Biodiesel Handbook PDF

THE
NOVOZYMES
ENZYMATIC
BIODIESEL
HANDBOOK
Refined Oil
Flexible
Feedstocks
THE NOVOZYMES ENZYMATIC BIODIESEL HANDBOOK
TABLE OF CONTENTS
Introduction 5
CHAPTER1. ENZYMATIC TRANSESTERIFICATION 7

Enzymatic catalysis 9
What is an enzyme? 10
Enzymes in biodiesel production 11
Enzyme handling 14
Enzyme safety 14
CHAPTER 2. NOVOZYMES ENZYMATIC BIODIESEL PROCESS 17

Equipment and unit operations 19
Control parameters for feed oil 20
Feed oil pre-treatment 23
Filtration 23
Phosphoric acid wash 23
Degumming 24
Water wash 24
Mineral acidity neutralization 25
Downstream refining and product polishing 27
Caustic wash 28
One-pot process 29
Resin catalyzed transesterification 31
Enzyme inactivation 31
CHAPTER 3. FEEDSTOCK FLEXIBILITY FOR ENZYMATIC BIODIESEL 33

General feedstock quality 34
Feedstock influences processing 35
How to compensate for changing feedstocks 36
Lipid composition 36
Surfactant concentration 37
Melting point 38
CHAPTER 4. LABORATORY TOOLS FOR QUALITY ASSESSMENT AND CONTROL 41

Tools for laboratoryoratory evaluation of feed oils 42
Analytical tools 42
Basic compositional analysis 43
Advanced analytical tools 43
Laboratory assays for enzymatic biodiesel 45
Settling test for emulsifiers 45
Laboratory pre-treatment assay 46
Mineral acidity assay 47
Mini batch conversion assay 48
Enzymatic activity assay 48
Brix water/MeOH/gly 49
CHAPTER 5. PROCESS OPERATION 53

Process startup and operation 54
Troubleshooting 57
Enzyme recycling strategies 59
CHAPTER 6. NOVOZYMES AS ENZYME SUPPLIER 63

Access to strategic engineering partners 64
Joint technical support 65
CONCLUSION 67
LIST 68
List of figures 68
List of tables 68
List of equations 68
List of appendices 69
4
INTRODUCTION
Introduction
The biodiesel industry has been under pressure over the past four to six
years due to rising concerns about the price and availaboratoryility of
usable feed stocks for chemical biodiesel production, as well as the food-
versus-fuel issue.
For these reasons, cheap low quality and non-food oils have long been
considered for biodiesel feedstock. However in chemical processing,
these feed materials often require difficult and costly refining in order
not to harm the chemical catalyst. In addition, chemical processes often
require harsh chemicals, high energy input and high stoichiometric
MeOH excess, all adding to the capital and operational expenses.
Enzymatic biodiesel has been considered a promising alternative for the

past 10-15 years in the scientific community. Only recently, however,
has this potential become available with Novozymes’ official launch of
Novozymes Eversa® Transform, an enzyme with proven performance as
part of the first commercial-scale enzymatic biodiesel process in the world.
This Enzymatic Biodiesel Handbook is intended for use as a practical

guide to enzymatic transesterification and as a real-life toolbox.
Here is a short summary of the key requirements for processing low-cost

feedstocks. These are covered in-depth within this document:
• Robust pre-treatment and mineral acid neutralization

• Reactant addition strategy and reactor mixing input
• Selection of enzyme recycling strategy
• Selection of polishing method
• Fast and reliable analysis tools for process monitoring and quality
control
All the information provided herein is advisory and it is recommended to

contact Novozymes directly for more information on the enzyme and the
enzymatic process.
5
CHAPTER 1. ENZYMATIC TRANSESTERIFICATION
6
CHAPTER1. ENZYMATIC TRANSESTERIFICATION
CHAPTER 1.
ENZYMATIC
TRANSESTERI-
FICATION
7
Biodiesel is a term used for fatty acid methyl esters (FAME) used as a fuel
source for automotive or other transport forms. The fatty acid methyl
ester precursor can be a free fatty acid (FFA), triglyceride (TG), diglyceride
(DG) or monoglycerides (MG). To a lesser extent, traces of other fatty
species like phospholipids, wax esters and others can contribute
marginally to the FAME production.
In order to reduce complexity, this document focuses on glycerides

and FFA, as these precursors contribute by fattening the bulk of FAME
production. In contrast, polar lipids are generally undesirable due to their
emulsifying properties.
Fig. 1 shows the chemical structure of TG, DG, MG and FFA. Here all
the fatty acids are exemplified as oleic acid. In real-life applications, any
feedstock will be composed of a distribution of different fatty acids of
various chain lengths and degrees of saturation.
O
O
A
O
O
O HO
O
OH B O
O
OH
O
C
O
O
D O
OH
Fig. 1: A: Oleic triacylglycerol (TG), B: Oleic diacylglycerol (DG), C: Oleic monoacylglycerol
(MG), D: Oleic free fatty acid (FFA)
In biodiesel terms, “transesterification” describes the reaction between

a glyceride molecule and methanol (MeOH) to form a partial glyceride or
free glycerin molecule and a FAME molecule. Strictly speaking, the reaction
between a FFA and MeOH is an esterification reaction, but for simplicity’s
sake, this document will use “transesterification” to describe both.
8
Fig. 2 shows the full reaction scheme from glycerides and FFA to FAME.
R1 O
O
HO R2
O
O k1 O O
+ R OH
+
k-1 R1
O R2 O O R
O O O
R3
R3
HO
HO k2
O OH O
+ R OH
+
k-2 R2
O R2 O O R
O O O
R3
R3
OH
HO
k3 O
+ R OH + R3
k-3 O R
OH
OH OH
O O
R3
OH O
+ R OH
R + H 2O
R O O O
Fig. 2: Reaction scheme of transesterification of acylglycerides and FFA
Biodiesel production by transesterification of glycerides and fatty acids

is a well-known reaction. Common chemical catalysts like sodium
hydroxide (NaOH), sulfuric acid (H2SO4) and sodium methoxide (NaOMe)
are widely used.
The challenge with chemical catalysis is that all variations of this process
require highly refined feed oil in order to perform. As long as refined soy
bean oil or similar is available at a decent price, this is not a problem. But
with rising prices of refined oil and the ongoing food-versus-fuel debate,
a plant’s process economy suffers and global production of biodiesel
declines as a result. Taking low quality oils with high FFA and other trace
contaminants into a chemical process requires costly refining, which
often limits conventional biodiesel production.
Enzymatic catalysis
Enzymatic technology enables biodiesel producers to circumvent these

obstacles and produce high quality biodiesel from waste and non-food
9
oils, as well as oils with any concentration of FFA from 0-100%. The
enzymatic catalyst functions independently of feed oil FFA concentration,
and remains efficient in a moisture-containing environment.
In addition the enzyme catalyst is highly specific, thus enzymatic
transesterification produces no unwanted side products like polymers
and oxidation compounds.
What is an enzyme?
Fig. 3. An enzyme is a molecule from a living organism consisting of a

folded strand of amino acids. In nature, enzymes are responsible for all
the biochemical reactions within living organisms, including breakdown
of food and tissue generation.
The functionality of any enzyme is usually limited to one specific

reaction, which naturally hinders any unwanted side reactions. Owing
to this specificity, the action of most enzymes can be compared to a key
in a lock. Like a key in a lock, only a specific set of substrates will fit into
the active site of an enzyme, and only one catalytic conversion can take place.
Substrates
Product
Enzyme Enzyme-substrate complex Enzyme
Fig. 3: Lock-and-key model of enzymatic catalysis
Novozymes is world leader in production of industrial enzymes. In

most cases, production starts with identification of a gene coding for a
specific enzyme, which is then spliced into the genome of a well-known
microorganism. This organism is then fed on sugar and other nutrients
and grown in large-scale fermentation processes that are designed
to maximize enzyme expression. The free enzyme is purified from the
fermentation broth and further processed. In the final formulation,
stabilizers and other additives may be used to ensure stability and shelf
life of the enzyme product.
10
Fig. 4 exemplifies the industrial production of enzymes.
Microorganisms
Raw materials
Formulation
Liquid products
Immobilized product
Purification
Fermentation
Microorganisms to be inactivated
Fig. 4: Step-by-step overview of enzyme production
Enzymes in biodiesel production
As biological molecules, most enzymes are water-soluble and generally

functional in a water-rich environment. To perform efficiently, an
enzymatic reaction system actually requires low levels of water to be
present, even though this is counter-intuitive to conventional thinking
regarding biodiesel production.
Water can be added to an enzymatic system as free water, with

wet MeOH, or it can be produced in situ by esterification of FFA. In
addition, water-soluble glycerin is the main side product of glyceride
transesterification and the consequence is a two-phase system with a
heavy phase (HP) consisting of water, glycerin and MeOH, and a light
phase (LP) consisting of glycerides, FFA and FAME with some dissolved
MeOH as well.
In a biodiesel system, the hydrophilic nature of the enzyme causes it to

go into the heavy phase (HP). The interactions between the enzyme and
the fatty reactants happen on the interphase layer between the light and
heavy phase, so the rate of reaction is dependent on both the overall
contact surface area of the light and heavy phases as well as the actual
concentration of enzyme on this interphase.
11
Fig. 5 shows a snapshot of the interactions between a suspended droplet

of heavy phase and the continuous light phase.
HO O
O
HO O
HO
O O O O
O O
O
O O
O
O
Fig. 5: Schematic representation of important interactions between the heavy and light
phases in an enzymatic biodiesel system
In a real-life application of enzymatic biodiesel production, a pseudo

three-phase system occurs, illustrated in Fig. 6.
(Note about Fig. 6: In real-life enzymatic biodiesel production, the feed

