steep liquor 0.01 0.01 0.01

DAP 0.02 0.02 0.02

Sorbitol 0.01 0.01 0.01

Cellulase 0.78 0.58 0.23

Cellulase transport 0.02 0.00 0.00

Caustic 0.05 0.05 0.06

Lime 0.03 0.03 0.03

Water 0.00 0.01 0.01

Subtotal 1.76 1.58 1.32

Utilities

Electricity 0.01 0.01 0.01

Steam 0.02 0.02 0.02

Waste 0.03 0.03 0.03

Subtotal 0.06 0.06 0.06

Fixed costs

Labor 0.09 0.11 0.11

Overheads & insurance 0.26 0.26 0.26

Capital/finance 0.61 0.61 0.61

Subtotal 0.96 0.97 0.98

SUM 2.78 2.61 2.36

ttttFor 70 000-tonne/year ethanol production

declare that for cellulosic ethanol production, 'the cost of producing enzymes was much higher than that commonly assumed in the literature.' Their paper4 cites costs

presented by other authors that range from $0.10 to 0.40 per gallon of ethanol; and it presents its own estimate of $0.68-1.47 per gallon. This study confirms that cellulase is indeed a significant cost (Tables 9 and 11).

This study models the current, available process approaches, finding that they have relatively comparable capital and operating costs, with the exception of the cost/ price of feedstock for the cellulase. This study then quantifies the differences, showing that this cellulase cost can be significantly reduced, from 0.78 to 0.58 to 0.23 $/gallon (Table 11), by shifting from the off-site to the on-site to the integrated approach of cellulase production. These are, respectively, 8% and 20% reductions in cash costs, and 7% and 19% reductions in full costs of cellulosic ethanol production.

Activity comparison of glucose- and cellulose-based cellulase

The preceding analysis finds integrated enzyme production to be lower cost than off-site or on-site. It also raises the question: How do the performances compare? Based on a blinded, benchmarked experiment (section on Experimental details), performance - in converting cellulose to monomers glucose and xylose - was compared of integrated enzyme versus off-site enzyme.

Sugar yields of integrated enzyme are better to or equal to that of off-site enzyme, across enzyme loadings ranging from 0.05 to 0.5%.* With neutral pre-treatment and 3-day enzyme exposure the performance is clearly better (Fig. 3); with acid pre-treatment it is about the same (Fig. 4).

A reason for the superior performance might result from the fact that the integrated enzymes are already produced on the feedstock that they are than used to hydrolyze. Hence, these enzymes are feedstock and process specific in comparison to the off-site enzyme which provides a compromise applicable for all processes and feedstocks, but for the price of a somewhat lower sugar yield.

Extra time does not help very-low loadings

One reaction to the high cost of enzyme could be to lower enzyme loadings and to raise enzyme exposure times, i.e., charge less enzyme to depolymerization but give it longer to react. So, how low can go we go? Based on a blinded,

*The enzyme loading in the preceding economic analysis was 1%. Analyses reported in the literature use loadings of 1-3%.

Figure 3. Sugar yield of integrated vs off-site enzymes (neutral pre-treatment).

Figure 4. Sugar yield of integrated vs off-site enzymes (acid pre-treatment).

benchmarked experiment (Experimental details section), 0.05-0.1% loadings are too low.

Very-low enzyme loadings - namely of 0.05% and 0.1% -improve their yields over time only modestly at best. Only in one of four cases, 0.1% enzyme after 7 days (Fig. 5), does the very-low loading even begin to reach the xylose yield achieved with a 0.5% loading. In all other cases (Figs 6-8) yields do not reach those of 0.5% loadings, even after 10 days of exposure.

Experimental detail

In the first experiment, cellulase was used to hydrolyze wheat straw pre-treated one of two ways: in pH neutral

0.1% Enzyme loading, neutral pretreatment

0 2 4 6 S 10

—Xylose, offsite enzyme -■- Xylose, integrated enzyme

Figure 5. Xylose yields, longer exposure, 0.1% enzyme loading, neutral pre-treatment.

0.1% Enzyme loading, neutral pretreatment

__________ __ 0.5% Integrated enzyme

-----------r-. 0.5% offsite envzme

-1— 7—— »

0 2 4 6

♦ Glucose, offsite enzyme ■ Glucose, integrated enzyme

Figure 6. Glucose yields, longer exposure, 0.1% enzyme loadings, neutral pre-treatment.

Figure 7. Xylose yields, longer exposure, 0.05% enzyme loadings, acid pre-treatment.

Figure 8. Glucose yields, longer exposure, 0.05% enzyme loadings, acid pre-treatment.

conditions and in acidic conditions. Samples of 200 mg (dry mass) of pre-treated straw were mixed with 1-0.1 mg enzyme (0.5-0.05% enzyme/substrate loading). Sodium acetate was added 100 mM up to 1 mL. The mixture was incubated for 3 days at 50°C. The supernatant was then analyzed by high-performance liquid chromatography (HPLC)

to determine its glucose und xylose content. The second

experiment was similar, except that enzyme loading was

0.2.0.1 mg, and the incubation was for 3-10 days.

References

1. Cherry JR and Fidantsef AL, Directed evolution of industrial enzymes: an update. Curr Opin Biotechnol. 14(4):438-443 (2003).

2. Fang X, Yano S, Inoue H and Sawayama S, Strain improvement of Acremonium cellulolyticus for cellulase production by mutation. J Biosci Bioeng. The Society for Biotechnology, Japan 107(3):256-261 (2009).

3. Brijwani K, Oberoi HS and Vadlani PV, Production of a cel-lulolytic enzyme system in mixed-culture solid-state fermentation of soybean hulls supplemented with wheat bran. Process Biochem 45(1):120-128 (2010).

