Bioenergy

Application sheet

Novozymes Cellic® CTec3 HS

- secure your plant's lowest cost
Cellic® CTec3 HS is a highly efficient cellulase and hemicellulase complex
that ensures a cost-efficient conversion of pretreated lignocellulosic
materials to fermentable sugars. The improvement in conversion
efficiency provided by Cellic® CTec3 HS over previous generations unlocks
new opportunities to optimize pretreatment, hydrolysis, and fermentation
processes to ultimately secure the lowest total cost of producing cellulosic
ethanol for your plant.

Benefits
 Significant reduction in the total cost of producing ethanol
 Great process and substrate versatility
 New opportunities to optimize conversion processes, including:
 Increasing biomass to sugar conversion
 Decreasing enzyme dosing
 Increasing total solids loading
 Reducing hydrolysis time
 Reducing the severity of pretreatment

Performance
Cellic® CTec3 HS contains proficient cellulase components boosted by proprietary enzyme activities, including
advanced AA9 molecules, improved β-glucosidases as well as a new array of hemicellulase activities, and
together, they improve the conversion efficiency of Cellic® CTec3 HS by at least 1.5 times over Cellic® CTec2.

Usage
Novozymes has partnered widely with leading industry players, and together, we have worked with the Cellic®
product family to optimize the cellulosic ethanol production process. We have gathered application
information using Cellic® CTec3 HS in various process configurations with different types of feedstock and
pretreatment processes, and the insights we have gained have helped to generate the general application
recommendations contained in this document.
It is recommended to dose Cellic® CTec3 HS according to the level of cellulose contained in the substrate to
allow for direct comparisons of enzyme efficiency on various biomass materials, including those from different
types of feedstock and pretreatment combinations. The suggested enzyme trial dosing levels for initial
investigation of a substrate are between 1% and 6% w/w (g-Cellic® CTec3 HS/g-cellulose). These data points
should then be used to generate a dose-response curve (enzyme dosing vs. percentage cellulose conversion).
Commercially feasible cellulose conversion depends on many factors, including feedstock cost, type of
pretreatment, plant capacity, process yields, and value of co-products.

In general, the lower end of the dosing range recommended above should serve as a target for what could be
considered commercially feasible cellulose hydrolysis. Additional testing is recommended to refine the dose
response curve and determine the effects of time, solids loading, cellulose conversion, and enzyme dosing.
Note that Cellic® CTec3 HS contains a significant amount of sugar, so a reference/blind sample should be
included if a high enzyme dose is applied (see note on product formulation below).

100%
90%
80%
Cellulose conversion

70%
60%
50%
40%
30%
20%
10%
0%
0 1 2 3 4
Enzyme dose
(g Cellic® CTec3 HS/100g-cellulose)

Fig. 1. Enzyme dose response for Cellic® CTec3 HS after 5 days of enzyme hydrolysis at 50°C and pH 5.0 of an unwashed
sample of dilute acid-pretreated corn stover (18% total solids loading). If 80% conversion is required, then the
recommended dosage for this feedstock under the hydrolysis conditions mentioned above would be ~3% w/w (30g-Cellic®
CTec3 HS/kg-cellulose)

Cellic® CTec3 HS is a highly efficient cellulase and hemicellulase complex that works on a variety of substrates,
including acid, auto-hydrolyzed, and alkaline pretreated substrates. Frequently, only Cellic® CTec3 HS will be
needed. However, if the pretreated feedstock of interest contains an appreciable amount of hemicellulose, it is
advisable to combine Cellic® CTec3 HS with other lignocellulytic enzymes to further boost overall conversion
yields and potentially reduce the severity of your pretreatment. The enzyme type and dosage recommendation
will depend on product, feedstock type, pretreatment technology, and processing conditions. For further
technical advice about your specific substrate and targets, please contact your technical representative from
Novozymes.

Considerations for enzyme application

Historically, cellulosic enzymes were significantly affected by feedback inhibition resulting from glucose
production, which led to ineffective conversion. Later generations of enzymes, including Cellic® CTec3 HS, were
developed to eliminate this effect on the enzyme complex. With the introduction of Cellic® CTec3 HS, there are
more options available for consideration when selecting optimal process layout and targeting cost-efficient
cellulose conversion. These include simultaneous saccharification and fermentation (SSF), separate hydrolysis
and fermentation (SHF), and a modified version of these referred to as hybrid hydrolysis and fermentation
(HHF).

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The hybrid HHF process can also be described as viscosity reduction and pre-saccharification followed by SSF
for ethanol production, and this setup can be configured in relation to overall process layout and conversion
targets. The impact of hydrolysis and fermentation conditions using Cellic® CTec3 HS on pretreated corn stover
is illustrated in figure 2 (enzyme and fermentation kinetics and final ethanol yields will depend on pretreated
substrate, fermentation organism, and process configuration).