oils will likely be of poor quality with significant color content. For
the sake of a clear visualization of the phase separation, clean refined
soy bean oil was used in Fig. 6 to illustrate the phase behavior of the
system.)
Fig. 6 shows a light phase with > 95% FAME on top and a clear phase
consisting of glycerin, water and MeOH on the bottom. In between the
light and heavy an emulsified interphase layer phases can be seen. This
layer consists of a mix of FAME, partial glycerides, glycerin, water and
MeOH. In addition, research has shown that 90-95% of the residual
enzymatic activity is tied to this layer.
Taking advantage of this fact, Novozymes has developed strategies for

recycling and re-use of a liquid enzyme formulation. By retaining the
interphase layer in the reaction system while draining both the primary
FAME product from the top of the tank and the secondary glycerin
side product from the bottom, a maximal utilization of the enzyme is
12
achieved with no or very little capacity loss between cycles. See the
section on Enzyme Recycling Strategies on page 59-61, and in Table 10
on page 60.
In full scale application, it has been shown

that an overall enzyme consumption of 0,2
wt% of oil or lower is achievable, making the
cost of the enzymatic process competitive to
conventional chemical technology.
Further strengthening the overall process

economy is the ability to use not only a lower
overall excess of MeOH but also the ability to
use wet MeOH, thereby significantly reducing
costs for MeOH rectification.
Finally, compared to conventional biodiesel

technology, a superior glycerin side product is
produced with virtually no salt content. High
purity technical grade glycerin is produced just
Fig. 6: Appearance and phase by removing water and MeOH, see Fig. 7.
behavior of crude enzymatic Further refining by resins and/or activated
soybean biodiesel carbon treatment can bring the glycerin
concentration well above 99% concentration,
making a high purity side product from a
waste starting material.
Fig. 7: Comparison of enzymatic and chemical glycerin quality. Courtesy of Piedmont
Biofuels
13
Enzyme handling
Being biological molecules, enzymes are generally stable at ambient

conditions but are sensitive to extreme conditions such as high
temperatures. At these extremes, the three-dimensional structure of the
enzyme molecule starts to unfold and the enzyme irreversibly loses all
catalytic activity.
The formulation of Eversa® Transform ensures a high stability and long

shelf life of the product. Eversa® Transform has a guaranteed six-month
shelf life at <10°C storage temperature and a guaranteed two-month
shelf life at <25°C storage temperature.
Enzyme safety
Like pollen from birch or grass, enzymes are protein molecules that in
some cases can cause allergenic sensitization. Like pollen, enzymes are
relatively large molecules and can only enter the blood stream and possibly
provoke an immune response through the respiratory system. Preventing
aerosol formation and using appropriate respiratory safety equipment will
prevent this from happening.
In case of skin contact, active enzymes can cause irritation. Therefore

protective gloves are required when handling the enzyme formulation.
Furthermore, enzyme spills should always be avoided, and any incident
should immediately be cleaned or washed away.
Please see Novozymes MSDS sheet and enzymatic biodiesel application

sheet in Appendix I and II for further information.
14
15
CHAPTER 2. NOVOZYMES ENZYMATIC BIODIESEL PROCESS
16
CHAPTER 2.
NOVOZYMES
ENZYMATIC
BIODIESEL
PROCESS
17
The Novozymes enzymatic biodiesel process has been developed in

close collaboration between Novozymes’ R&D, leading global process
engineering companies and commercial-scale industrial biodiesel
producers.
This close collaboration has resulted in an efficient and robust process

design for enzymatic transesterification. Fig. 8 shows a process diagram
of the enzymatic biodiesel process.
Novozymes Eversa®
Feedstock Biodiesel
Methanol
Enzymes
Water
Oil
pretreatment Polishing
FAME after reaction

Separation of crude
Flash
dryer
Pretreated oil
Heavy phase
Enzyme reactor
Fig. 8 Enzymatic biodiesel process
It is important to mention that Novozymes, as the enzyme supplier,

cannot take responsibility for the engineering of a commercial-scale
enzymatic biodiesel plant. Plant engineering, commissioning and/or
retrofitting should preferably be done in collaboration with an external
engineering company with expertise in this area.
Alternatively, the engineering can be done internally but Novozymes

highly recommends employing an experienced engineering company.
Novozymes’ engineering partners have extensive experience with the

process development and operation, which will help ensure a functional
and optimized process design at a reasonable cost. This will also give
access to a joint technical service support program from both Novozymes
and the engineering partner, which will ensure fast and reliable start-up
and troubleshooting of all aspects of the enzymatic biodiesel process.
18
Equipment and unit operations
Table 1 shows a list of the required equipment and unit operations

necessary to run the enzymatic biodiesel process. Every item in Table 1
corresponds to the process layout shown in Fig. 8.
Item Comments
5 micron filtration and water wash is recommended for all feed oils.
Enzymatic and/or acid degumming is recommended for feed oils high
1 Feed pre-treatment in non-hydratable phospholipids. Mineral acid neutralization by NaOH
addition is required for all feed oils as the last pre-treatment step
before enzymatic reaction.
2 Feed oil tank Contains and preheats the pre-treated feed oil.
2% water is required in the reaction. It can be added as free water
3 Water tank in the feed stream or directly into the reactor. Enables direct use of
rectified wet MeOH from the process, see items 6 and 12 below.
A separate enzyme tank might be an advantage for very large-scale
productions. In most cases the enzyme can be stored in and added
4 Enzyme tank directly from the shipping containers. For long-term storage > 6
months, a storage temperature of < 10 °C is recommended. Dilution
of enzyme with water is not recommended.
MeOH tank for adding MeOH to the process. A separate dosing
pump is required to manage the MeOH flow to the process.
Commonly the water concentration in the process is controlled by
5 Methanol tank
having two MeOH tanks with wet and dry MeOH respectively. Wet
MeOH is rectified and recycled from the product and side product
streams, see items 11 and 12 in this list.
Main reactor of the process and key factor for sizing the rest of the
process. A stainless steel tank is recommended with height/diameter
ratio of 1,0 to 1,5 and a sloping or cone-shaped bottom. The reactor
should be fitted with a bottom outlet.
6 Enzyme reactor Good temperature control is required. Depending on the ambient

climate, heating or cooling of the reactor might be necessary.
Level control is also recommended to avoid overflow. Biodiesel outlets
and sample ports can be placed at several points along the vertical
axis of the reactor to ensure efficient product draining and QC
sampling.
The rate of the enzymatic reaction is proportional to the interphase
surface area between the light and heavy phases in the enzymatic
biodiesel system. Intensive mixing is the best way to decrease the
heavy phase droplet size and thereby increase the surface contact
area. Be aware, however, that oil/water systems can emulsify
irreversibly if energy input is too high so it is important to maintain a
rough balance.
7 Mixing Several effective options exist for intensive mixing. One solution is
a nozzle-type mixing device called eductors, which are being used
successfully in commercial enzymatic plants running today. A set of
eductors fitted to a circulation loop around the main reactor can give
a high volumetric turnover rate for primary mixing in the enzymatic
biodiesel process. With eductors, the mixing intensity in the reactor
should turn over the entire volume every 10-20 minutes to achieve
good reaction rates. Drawing the circulation stream from a bottom
outlet prevents pockets of precipitated heavy phase from forming.
The key purpose of the primary settler is to separate crude biodiesel
from the heavy phase.
Gravimetric settling in the reactor is possible and offers a simple

but slow process. It will take up capacity in the reactors and can be
affected negatively by excess surfactants in the feed oil.
8 Primary separator
Settlers and centrifuges are faster and more reliable, but require
CAPEX and OPEX to install and operate.
A secondary separator can be beneficial to separate the heavy phase

into clear glycerin and interphase layer.
Table 1 Key unit operations and technical requirements of the enzymatic biodiesel process
19
Table 1 does not contain auxiliary equipment like pumps, valves and
piping. For a detailed PID diagram, we highly recommend using the
services of one of Novozymes’ local engineering partners. Novozymes
collaborates with several global engineering companies who know the
ins and outs of the process and can design and instrument a process
according to local needs and opportunities.
Control parameters for feed oil
Table 2 shows a list of analyses to be used as control parameters for

efficient operation. Each analysis method and control tool will be
described in detail in Section 4 on page 41, describing laboratory tools
and analyses.
Feed oil
Purpose Tools and methods Specifications
analysis
Before pre-treatment
Determines TG,
DG, MG and FFA
Feed oil
concentration to NIR/GC/HPLC, FFA titration No limits
composition
estimate required
MeOH addition load
Determines untreated
Feed oil settling Feed oil settling test Min. 45ml oil/15 min.
settling time
Feed oil un- Determine how much
Un-SAP method Max. 2%
saponifiables oil is convertible
Feed oil water Determine the amount
Titration method (Karl Fisher) Max. 0,5%
content of water
Determine the amount
Feed oil solid
of particles suspended Centrifuge laboratory test Max. 0,1%
content
in the oil
After pre-treatment
Recommended feed
Feed oil soap
oil soap after pre- Soap titration Max. 500 ppm
level
treatment 50-500 ppm
Recommended feed
Feed oil moisture
oil moisture after pre- Karl-Fischer titration Max. 7000 ppm
level
treatment
Phosphorous can
Feed oil challenge separation
ICP/P-NMR Max. 50 ppm
phospholipids if enzyme emulsion is
recycled
Determines pre-treated
Feed oil settling Feed oil settling test Min. 45ml oil/15 min.
settling time
Outcome of NaOH
NaOH See detailed section
assay gives NaOH load NaOH assay
determination on this
to reactor
Table 2 Strongly recommended feed oil analyses before and after pre-treatment
Table 2 describes the minimum analysis required to approve a pre-treated