4. Klein-Marcuschamer D, Oleskowicz-Popiel P, Simmons B and Blanch HW, The challenge of enzyme cost in the production of lignocellulosic biofuels. Biotechnol Bioeng 109(4):1083-1087 (2012).

5. Barta Z, Kovacs K, Reczey K and Zacchi G, Process design and economics of on-site cellulase production on various carbon sources in a softwood-based ethanol plant. Enzyme Res Jan:734182 (2010).

6. Humbird D, Davis R, Tao L, Kinchin C, Hsu D, Aden A et al., Process Design and Economics for Biochemical Conversion of Lignocellulosic Biomass to Ethanol. National Renewable Energy Laboratory Golden, Colorado, 136.pp (2011).

7. Sorensen A, Teller PJ, Lubeck PS and Ahring BK, Onsite enzyme production during bioethanol production from biomass: screening for suitable fungal strains. Appl Biochem Biotechnol 164 (7):1058-1070 (2011).

8. Hong Y, Nizami A, Bafrani MP and Saville BA, Impact of cellulase production on environmental and fi nancial metrics for lignocellulosic ethanol. Biofuels Bioprod Biorefining 7:303-313

(2013).

9. Khokhar Z-U, Syed Q-A, Wu J and Athar MA, Onsite cellulase production by Trichoderma Reesei 3EMS35 mutant and same vessel saccharification and fermentation of acid treated wheat straw for ethanol production. EXCLI Journal 13:82-97

(2014).

10. Mazzoli R, Development of microorganisms for cellulose-bio-fuel consolidated bioprocessings: Metabolic engineers' tricks. Comput Struct Biotechnol J 3:e201210007 (2012).

11. Horn SJ, Vaaje-Kolstad G, Westereng B and Eijsink VGH, Novel enzymes for the degradation of cellulose. Biotechnol Biofuels 5(1):45 DOI: 10.1186/1754-6834-5-45 (2012)..

12. You C, Chen H, Myung S, Sathitsuksanoh N, Ma H, Zhang X-Z et al. Enzymatic transformation of nonfood biomass to starch. Proc Natl Acad Sci USA 110(18):7182-7187 (2013).

13. Gomes D, Rodrigues AC, Domingues L and Gama M, Cellulase recycling in biorefineries—is it possible? Appl Microbiol Biotechnol 99(10):4131-4143 (2015).

14. Munjal N, Jawed K, Wajid S and Yazdani SS, A constitutive expression system for cellulase secretion in escheri-chia coli and its use in bioethanol production. Synthetic Biology and Biofuels Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg,

New Delhi, India: Public Library of Science; PLoS One 10(3): 1-17 (2015).

15. Delabona PDS, Farinas CS, da Silva MR, Azzoni SF and Pradella JGDC, Use of a new Trichoderma harzianum strain isolated from the Amazon rainforest with pretreated sugar cane bagasse for on-site cellulase production. Bioresour Technol. 107:517-521 (2012).

16. Pullan ST, Daly P, Delmas S, Ibbett R, Kokolski M, Neiteler A et al., RNA-sequencing reveals the complexities of the transcriptional response to lignocellulosic biofuel substrates in Aspergillus niger. Fungal Biol Biotechnol 1(1):3 (2014).

17. Tao L, Schell D, Davis R, Tan E, Elander R and Bratis A. NREL 2012 Achievement of Ethanol Cost Targets : Biochemical Ethanol Fermentation via Dilute-Acid Pretreatment and Enzymatic Hydrolysis of Corn Stover. National Renewable Energy Laboratory Golden, Colorado (2014).

18. biochemtex. Crescentino: World's first advanced biofuels facility .[online]. Available at: http://www.biofuelstp.eu/pres-entations/crescentino-presentation.pdf [February 09, 2016].

19. Wu M, Wang M, and Huo H, Fuel-Cycle Assessment of Selected Bioethanol Production Pathways in the United States. Argonne National Laboratory, US Department of Energy, OSTI, Oak Ridge, TN, USA. (2006).

20. MacLean HL and Spatari S. The contribution of enzymes and process chemicals to the life cycle of ethanol. Environ Res Lett 4 (1):014001 (2009).

21. Lee D, Owens V, Boe A, Jeranyama P. Composition of Herbaceous Biomass Feedstocks. 2007.

22. Dunn JB, Mueller S, Wang M and Han J, Energy consumption and greenhouse gas emissions from enzyme and yeast manufacture for corn and cellulosic ethanol production. Biotechnol Lett 34(12):2259-2263 (2012).

23. Saville BA. Personal Communication. 2014.

24. Hsu DD, Inman D, Heath G a, Wolfrum EJ, Mann MK and Aden A, Life cycle environmental impacts of selected U.S. ethanol production and use pathways in 2022. Environ Sci Technol 44(13):5289-5297 (2010).

25. Nielsen PH, Oxenboll KM and Wenzel H, LCA Case Studies cradle-to-gate environmental assessment of enzyme products produced industrially in denmark by Novozymes A / S. Int J LCA 12(2006):432-438 (2007).

Eric Johnson

Eric Johnson is Managing Director of Atlantic Consulting, Editor-in-Chief Emeritus of Environmental Impact Assessment Review, an editor of the Journal of Health and Pollution, and a Technical Advisor to two NGOs, Green Cross and the Blacksmith Institute. He serves on the CEN/ISO Committees that are developing 'sustainability' criteria for biofuels. Technology, economics and environmental impacts of fuels, chemicals and plastics - these are key areas of his expertise. He is a chemist, who began his career as an editor of Chemical Engineering and Chemical Week magazines. He has been nominated by Switzerland's federal government as an IPCC inventory assessor.