SSF HHF SHF

100%
90%
80%
Relative ethanol titer

70%
60%
50%
40%
30%
20%
10%
0%
Time

Fig. 2. Ethanol yield under varying hydrolysis conditions using Cellic® CTec3 HS at pH 5.0 and 50 °C on unwashed dilute
acid-pretreated corn stover at 18% total solids loading. The yeast was pitched at different times, as indicated by initiation
of ethanol production. The ethanol yield will vary depending on the substrate, enzyme dosing, yeast pitch, and hydrolysis
configuration. In this example, an SSF configuration does not achieve the same yields as the options that include a
dedicated hydrolysis step prior to fermentation. The process time available for hydrolysis and fermentation will dictate the
options available

Optimum pH and temperature

Optimal performance of Cellic® CTec3 HS occurs at a temperature range of 50-55°C and at pH 4.75-5.25, as
illustrated in figures 3 and 4. The optimal conditions can vary with specific pretreated substrates and process
conditions (e.g. solids content and hydrolysis time); however, our recommendations are to conduct hydrolysis
within the range of conditions specified above.

Please consult with your Novozymes representative for further guidance on identifying optimal conditions for
your specific substrate and process. We recommend that you monitor and control pH and temperature within
the ranges suggested above in that stage of your process where enzymes are applied, as major deviations
could have an impact on hydrolysis kinetics and final conversion yields. Complex enzyme systems may be
irreversibly damaged by high temperature and strong acidic or alkaline conditions. Due to this fact, we strongly
recommend that you adjust pH and temperature to proper operational conditions prior to enzyme addition in
order to ensure optimal conversion of your pretreated substrate.

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 120%
110%
100% 100%

Relative performance
90%
Relative performance

80%
80%
70% 60%
60%
50% 40%

40%
20%
30%
20% 0%
25 30 35 40 45 50 55 60 65 70 75 2 4 6 8
Temperature °C pH value

Fig. 3. Relative product performance of Cellic® CTec3 HS Fig. 4. Relative product performance of Cellic® CTec3 HS
with respect to reaction temperature for unwashed acid- with respect to pH of reaction for unwashed acid-pretreated
pretreated corn stover (PCS) at 18%TS for 3-5 days; pH 5 for corn stover (PCS) at 18%TS for 3-5 days; temperature
all data points 52.5°C for all data points

The product
Biomass substrates are composed of lignin and a number of mutually entangled and chemically bonded
carbohydrate polymers that require multiple enzymes working together synergistically for complete hydrolysis.
For this reason, the performance of a complex multicomponent enzyme product such as Cellic® CTec3 HS
cannot be evaluated based on the activity of a single enzyme or using single polymer substrate such as filter
paper, Avicel or carboxymethyl cellulose (CMC). Therefore, Novozymes has developed a performance activity
assay based on acid-pretreated corn stover for our internal quality control, so we can deliver high performance
products to our customers in a consistent manner. The method uses a fluorescence enhancement marker and
measures the extent of cellulose hydrolysis relative to a standard enzyme concentration for quantification of
Biomass Hydrolysis Units - BHU(2)-HS. Cellic® CTec3 HS contains chemical formulations for improved storage
stability, including glucose and/or sucrose. These compounds may affect the interpretation of hydrolysis data
analysis when Cellic® CTec3 HS is dosed at high levels.

There is more information about the products available at Novozymes Market.

Safety, handling and storage

For best product performance, Cellic® CTec3 HS should be stored at cool temperatures in closed containers
protected from sunlight. The product has been formulated for optimal storage stability; however, enzymes
gradually lose their activity over time. The recommended storage conditions are 0-10°C (32-50°F). Prolonged
storage time and/or adverse conditions such as higher temperature may lead to a higher dosage requirement.

Safety, handling, and storage guidelines are provided with all products. Please contact your Novozymes
representative for this information.

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Additional information
Our committed expert technical and commercial teams will be pleased to help you commercialize cellulosic
ethanol production while always keeping your specific goals in mind. For further information, please contact
your Novozymes representative. Join us on the path to commercializing cellulosic ethanol!

About Novozymes Novozymes Latin America Ltda. Novozymes A/S

Novozymes is the world leader in biological solutions. Together with customers, partners Rua Professor Francisco Ribeiro, Krogshoejvej 36
and the global community, we improve industrial performance while preserving the 683 2880 Bagsvaerd
planet’s resources and helping build better lives. As the world’s largest provider of Parana (PR) Denmark
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Apr 18, 2017 · Luna No. 2017-13750-03

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written agreement between the customer and Novozymes to such effect, information provided in this document “AS IS” is of good faith and does not constitute a representation or
warranty of any kind and is subject to change without further notice. novozymes.com