feedstock.
20
It is highly important to perform these initial feedstock analyses

in order to determine critical operational factors like NaOH, MeOH
and moisture loads before adding a particular feedstock into the
reactor.
In addition, the settling test will shed light on the settling performance of
a feed oil and thereby on the performance of the pretreatment process,
which should eliminate poor settling feedstocks. These analyses are usually
performed in a QC laboratory by laboratory technicians. For more details,
see Section 4 on page 41, describing laboratory tools and analyses.
Table 3 lists the key operating conditions for the enzymatic

transesterification process.
Numerical limits are quoted for each parameter. The moisture, enzyme
and MeOH loads are determined for any particular feedstock before the
process is started.
The MeOH load is calculated based on the glyceride composition of the

feed oil.
The total water load of 2 wt% of oil has several contributing factors.
The feed oil will contain moisture residues from the pre-treatment
process (500-5000 ppm) and the NaOH required for acid neutralization
will also contribute to the moisture. In addition the esterification of
FFA will produce one molecule of water, so for feed oils with high
FFA the moisture required might be fully produced via esterification. If
water addition is required to meet the 2 wt% load it can be added as
free water or as a component of a wet MeOH stream from a MeOH
rectification plant.
The reaction temperature of 35°C/95°F needs to be monitored and

controlled throughout the process. The enzyme will be affected
negatively if the temperature increases above 40°C/105°F and will be
seriously harmed at temperatures > 45°C 115°F.
The final entry in Table 3 is the enzymatic activity. This parameter comes
into play only for those processes running with enzyme recycling and
is a deciding factor for predicting the enzyme load for the next batch
in a series. The limit of 15% FFA quoted in Table 3 serves as a general
guideline. For a fuller discussion of the enzyme activity, see the section on
Enzyme Recycling Strategies on page 59-61, and Table 10 on page 60.
21
The data needed for calculating the final process parameters are
created using the tools described in Table 2. The reason for monitoring
these parameters is to start up with and maintain optimal processing
conditions for fast reaction and optimal enzyme productivity. These
analyses are most likely performed by process operators in a laboratory
for process monitoring and control. For more detail, see Section 4 on
page 41, describing laboratory tools and analyses.
Operating
Set point Tools and methods
parameter
moisture contributions from feed oil, 1% NaOH, FFA
Total water load 2 [wt% of oil]
esterification and wet MeOH + free water
Total MeOH load 1,5 mol eq. NIR + volumetric flow control on MeOH pump
0,04-0,7 [wt% of feed
Total enzyme load Volumetric flow control on enzyme pump
oil]
Reaction
35°C/95°F Temperature probe/feedback control
temperature
Enzymatic activity > 15 wt% FFA @1h Enzymatic activity assay
Table 3 Standard operating conditions for the enzymatic biodiesel process
Table 4 shows some critical and useful process monitoring and control
tools. The reason for monitoring these parameters during the reaction
is to continually monitor the balance of the ingredients in the reactor,
as well as determine the progress of the reaction and decide when
the current reaction cycle has gone to completion. These analyses are
most likely performed by process operators in a process monitoring and
control laboratory. For more detail, see Section 4 on page 41, describing
laboratory tools and analyses.
Process monitoring and

Control point Recommended tools
control
Temperature in reactor 35°C/95°F Temperature probe/feedback control
Water in heavy phase 18-25 [wt%] NIR, Karl Fischer
MeOH in heavy phase 13-16 [wt%] NIR, refractive index
Glycerin in heavy phase ∆ [wt%] NIR, refractive index
MeOH: water ratio in heavy

<1 NIR, Refractive index
phase
Soap in heavy phase 500-3000 ppm Soap titration
FAME ∆ [wt%] NIR/GC/HPLC
TG, DG, MG/ Bound Glycerin ∆ [wt%] NIR/GC/HPLC
FFA ∆ [wt%] FFA titration
BG ∆ [wt%] NIR/GC/HPLC
Free glycerin ∆ [wt%] NIR
Table 4 Important process control and monitoring parameters
22
Feed oil pre-treatment
Most feed oils require some level of pre-treatment for optimal reaction
in the process. With a range of feed oil qualities from refined soy bean
oil to brown grease and PFAD, any number of pre-treatment procedures
can be beneficial. Included here are the most commonly applicable
pre-treatment processes. Please keep in mind that the choice of pre-
treatment options in a specific plant depends largely on the nature,
composition and quality of the feed oil at this location. The list below
describes a wide range of pre-treatment options, of which some will
be more or less important for each type of feed. In addition some pre-
treatment steps can be used advantageously in combination or sequence
in order to optimally utilize equipment and residence times.
Specific methods for laboratory evaluation of the techniques described
below can be found in Section 4 on page 41, describing laboratory tools
and analyses.
Filtration
A filtration of the incoming crude feed oil is highly recommended as a

first pre-treatment step to remove particulates from the feed. A cascade
of 100 micron–50 micron-5 micron bag filters will ensure minimal
transfer of particulate material to the reaction system, but other filtration
options can be used as well.
Phosphoric acid wash
A phosphoric acid wash can be used in place of the water wash

described above. This can be very beneficial for the process performance
especially for feed oils with high levels of phospholipids. Phospholipids
can slow down the enzymatic reaction by occupying and thereby
blocking a part of the interphase surface area. They can also act as
powerful emulsifiers and severely affect the phase behavior of the
system after reaction completion. In some cases extensive emulsification
can hinder gravimetric separation of the crude biodiesel and the heavy
phase. It can also affect the secondary separation of clear glycerin and
interphase layer and make enzyme recycling troublesome. Beyond the
enzymatic process, phospholipids can cause problems with passing cold
soak filtration and cloud point specification of the final product, so these
need to be removed from the system downstream if not upstream.
23
It is crucial that any excess of phosphoric acid in the oil is

neutralized before the enzymatic biodiesel reaction.
A wash with 5 wt% of a 1% (10 g/L) phosphoric acid solution

should extract both hydratable and to some extent non-hydratable
phospholipids from the crude feed oil. If phosphoric acid wash is used,
it is recommended also to have a water wash immediately downstream
of the acid wash. This will remove most residues of phosphoric acid and
limit the NaOH load required for adjusting the pH of the system.
Degumming
With oils containing very high levels of non-hydratable phospholipids,

a degumming pre-treatment step might be beneficial. Chemical
degumming will remove the phospholipids and some entrained oil with
an associated yield loss. Enzymatic degumming will not only remove
phospholipids but will entrain less oil in the gum phase and add to the
yield of the reaction by converting the phospholipid to an FFA, which
goes into the oil phase, and a lyso-phospholipid, which goes into the
water phase.
Degumming is recommended only for troublesome oils, and in cases

where a valuable lecithin product can be extracted from the feed oil and
sold as a by-product.
Water wash
Depending on the feed oil quality, a simple water wash with 5 wt%
process water at 70-80°C for 1-2 hours is also highly recommended
to remove any hydrophilic surfactants and other non-convertible
contaminants from the feed oil. If a phosphoric wash or degumming
step is used, a water wash is required to remove residues of phosphoric acid.
Centrifugation is recommended for separation of the washing water, but

gravimetric settling can be sufficient for separation of the washing water.
24
Depending on the efficacy of the water separation, the feed oil will
typically contain 500-5000 ppm water. This water should always be
included in the calculation of the final water load to the reactor.
Mineral acidity neutralization
Mineral acidity neutralization of the feed oil is the number one

pre-treatment. This is crucially important for any type of feed oil,
independent of its source.
Experience from Novozymes and our enzymatic biodiesel customers

clearly shows that excess mineral acidity in the feed oil is by far the
number one cause of poor enzyme performance. This highlights the
importance of this parameter. Enzymes are biological molecules and are
most stable under near-neutral conditions.
Mineral acidity in the feed oil can come from many different sources. The
most common source is carryover of phosphoric or sulfuric acid from the
initial oil refining, but other factors can also affect the oil pH.
It may seem strange to talk about pH in an oil system, as pH is a property

of an aqueous system. In addition, most mineral acids have very little
solubility and therefore very low concentrations in oil, so what is the point?
To explain, please consider Fig. 9, which is very similar to Fig. 5. It is

intended to illustrate the point that any acidic residues (H+) in the feed
oil will dissolve in the heavy phase droplets of the enzymatic biodiesel
system. Even low concentrations of mineral acid can cause the effective
pH within the heavy phase droplet to be very low, which in turn causes
irreversible inactivation of the enzyme.
This explains why the FFA acidity does not adversely affect the enzyme,
as FFA is dissolved in the oil phase. Furthermore, the average pKa of fatty
acids is about 4,9 which is acceptable for the enzyme.
25
HO O
O
HO O
HO
O O O
H+ O
H+
O O
H+
O
O O
O
O
Fig. 9 Localized concentration of strong acidity in heavy phase droplet
It is therefore critically important to neutralize any mineral acidity

in the feed oil with dilute NaOH (2 wt% solution) before it is
mixed with the enzyme.
The current best method to evaluate the NaOH requirement of any feed
oil is to perform a multiple batch assay where 2-5 levels of NaOH addition
are tested for one hour in an oil hydrolysis reaction. The best of these
levels is then used to calculate the overall NaOH addition to the full-scale
reaction. See section 4, page 41 for further detail about mineral acid
neutralization and Appendix III for a Standard Operating Procedure (SOP).
Alternatively a two-step titration method can be used to determine the

required amount of NaOH to add to the feed oil.
The amount of NaOH required is typically in the range of 50-100

ppm added as a 2% solution. Extensive mixing during addition of the
NaOH solution is very important in order to achieve the desired mineral
acid neutralization with minimal FFA saponification. In addition, it is
important to consider the water added along with the NaOH solution in
the overall water balance of the reaction system of 2wt% of feed oil.
26
Downstream refining and product polishing
The bottom of Table 5 shows examples of the composition of crude

enzymatic biodiesel coming out of a process using cooking oil (UCO), corn
oil (CO) and palm oil fatty acid distillate (PFAD). The processing conditions
for each system are also listed.
Feedstock Scale Process conditions

Reaction Water % Methanol,
FFA % of oil Dosage
time, hours of oil eqv
UCO 1 6000 gal 12.1 1% 23 2 1.5
UCO 2 6000 gal 10.1 1% 20 2 1.5

1%, 4
Corn oil 1 1L 8.9 22
batches
1%, 4
Corn oil 2 1L 9.1 22
batches
PFAD 1 25L 85 0.2% 27 1 2.2
PFAD 2 1L 86.8 0.45 24 2.25 1.5
Feedstock After enzyme reaction
FFA % of oil FFA MG DG TG Bound Gly
UCO 1 12.1 2.2 0.34 1.2 0.60 0.28

UCO 2 10.1 2.5 0.32 1.40 0.55 0.31
Corn oil 1 8.9 1.4 0.46 0.20 0.02 0.15
Corn oil 2 9.1 1.3 0.45 0.28 0.02 0.16
PFAD 1 85 2.7 0.9 0.3 0.1 0.29
PFAD 2 86.8 2.83 0.15 0.25 0.08 0.081
Table 5 Reaction conditions and composition of crude enzymatic biodiesel from various
feedstocks
Similar to conventional biodiesel production, the crude biodiesel coming

out of the enzymatic process will require downstream refining and
polishing in order to meet ASTM specifications for B100 quality biodiesel.
Typically enzymatic biodiesel is produced from various low quality feed

materials with high content of unsaponifiable matter contamination. In
addition to the primary reactants and products shown in Table 5, the
crude biodiesel can also contain compounds like polymeric glycerides,
oxidation products, color compounds, residual protein and other non-
fatty organic surfactants.
The simplest and most efficient way to address this wide variety of
contamination and to ensure stable quality of the final biodiesel product
is product polishing followed by distillation. Many biodiesel producers
27
will have distillation already in place, but if not, it is highly recommended

to consider investing in a distillation process to avoid future problems
with cold soak filtration, sulfur content, bound glycerin and other
undesirable variations.
Even though the distillation will also take care of residual bound glycerin,
any FFA in the crude biodiesel will go out of the top together with the
methyl esters. In addition, the distillation bottom is not suitable for
recycling back to the front of the enzymatic process as it will accumulate
organic surfactants and other compounds that will hinder phase
separation in the main reactor.
Which downstream refining option to choose depends on factors like

feed oil identity, cost of retrofitting or construction of refining process,
local cost, availability of energy and chemicals and many other factors.
It is therefore very difficult to give general recommendations about
the choice of downstream refining process. It is highly recommended
to involve an experienced engineering company in the evaluation and
commissioning of the optimal downstream refining process for your
plant.
Below, four alternatives are described with brief discussions of the pros
and cons.
Caustic wash
Caustic washing of vegetable oil has been used for more than 100 years
to remove FFA and produce soap. FFA and FAME cannot be separated by
distillation. Instead, FFA can be physically removed by converting them
into soap and washing them out with water. The caustic requirement is
based on a QC measurement of the FFA in the crude biodiesel, which
is converted to an absolute molar load of NaOH or KOH required for
FFA neutralization. Typically an additional 10% molar excess caustic is
used to ensure complete neutralization of FFA. The NaOH is added as
a 0,4-2 wt% solution and stirred with the oil for 2 hours at 60°C to
allow for the full reaction. The water soap phase is separated by settling
or centrifugation before the biodiesel is washed with water to remove
residual soap dissolved in the FAME phase.
28
3.5
2.5
% 2
1.5
0.5
0
After TE After CW
FFA TG DG MG
Fig. 10 shows the FFA and glyceride concentrations in crude enzymatic biodiesel after
transesterification (TE) and caustic wash (CW).
First of all, Fig. 10 shows that the caustic wash is efficient at removing
the FFA, which is reduced from about 2% to < 0,1%. Secondly it is
also observed that the caustic wash also has a significant impact on the
MG content, which is reduced from 0,9% to < 0,3%. TG and DG are
relatively unchanged.
The advantage of caustic washing is that it is a well-known technology

and efficient at removing not only FFA but also MG. In addition, the soap
stock can be acidulated and fed back to the front of the process and
thus help to minimize the yield loss.
The main disadvantage of traditional caustic wash is the fact that soap
has increased solubility in methyl ester compared to glyceride oil. It can
therefore be challenging to wash out the soap without entraining methyl
ester in the water phase. This methyl ester can be recovered at a later
stage but this requires additional equipment and processing.
One-pot process
What we call the “one-pot process” is a polishing method designed for

an enzymatic process using a one-time-use enzyme strategy.
This carries several advantages compared to normal caustic wash.

Firstly there is no seed for primary phase separation after the enzymatic
conversion and no need for auxiliary reactors for the polishing reaction.
29
Secondly there is a reduced loss of FAME emulsified into the enzyme/

water emulsion phase because glycerol and methanol help break the
emulsions and reduce levels of soaps in the FAME phase. Lastly there
is a reduced cycle time because separation can take place at higher
temperatures, e.g., 60°C/140°F.
The one-pot polishing reaction is carried out immediately after the

enzymatic conversion has finished. Once the primary enzymatic
conversion targets have been met, 1,15 molar equivalents of NaOH to
FFA are dosed as 2% (w/w) solution of NAOH. This is mixed directly into
the reaction mixture, the temperature is adjusted to 60°C and held at
that temperature for 30 minutes. Afterwards the mixing is stopped and
the reaction is left to phase separate by gravity settling or alternatively
decanting or centrifugation.
It bears repeating that this process is only suitable for single-time use
strategies for enzyme recycling. The benefits come from the yield
increase, low soap-content FAME phase and reduced cycle time/higher
throughput, which in turn pay for the one-time use of the enzyme
catalyst.
Fig. 11 and 12 show the FAME and FFA concentrations during enzymatic
transesterification Fig. 11 and during one-pot polishing Fig. 12.
100.0 6.0
90.0
5.0
80.0
FAME concentration [wt%]
FFA concentration [wt%]
70.0
4.0
60.0
50.0 3.0
40.0
2.0
30.0
20.0
1.0
10.0
0.0 0.0
0 5 10 15 20 25 30
Reaction time [h]

FAME FFA
Fig. 11 FAME and FFA concentrations during enzymatic transesterification
30
98.0 1.40
97.5
1.20
97.0
FAME concentration [wt%]
FFA concentration [wt%]

96.5 1.00
96.0
0.80
95.5
0.60
95.0
94.5 0.40
94.0
0.20
93.5
93.0 0.00
0 0.5 1 1.5 2 2.5
Reaction time [h]

FAME FFA
Fig. 12 FAME and FFA concentrations during one-pot polishing
Resin catalyzed transesterification
Acidic resins are currently being used in enzymatic biodiesel polishing.

After separation of the heavy phase, the crude biodiesel enters a series
of columns. The first column is a guard column that ensures that no free
glycerin and other contaminants (i.e., metal ions) enter the resin columns.
The acidic sites on the resin catalyze the esterification of residual FFA
with MeOH. This has the advantage of being a continuous process with
an increased biodiesel yield because the polishing is a conversion instead
of a physical separation.
Enzyme inactivation
In order to avoid backwards reaction of FAME to FFA, it is very important

to inactivate any enzyme residuals after enzymatic reaction. Usually, all
of the enzyme is not fully inactivated in the caustic wash step and it is
recommended to inactivate the enzyme after final water washing step by
flash drying at temperatures above 120°C.
NB: Addition of water during the washing step can initiate a reverse
reaction, so it is highly recommended to perform the water washing
step and the following flash drying step in a continuous set-up in order
to control the residence time when active enzyme, water and FAME are
present.
31
CHAPTER 3. FEEDSTOCK FLEXIBILITY FOR ENZYMATIC BIODIESEL
32
CHAPTER 3.
FEEDSTOCK
FLEXIBILITY
FOR ENZYMATIC
BIODIESEL
33
In contrast to most conventional biodiesel technologies that require a

high-quality feed oil like refined soy bean oil, enzymatic biodiesel can be
made from basically any type of fatty material consisting of mainly fatty
acids and glycerides.
One of the key features that gives the enzymatic biodiesel process its
competitive edge is the fact that it functions independently of the FFA
concentration in the feed oil. This feature of the enzymatic process
circumvents the costly and messy chemical oil pre-treatments like
sulfuric acid esterification and thermal glycerolysis commonly used in
conventional biodiesel pre-treatment.
Feedstock flexibility also provides the enzymatic biodiesel producer with

the option of utilizing a broad and versatile range of feed oils. This allows
him or her to browse the market for the currently most profitable feed
material and take advantage of the fluctuations in the feed oil prices.
General feedstock quality
In Table 6, a number of common feed oils particularly suitable for

enzymatic biodiesel are described in general terms, as well as some
important pros and cons.
In addition the generally recommended pre-treatment steps are listed for

each of the feed oil examples to help the producer evaluate which oil is
currently the most profitable in their particular process.
However, it is important to keep in mind that there can be large deviations

from the standard in both quality and composition of each oil within a
particular group. General comments and recommendations for specific oils
can therefore only be advisory and used as a general rule of thumb.
34
Common feed materials for enzymatic biodiesel
Type Pros Cons Pretreatment
Before pre-treatment
Waste to fuel, feed
Increasing price, limited Filtration, water
collection systems in
Used cooking oil volume. Content of wash, mineral acid
place, potentially high
polymeric glycerides neutralization
quality glycerin
Bolt-on process for Waxes and sterylglycosides
Settling/decantationa
bioethanol plant, and phospholipid content,
Distillers corn oil water wash, mineral acid
potentially high-quality surfactant carryover from oil
neutralization
glycerin extraction
High sulfur content, high
Animal/rendered Price, no food oil Water wash, mineral acid
protein content, high melting
fat competition neutralization
point
Extreme mineral acidity,
Waste to fuel, bolt-on
very dark color, high Water wash, mineral acid
Acid oil process for oil refining
unsaponifiable content, neutralization
plant
possible high sulfur
High sulfur content, high
Settling/decantationa,
Price, waste to fuel, fast protein content, high
Brown grease filtration, water wash,
reaction unsaponifiable content,
acid neutralization
limited volume available
No glycerolysis, low
contamination, fast Mineral acid
High melting point ->fed
FAD reaction, bolt-on neutralization, pre-
batch/continuous process
process in oil refining heating
plant
Large volume available,
High melting point, food oil Filtration, mineral acid
CPO micronutrient recovery,
competition neutralization
high quality glycerin
Filtration, water
Crude vegetable wash enzymatic
High methyl ester yield Price, food oil competition
oil degumming, mineral acid
neutralization
Extreme variance in oil quality
Reclaimed oil Filtration, water wash/
Price, waste to fuel and nature and concentration
and slurries phosphoric acid washb
of oil contaminants
a
Settling or decantation can be useful to remove precipitates and/or macro particulates
b
Choice of water wash or enzymatic degumming depends on the specific feed oil
Table 6 Overview of typical enzymatic biodiesel feedstocks
Feedstock influences processing
Most feedstocks suitable for the enzymatic biodiesel process have great
variations in composition, quality, and physical appearance. Used cooking
oil for instance is mainly composed of TG whereas PFAD has > 80% FFA.
Distillers corn oil can be high in phospholipids. Acid oils can have very
high content of mineral acids. Animal fats are commonly high in sulfur
caused by protein residues in the oil. Finally oils with a high content of
saturated or short chain fatty acids commonly have a melting point above
the recommended enzymatic processing temperature of 35°C.
35
It is therefore clear that the choice of feed oil influences the process
and processing conditions. Care needs to be taken in order to establish
stable, reliable operations producing satisfactory product quality, given
that the process sources versatile feedstocks from any number of
suppliers. Again it is highly recommended to utilize the competencies
and experience from a trusted engineering partner to ensure that you
get a robust process capable of handling versatile feedstocks with
minimal process changes.
How to compensate for changing feedstocks
Ideally any process should maintain as constant a feed flow as possible.

With enzymatic biodiesel, that is not generally possible, as the feedstocks
are so versatile in their nature.
Below is a brief description and discussion of the most important

parameters to take into account when adding any feed oil into the
process.
Lipid composition
The concentration of various fatty components in the oil, i.e., TG, DG,
MG and FFA - make up the lipid composition. The lipid composition can
be measured using a variety of techniques like GC, HPLC, NIR and FFA
titration, see “Advanced analytical tools” in Section 4, page 43.
Controlling the water concentration

The FFA concentration in the feed oil dictates the net production of
water in the FFA esterification reaction, and this needs to be taken into
account to match the 2% overall water concentration recommended
for the system. Regarding water it is also important to take into account
the water dissolved in the feed oil, the water added with NaOH pre-
treatment and finally any water introduced via a stream of wet MeOH
going into the process.
Bear in mind that for instance PFAD with > 80% FFA will have a net
excess of water produced via esterification. In such a case it is necessary
to investigate what, if any, effects the increased water has on the
conversion and equilibrium position of the reaction. In some cases,
conversion remains satisfactory and no rectifying action needs to be
36
taken. In other cases, FFA is high in the reaction product and water
needs to be controlled.
If necessary the water balance can be offset by increasing MeOH or

adding a hydrophilic component like glycerin to the heavy phase to
act as a water sink to reduce the water activity in the system. It is also
possible to continuously bleed a proportion of the heavy phase to reduce
water. In this case MeOH and possibly enzyme can be lost with the heavy
phase bleed off and needs to be rectified.
Appropriate overall MeOH load

In addition to controlling the water concentration, the molar glyceride
and FFA profile of the feed oil also determine the appropriate overall
MeOH load to add to the process.
Surfactant concentration
Surfactants in the feed oil can hinder enzymatic reaction by occupying

the interphase surface area during the reaction. In addition, they can
greatly influence the settling and/or separation of the heavy and light
phases after reaction completion.
Because so many possible low quality feed oils are used the enzymatic
biodiesel process, there is a wide window of surfactants and surfactant
concentrations possible for any feed oil. For this purpose, however, the
class ‘surfactants’ can be viewed as one ‘black box’ containing any non-
convertible emulsifiers and surfactants in the feed oil.
Taking advantage of this simplification, it is possible to use a simple

shaking and settling test both to estimate the need for pre-treatment of
any incoming feed oil, and also to evaluate the efficacy of the current
pre-treatment process. This last is accomplished by testing the pre-
treated feed oil and comparing with the incoming feed before pre-
treatment. See on page 41, Laboratory tools for quality assessment and
control, specifically the section on the Settling test, page 45.
37
Melting point
The enzyme has maximum productivity at 35°C and it becomes severely

unstable already at temperatures above 40°C.
Generally it is recommended to run with oils that are liquid at 35°C.

That would seem to put severe restrictions on feed oils like PFAD, CPO
and animal fats as these commonly have melting points above 40°C, but
fortunately that is not the case.
High temperature melting fats can be used without difficulty as long

as you consider the melting point from the beginning, when designing
the process. In some cases high melting feed oil can be mixed with
other low melting oils, recycled crude biodiesel or even MeOH to reduce
the melting point to < 35 °C. This enables fully normal processing as
described in the sections above.
In other cases where no low melting oils are available or mixing is

undesired, the process needs to be designed for the purpose.
The hurdle of melting point can be overcome using a feed batch or

continuous process. It has been shown that adding PFAD at 55°C to a
biodiesel reaction maintained at 35°C is possible without adverse effects.
At steady state, a constant concentration of FAME and MeOH exists in
the reaction medium, and this enables the system to partly dissolve and
partly convert the incoming feed oil instantly before it gets a chance to
re-solidify in the reactor.
In a fed batch set-up, the total time for feed oil addition would be 6-10
hours and the overall reaction time 20-30 hours. In a continuous process
the residence time at steady state will be 20-24 hours.
38
39
CHAPTER 4. LABORATORY TOOLS FOR QUALITY ASSESSMENT AND CONTROL
40
CHAPTER 4.
LABORATORY
TOOLS FOR
QUALITY
ASSESSMENT
AND CONTROL
41
In collaboration with our industrial partners, Novozymes has developed

and adapted a number of practical and analytical tools to support
enzymatic biodiesel producers through the most important steps in the
process, all the way from feed oil evaluation to product quality control.
Similar to the section on page 27 on downstream refining and

product polishing , this section will not cover the full range of tests
and qualifications to meet full ASTM specifications for B100 biodiesel.
Rather, the focus will be on how to ensure a reliable enzymatic reaction
process and a stable product quality through testing and monitoring key
parameters at key stages on the path from feed oil to B100.
Tools for laboratoryoratory evaluation of feed oils
This section contains recommendations for analytical techniques

necessary for running an efficient enzymatic biodiesel production and
evaluating various parts of the process.
Tables 2-4 gave an overview of when and where to use various analytical
tools to operate and control the enzymatic biodiesel process. This section
gives further insights into why it is so important to use the correct
analytical tools, how to interpret the results and make the right decisions
based on analytical data.
Analytical tools
Most established biodiesel producers and general oil processing

operations will already have some or most of the analytical equipment
described in this section and the expertise to operate it. For greenfield
start-ups this section can serve as a quick guide to some of the
important analytics.
Please note that this handbook covers only the tools required for handling
the part of the biodiesel process directly relating to the enzymatic
conversion. In order to control non-enzyme related aspects of the
downstream process like product polishing and ASTM specification,
additional tests and equipment may be required. Novozymes recommends
using experienced engineering partners for advice on these topics.
42
Furthermore, the procedures described in this section and in Appendices

III-VII are to be used at your own risk. The procedures described have
been tested and proven in commercial-scale enzymatic biodiesel
production. Nonetheless Novozymes cannot take responsibility for
damaged equipment or other damage caused by following the
instructions provided here.
Basic compositional analysis
Some basic analytical tools are required for feedstock analysis, process
control and product validation. The analytical methods mentioned in
Table 7 are well known, widely published techniques so they will not be
described in detail here.
Target Method Comments

AOCS standard method Ca 5a-40 “Free Fatty
FFA conc. [oleic wt%] Colorimetric titration
Acids”
AOCS standard method Cc 17-95 “Soap in oil
Soap conc. [ppm] Colorimetric titration
titrimetric method”
Water conc. [ppm] Karl-Fischer titration Equipment supplier methodology
Table 7 Basic analytical tools for feedstock analysis, process control and product validation
Advanced analytical tools
Chromatography
In addition to the basic techniques above, additional more advanced
analytical tools are required to operate a biodiesel plant.
In order to run the process optimally, you will need to gain additional
information about the composition of the feed oils and the reaction
system as it progresses. Gas Chromatography (GC) and High Pressure
Liquid Chromatography (HPLC) are well known techniques to analyze
glyceride composition and ester content in a lipid system. Biodiesel
chromatographic techniques are very useful because they can give great
accuracy and reproducibility. Most biodiesel producers rely on GC to pass
final certification of their B100 product, and it is generally recommended
to have one in place for this purpose.
A number of equipment manufacturers can supply the right column

setup and methodologies for GC, HPLC and other chromatographic
methods. In addition, several methodologies have been published. It is
43
outside the scope of this section to describe in detail how to operate

chromatographic analysis.
Chromatography is widely used in the biodiesel industry but it does

have a few downsides. The equipment can be very costly to buy and
operating costs can be considerable for maintenance, consumables and
waste associated with running chromatography. In addition and quite
importantly, there is an unavoidable and substantial time lag between
the time a sample is taken and when the analytical result is ready.
Spectroscopy
Spectroscopic methods generally use the absorbance of light of
the different components in a lipid material to determine its overall
composition. Especially suitable for a biodiesel producer is Near Infrared
Spectroscopy because it is good at handling inhomogeneous liquids.
A beam of near infrared light is bounced off a sample surface and the
scattered light bouncing back is recorded. This data is then Fourier
Transformed and compared to a library of reference spectra to give an
estimation of the composition of the original sample.
In addition to the concentration of the fatty components of the biodiesel

system, NIR can also be used to measure MeOH in the light phase of
the system and also measure glycerin, water and MeOH concentration
in the heavy phase. MeOH concentration and water ratio measurement
is extremely important as a control parameter for the enzymatic
conversion, see Table 4.
An alternative ‘low tech’ method for heavy phase water, glycerin and
MeOH determination is given on page 49 in the section “Brix water/
MeOH/gly.”
The immediate and vital advantage of the NIR technique is the fact that
it is a near real- time analysis. Sampling, sample preparation, loading,
analysis and results creation can be done in a matter of minutes, giving
a very good image of the current situation in the reactor. Another great
advantage is that there is little or no sample preparation required and no
moving parts. This enables the operators to monitor a large number of
variables in different simultaneous reactions with minimal effort.
The accuracy of the NIR technique depends entirely on the volume and
quality of the reference spectra library. It is possible to create an entire
library from scratch and most equipment suppliers offer to assist in the
model creation and validation. However this can be a very lengthy and
44
costly process and a great number of samples are required in order to be

able to accurately calibrate and validate the model.
Instead, Novozymes recommends choosing a supplier that offers access

to a validated and updated online reference library (e.g., Eurofins). Some
suppliers also offer package deals with access to both their reference
library as well as their ready-made and certified models for in-process
biodiesel analysis and B100 biodiesel certification.
The downside of the NIR technique is the cost. The equipment is

rather costly and in exchange for reference library access and method
validation, the supplier will charge a monthly fee. Nonetheless
Novozymes’ opinion is that any producer considering starting an
enzymatic biodiesel process who invests in a NIR tool including reference
libraries and models will find that it pays off generously in the long run.
Laboratory assays for enzymatic biodiesel
A number of tests specifically designed for the enzymatic biodiesel

process are necessary for feedstock evaluation, pre-treatment assessment
and enzyme activity. None of the below tests require advanced
equipment. Some require support from the analytical techniques
described in the section above.
For each method below the principle and background for the assay is
described. For details and Standard Operating Procedures for each assay
please see Appendices III-VII for full SOPs of the methods described.
Settling test for emulsifiers
In order to address the huge group of compounds identified as

‘surfactants’ without singling out any specific species, a simple settling
assay has been developed, see Appendix IV for a SOP of the method.
Rather than identifying and quantifying any one target surfactant, the
overall effect of the current ‘surfactant’ concentration is estimated.
Being amphophilic molecules by nature, surfactants will tend to appear

on the interphase between water and oil. By vigorously mixing an oil
sample with a specific volume of water and recording the time it takes
the water to settle back out, the severity of any surfactant can be
45
estimated by comparing the measured settling time with a pre-defined

‘worst acceptable’ time. This procedure is sufficient to categorize the
results as ‘good’, ‘acceptable’. or ‘bad’.
This simple settling assay is of key importance to evaluate the

performance of the plant pre-treatment process before the pre-treated
feed oil enters the reactor. If the settling time of the pre-treated feed oil
is more than the acceptable limit, the pre-treatment is not achieving its
purpose and this can cause severe difficulties with reactor settling and/or
separation after the reaction is complete.
Any feed oil that has not passed the settling test before it enters
the reactor should not be processed - unless the cause is fully
understood and can be rectified.
In addition to mandatory testing of the pre-treated feed oil, the settling

test can also be useful for testing incoming feed oils from the suppliers
to estimate the level of pre-treatment required for that oil.
Please note, however, it is recommended to pre-treat all feed oils going

into the enzymatic process. So the initial feed oil shaking test is just an
indication of pre-treatment requirements at most. Please do not use this
test to skip or avoid pre-treatment. Instead the incoming feed oil settling
test result should be compared with the result of that same oil after
pre-treatment to gain additional information about the severity of the
‘surfactants’ in the oil and the efficacy of the pre-treatment process to
handle them.
Laboratory pre-treatment assay
Doing the shaking test with each incoming feed oil will give variable
and often relatively bad results. Instead of rejecting a feed oil that does
not pass the initial settling test without further notice, the effects of the
pre-treatment process can be tested in the laboratoryoratory. This should
give a clear answer as to how this feed oil will perform in the enzymatic
biodiesel process after it has been properly pre-treated.
The laboratoryoratory pre-treatment assay is a two-step method that

simulates a ridged full-scale pre-treatment process in laboratoryoratory
46
scale. The methodology consists of the initial feed oil settling test,
followed by a water wash with 5 wt% water, and finally a second
settling test to evaluate the effects of pre-treatment.
If the second settling test does not meet the acceptable time limit, the
feed oil should be rejected and sent back to the supplier. For a full SOP
see Appendix V.
Mineral acidity assay
It has been highlighted several times so far but cannot be overstated

that enzymes are extremely sensitive to mineral acidity in the feed oil
and process. Therefore any possible sources of contamination with
strong mineral acid should be completely eliminated from any enzymatic
biodiesel setup in any scale from laboratoryoratory assays, to pilot trials,
to full-scale operation.
In order to accurately determine the NaOH load required to neutralize

mineral acidity in any feed oil, it is necessary to run a series of 1 hour
enzymatic reactions into which varying amount of NaOH is added to
each reaction. The typical NaOH load to an industrial process is 10-500
ppm so it is recommended to perform the tests within this area.
The basic principle is to track the progress of an enzymatic biodiesel

conversion in each system for one hour and plot the end point results as
a function of the NaOH added to each reaction. Typically what is seen
is a curve that starts low at low NaOH but increases to a certain level
before it becomes flat at higher NaOH loads. The optimal NaOH load
is determined as the load at which the curve started to become flat.
This ensures full neutralization of mineral acidity in the oil as well as a
minimal soap by-product formation.
Bear in mind that soap is not bad for the enzyme as such, but large
excesses of soap in the reactor can cause severe problems with
settling and yield loss in downstream refining, so it should be kept at a
minimum. For a full SOP see Appendix III.
47
Mini batch conversion assay
In order to fully determine the performance of any feed oil in the full-
scale process, it is recommended to perform laboratoryoratory mini
batch enzymatic transesterification assays with any fresh incoming
feed oil. The batch assay closely mimics the conditions of the full- scale
process in order to get the most realistic results possible. The mini
batch assay will capture any unsuspected issues with the feed oil that
negatively influence the enzyme or the enzymatic process that is not
captured in the other analysis and tests described above.
The downside of running mini batches is that the reaction takes 20 hours
plus preparation and analysis. This directly translates into holding time of
that particular feed oil in the feed oil storage tanks and a minimum 24-
hour lag period for feed oil turnover.
Still it is highly recommended to perform the mini batch assay whenever

starting up and troubleshooting a new process. As the crew gets more
experienced the frequency can possibly be reduced during normal
operation. However, particularly when switching to a new feed oil identity
or in the case of a first shipment from a new and unknown feed oil
supplier, it is highly recommended to evaluate the feed oil using the mini
batch assay. For the full SOP of the mini batch assay see Appendix VI.
Enzymatic activity assay
Some processes reclaim the used enzyme rag layer after a completed
batch and recycle it back up to the front of the process for the next
batch. Other processes employ a single use of the enzyme. The choice
of enzyme recycling strategy depends on several factors. Three different
strategies for enzyme recycling are described in the section on enzyme
recycling on page 59.
In all situations, it is very important to know what the current enzymatic

activity is in the reactors at all times in order to gauge if the enzyme
dosage to meet production requirements needs to be adjusted.
Enzyme activity is a measure of how efficiently the enzyme is working.

Similar to the mineral acidity assay described above, the enzymatic
activity assay tracks an enzymatic conversion (typically hydrolysis) in a 1
hour batch assay.
48
In practice, a representative sample of the reaction medium is drawn

from the reactor and spun in a centrifuge to separate the heavy phase
from the light phase. The light phase is decanted and the remaining
enzyme rag layer/glycerin is re-homogenized. A specific volume of
sample of this homogenous heavy phase is then drawn and added to a
model reaction system containing refined oil and water.
After stirring for 1 hour at 35°C, the reaction is stopped and analyzed
for FFA to determine the increase in FFA compared to the starting FFA
concentration in the refined oil.
Depending on the FFA level recorded for the reaction sample assay, the
enzymatic activity can be determined ‘good’, ‘acceptable’, or ‘bad’.
In the case of ‘good’ there is no need to take action. In the case of
‘acceptable’ the reaction should be monitored closely to see if it is
slowing down. If the rate of conversion in the process is not satisfactory
after 1-2 hours the enzyme should be replenished to compensate for the
loss of activity.
In the case of ‘bad’ there is no or little enzyme activity in the sample.

When this happens it is recommended if possible to re-test the activity.
If the results persist the reactor should be stopped and the cause of the
unexpected enzyme activity should be identified and rectified before
restarting the batch. Alternatively the entire bad reaction volume can
be forwarded to a holding tank for storage, analysis and re-processing.
This frees up the reactor for a fresh load of feed oil and decreases the
downtime of the plant.
Brix water/MeOH/gly
It is stated above that the ratio of water: MeOH in the glycerin phase
should be ≥ 1 at all times. Because this is such an important control
parameter, Novozymes has developed an alternative method for
analysis based on Karl-Fischer (KF) water determination and Brix-type
refractometer. The advantage for this method is that it involves a much
lower investment in equipment compared to the NIR spectroscopy
method described above.
In order to determine the water and MeOH in the glycerin phase using
KF and refractometry, it is first required to have a clear glycerin phase
sample from the reactor. It is therefore necessary to centrifuge the
49
reaction sample and extract ONLY the clear bottom part for the analysis.
To get the water: MeOH ratio, one sub-sample of clear glycerin phase is
first analyzed for water concentration by KF titration. Once this figure is
available, the glycerin in the sample is measured by Brix refractometer or
similar and the MeOH is then found using Equation 1:
%MeOH = 108,2 - 1.46 X Brix - %water (KF) (1)
Equation 1: MeOH wt% by Karl-Fischer and Brix refractometry
Equation 1 is an empirical expression for MeOH concentration. The

model has been validated and confirmed with an almost exact linear
correlation, see Fig. 13.
y=1.4557 X - 7.8024
80
R2=0.9884
75
70
% Glycerol
65
60
55
50
45 47 49 51 53 55 57 59
Brix
Fig. 13 Correlation of glycerin concentration and Brix measurements
For a full SOP of the Brix water/MeOH/gly method see Appendix VII.
50
51
CHAPTER 5. PROCESS OPERATION
52
CHAPTER 5.
PROCESS
OPERATION
53
This section describes in detail how to manage and operate an enzymatic

biodiesel process. A number of operational strategies are possible
depending on the specific process but some general terms are valid for
any enzymatic biodiesel process.
After pre-treatment, the next step is the enzymatic biodiesel reaction

itself. The enzymatic biodiesel process is a feed oil independent batch
transesterification process able to run with 0-100% FFA and with
recycling of the liquid enzyme catalyst.
In terms of enzyme recycling, there are several ways to operate the

process, and these will be described in detail in on page 36. Common
for the different strategies is the overall process layout and startup of the
first batch in a series.
A start-up batch will be described below to exemplify the enzymatic

biodiesel process as shown in Fig. 8 and Tables 1-4.
Process startup and operation
In all sections below it is assumed that the feed oil has been
appropriately pre-treated to remove troublesome surfactants influencing
the interphase surface area occupation and the reaction settling.
Before this pre-treated feed oil enters the main enzymatic biodiesel
reactor, NaOH is added to the pre-treated feed oil to neutralize mineral
acidity. The pre-treated and neutralized feed oil is then preferably mixed
with the liquid enzyme using a circulation stream in a static mixer before
the feed stream enters the reactor.
Once the reactor is filled, the reaction is agitated vigorously to distribute

the enzyme and water through the oil volume. In addition to oil and
enzyme, the reaction requires 1,5 molar equivalents of MeOH overall as
reactant and 2 wt% water to maintain enzymatic activity.
If necessary, water can be added to the feed stream or to the reactor.

Some feed oils contain substantial amounts of water already. The pre-
treatment will also leave behind residues of water in the feed oil, and this
54
should be taken into account. Lastly, any feed oil with FFA > 40% will
produce > 2% water during esterification, so these only initially require a
little water to get started and no water addition during the reaction.
The total of 1,5 eq. of MeOH should be added directly to the reactor,
but it is necessary to limit the flow rate of MeOH in order to keep the
current MeOH concentration at any time below 16% in the heavy
phase. Generally it is recommended to add MeOH during 6-10 hours of
reaction time depending on the conditions and identity of feed oil and
the current enzyme activity. Adding MeOH too quickly will increase the
MeOH concentration dramatically in the relatively small heavy phase
volume. This will irreversibly inactivate the enzyme and the batch will
come to a complete stop.
At low enzymatic activity it might be necessary to dose MeOH over a 20

hour period in order to prevent inactivation by MeOH poisoning.
Once the reaction is running, the overall MeOH level can be monitored
by sampling the reaction medium, followed by centrifugation to
separate the phases, and then measure the MeOH concentration in the
clear glycerin phase alone. Highly useful near-real-time analysis tools
are available for this and other process control operations, which are
described in detail in the section on Brix water/MeOH/gly on page 49
above and Appendix VII.
Fig. 14 shows the conversion of the main components of the feed oil
during a reaction of a hightriglyceride material in the enzymatic biodiesel
process under standard conditions. The overall reaction time typically
falls within the window of 20-24 hours, but many factors can affect this
depending on the operation conditions and the nature and quality of the
feedstock material.
High levels of surfactants and non-convertible material can occupy

significant portions of the oil/glycerin interphase surface area and
thereby limit the mass transfer across the interphase, thereby slowing
the reaction. On the other hand, owing to the two-step hydrolysis-
esterification mechanism of the enzymatic catalysis of glycerides,
feed oil with high FFA tends to react faster in a one-step esterification
mechanism. More details of specific feedstocks can be found in Section
3 above “Feedstock flexibility for enzymatic biodiesel” on page 33.
55
70
60
50
40
%
30
20
10
0
0 5 10 15 20 25
Reaction time [h]
FFA MG DG TG
Fig. 14 Typical reaction curve with TG, DG, MG, FFA and FAME
Once the reaction is running and the conversion is progressing, the

reaction should be monitored for the parameters described in Table 4,
reproduced here as Table 8:
Process monitoring and control Control point Recommended tools
Temperature in reactor 35°C/95°F Temperature probe/feedback control
Water in heavy phase 18-25 [wt%] NIR, Karl Fischer
MeOH in heavy phase 13-16 [wt%] NIR, refractive index
Glycerin in heavy phase ∆ [wt%] NIR, refractive index
MeOH: water ratio in heavy phase <1 NIR, Refractive index
Soap in heavy phase 500-3000 ppm Soap titration
FAME ∆ [wt%] NIR/GC/HPLC
TG, DG, MG/ Bound Glycerin ∆ [wt%] NIR/GC/HPLC
FFA ∆ [wt%] FFA titration
BG ∆ [wt%] NIR/GC/HPLC
Free glycerin ∆ [wt%] NIR
Table 8 Process control parameters
Of the parameters described in Table 8, it has been established that the

ratio of water: MeOH in the glycerin phase is a critical factor for good
enzymatic productivity. In order to keep the rate of reaction as high as
possible, it is necessary to monitor the MeOH and water concentration in
the heavy phase separately and control these two parameters to meet
the recommended concentration windows in Table 8.
56
Monitoring the glyceride and FFA conversion will provide the end point
of the enzymatic reaction. Variations for different feed oils will occur, but
in general the reaction should be stopped when the total sum of
glycerides (TG+DG+MG) is below 3% overall and the FFA is below 2,5%.
Troubleshooting
In the case of unfavorable observations made during the enzymatic

process, a troubleshooting methodology can be helpful to identify and
rectify any parameters operating outside their acceptable limits. Table 9
shows some common observations arising from poor process
performance. Each observation is correlated to a number of specific
process parameters. Suggestions are given as to how to rectify the
situation.
Observation Potential cause Correction

Check activity in assay.
Enzyme activity low
Add more enzyme.
Check oil in mini assay.
Acid in the oil
Check and correct NaOH addition.
Acid in the methanol Control methanol quality.
Soap is too high Reduce caustic addition.
Temperature out of control Adjust temperature/check it is OK.
Reaction is slow/stalling Methanol dosage too low Increase until 16% in heavy phase.
– insufficient decline in
glycerides Shut off methanol dosage and reduce
concentration in heavy phase by adding
water/glycerin.
Methanol dosage too high
High methanol might have inactivated
enzyme.
Reduce heavy phase and restart.
Too much unsaponifiable in oil Check oil quality in un-SAP test.
Check mixers. Is methanol well mixed

Mixing is inadequate
in?
Water content too low Add water (0,5-1,0%).
Too much water in the reactor Add methanol and/or glycerin.

FFA too high at equilibrium
Methanol too low Add methanol.
Too much enzyme added Reduce addition rate.
Reaction is too fast Temperature too high Cool.
Methanol dosage too high Reduce addition rate.

Insufficient conversion – high
Allow for higher conversion.
MG
Settling too slow Emulsifiers in feed (e.g., Improve pretreatment and/or reduce
phospholipids, Tween 80) recycle number.
Temperature too low Heat up.
Improve heat treatment in downstream
FFA is increasing during
Active enzyme in the product process, or water wash with low
storage of the (crude) FAME
holding time.
Table 9 Troubleshooting guide
57
Separation and heavy phase management
After the transesterification reaction is complete and conversion targets

have been met, the system is separated into its two main phases of
biodiesel and glycerin. The separation technique can be simple passive
gravimetric settling in the reactor or active settling via continuous
settlers, coalescence filters, centrifugation and others.
It is clear that surfactants in the feed oil affect the settling, and
in addition soap, DG and MG will act as emulsifiers and also limit
the settling rate. The effect of DG and MG should be limited if the
conversion targets are met during primary conversion.
Increased temperature will also increase the settling rate, but

temperatures above 40°C will irreversibly inactivate the enzyme, so heat-
assisted settling should only be applied in a single-use enzyme strategy,
or in the last batch of the reaction series, see Table 10.
Independent of the choice of separation techniques, the enzymatic

biodiesel process takes advantage of the fact that the product of the
reaction is a two-phase system. The biodiesel is in the light phase, while
the water, glycerin and MeOH are in the heavy phase.
Once the reaction has passed primary settling and the biodiesel phase
is clear of suspended glycerin, it can be pumped out of the reactor and
downstream towards the rectification and polishing operations. More
on that in the section “Downstream refining and product polishing” on
page 27.
In the settled reaction, the enzyme will have an affinity for the interphase
layer between the light and heavy phase. In practice a third intermediate
pseudo phase will emerge in the reactor upon secondary settling. This
intermediate phase will typically contain > 90% of the residual active
enzyme, and will be stabilized by natural emulsifiers in the system like
partial glycerides, phospholipids and soap residues among others, see
Fig. 6.
This can be utilized in the process as the clear glycerin phase can now
be drained from the bottom outlet of the reactor or secondary settler
with minimal loss of active enzyme. This will also prevent a build-up of
glycerin in the reactor and avoid the resulting capacity loss.
58
Theoretically it should be possible to continue the loop indefinitely. In

practice however, non-convertible emulsifiers and other contaminants
have a tendency to accumulate in the interphase layer regardless of
the efficiency of feed pre-treatment. Over time the accumulation can
become so severe that reaction rate and settling time are severely
affected.
Therefore it is necessary either to bleed off a part of the interphase layer

continuously, or to choose a point at which the entire intermediate
phase is discarded and a new reaction series is started, see the section
below on enzyme recycling strategies and particularly Table 10.
Enzyme recycling strategies
Optimal utilization of the enzyme is ensured by using the techniques

described above and maintaining the active enzyme in the reactor. There
will, however, always be a natural decay of active enzyme as well as an
enzyme activity loss caused by processing conditions. With good process
operation, up to 95% of enzymatic activity can be retained from batch
to batch and recycled in the process. In the case of a single use strategy,
the need for enzyme recycling is avoided and the overall processing is
much simpler in terms of enzyme handling.
For any multiple use enzyme strategies, the addition of enzyme is

necessary to each subsequent batch following the startup batch. This
will avoid gradually losing all active enzyme over time. An enzyme load
of 0,7 wt% is recommended for the first batch in a series where reaction
time <20 hours is desired. Because so much enzymatic activity is retained
in the reactor between batches, only a small overhead of additional
enzyme is required. Typically 0,04 % enzyme is added to each batch in a
series following the startup batch. Over a series with four recycles of the
enzyme phase, the overall enzyme consumption can theoretically be as
low as 0,21 wt% enzyme per batch.
Table 10 describes in detail each of the methods for heavy phase

management and enzyme recycling, including pros and cons of each
strategy.
59
Reactor system operation Estimated enzyme use Pros/cons

• Same process every batch
• No cycling-up of emulsifiers, etc.
One-time use - 0,3% • Easier settling by temperature
Centrifuge separation 0,3% in every batch increase
• Higher volume FAME/batch
• Long reaction time
• No problems with enzyme
recovery from rag.
0,26%
3 batches - • Easy to separate
Dosage 0,7% in batch one
Recycle all heavy phase - • High recovery of surplus methanol
and 0,04% in following
Tank separation in heavy phase
batches
• Different mass balance by every
batch
0,21%
• As in the 3 batch strategy +
4 batches - Dosage 0,7% in first batch
• Bleed off glycerin after batch 2
Tank separation and 0,04% in following
or 3
batches
Table 10 Enzyme recycling strategies with pros and cons
The enzyme handling strategies described in Table 10 are listed in order

of increasing complexity. In a single use strategy, there is no need for
interphase layer handling and recycling. In addition, heat can be used
to drive the settling after reaction because there are no concerns about
inactivating residual enzyme. This simplifies the process design and
operation immensely but has the downside of a slightly higher enzyme
consumption of ~3 kg/MT.
The 3 batch strategy is run with three times recycling of the entire
heavy phase with 0,7% enzyme in the first batch and additional 0,04%
enzyme added after each consecutive batch. This also simplifies the
enzyme handling as there is no bleed off of glycerin and no rectification
of the enzyme interphase layer. There is also a saving in MeOH
rectification cost and slightly reduced overall enzyme consumption with
this strategy.
The downside is that with the accumulating volume of heavy phase,

mixing becomes more difficult in cycles 2 and 3. In addition there is a
different mass balance of the system for each batch in the cycle, making
automated control more difficult.
The final option in Table 10 is four time recycling, with intermediate

bleed off of clear glycerin. This operational strategy is similar to the three
time recycle strategy but after the second or third cycle, the reactor is left
to settle and most of the clear glycerin is bled off before the fourth cycle
is started.
60
This strategy has most of the same pros and cons of the three time
recycling strategy. There is however a better enzyme utilization and
better overall capacity of the plant when running 4 instead of 3 cycles.
The final choice of enzyme recycling strategy depends on factors such

as feedstock identity, energy cost, equipment availaboratoryility, process
integration and many more. In order to evaluate and choose the
optimal and most cost efficient strategy in a specific plant, Novozymes
recommends using experienced engineering companies. Novozymes’
development partners know the process and details of enzyme handling
in relation to a specific process setup and can provide any further details
required.
61
CHAPTER 6. NOVOZYMES AS ENZYME SUPPLIER
62
CHAPTER 6.
NOVOZYMES
AS ENZYME
SUPPLIER
63
By choosing Novozymes as your enzyme supplier, you gain world class

innovation and technical support, both now and years in the future.
Novozymes sold its first enzyme product in 1941, and its first enzyme for
esterification in 1985. By 2012, Novozymes’ solutions were used in over
100 oils and fats processing plants globally. By mid-2014, more than
one third of US fuel ethanol was produced using Novozymes solutions.
In 2014, Novozymes launched Eversa® Transform, the first enzymatic
solution for biodiesel production. In years to come, you can expect to see
additional, ongoing innovation.
As a Novozymes customer, you will receive a guaranteed steady supply

of our enzyme product to optimally maintain your operation. We have
dedicated production facilities in the US, EU and China with the capacity
to meet current and future demands.
Eversa® Transform is approved for use in most countries throughout

the world, but if in doubt, please contact your local Novozymes
representative for further information. You can also contact us through
www.novozymes.com/Solutions.
Access to strategic engineering partners
Early in the development and initial implementation phase of

Novozymes’ enzymatic biodiesel process, we quickly realized that we
would need support. We found that the task of introducing a radically
new technology into a mature industry like biodiesel production would
require a strong process engineering capability.
Novozymes has therefore entered into strategic partnerships with a

number of well-known engineering companies ranging from local,
regional, and up to global presence in the field.
Novozymes is fully confident that utilizing the knowledge and experience

developed between Novozymes and our partners will enable the
biodiesel producer to commission, install, start and operate an enzymatic
biodiesel plant correctly and efficiently from day one. Novozymes
therefore strongly recommends that you allow us to connect you with
the best possible engineering partner in your region.
64
Joint technical support
Investing in a plant using one of our engineering partners will not only
ensure that you get the right equipment and technologies in place, it will
also ensure that you get proper training and operation guidelines on all
equipment. In addition, Novozymes technical service team works closely
with our partners, which ensures direct access to a full and experienced
technical service team to address all aspects of the enzymatic process
and its technical and practical operation.
65
66
CONCLUSION CONCLUSION
CONCLUSION
This handbook explains most technical and practical considerations
necessary to consider when evaluating, designing, building, starting up
and operating an enzymatic biodiesel process.
To summarize the main points, the key requirements for processing low-
cost feedstocks are:
• Robust pre-treatment and mineral acid neutralization

• Reactant addition strategy and reactor mixing input
• Selection of enzyme recycling strategy
• Selection of polishing method
• Fast and reliable analysis tools for process monitoring and quality
control
All the information provided herein is advisory and it is recommended to

contact Novozymes directly to get more information on the enzyme and
the enzymatic process.
Novozymes will also gladly facilitate contact with our engineering

partners, whose inputs and process design we believe are required to
establish and run a successful and profitable enzymatic biodiesel process
from day one.
Novozymes remains dedicated to delivering the most advanced and cost

efficient technologies while maintaining the highest level of customer-
friendly technical service. We live and learn together with our customers
and would therefore very much appreciate any comments you might
have to improve this document and its applicability.
67
LIST
LIST
List of figures
• Fig 1: Oleic triacylglycerol (TG), B: Oleic diacylglycerol (DG), C: Oleic

monoacylglycerol (MG), D: Oleic free fatty acid (FFA)
• Fig 2: Reaction scheme of transesterification of acylglycerides and FFA
• Fig 3: Lock and key model of enzymatic catalysis
• Fig 4: Step-by-step overview of enzyme production
• Fig 5: Schematic representation of important interactions between the heavy
and light phases in an enzymatic biodiesel system
• Fig 6: Appearance and phase behavior of crude enzymatic soy bean biodiesel
• Fig 7: Comparison of enzymatic and chemical glycerin quality. Courtesy of
Piedmont Biofuels
• Fig 8: Enzymatic biodiesel process drawing
• Fig 9: Localized concentration of strong acidity in heavy phase droplet
• Fig 10: Column diagram of FFA/MG/DG/TG in crude biodiesel before and
after CW
• Fig 11: FAME and FFA concentrations during enzymatic transesterification
• Fig 12: FAME and FFA concentrations during one-pot polishing
• Fig 13: Correlation of glycerin concentration and Brix measurements
• Fig 14: Typical reaction curve with TG, DG, MG, FFA and FAME
List of tables
• Table 1: Key unit operations and technical requirements of the enzymatic

biodiesel process
• Table 2: Required and recommended feed oil analysis after pre-treatment
• Table 3: Standard operating conditions for the enzymatic biodiesel process
• Table 4: Important process control and monitoring parameters
• Table 5: Reaction conditions and composition of crude enzymatic biodiesel
from various feed stocks
• Table 6: Overview of typical enzymatic biodiesel feed stocks
• Table 7: Basic analytical tools for feed stock analysis, process control and
product validation
• Table 8: Process control parameters
• Table 9: trouble shooting guide
• Table 10: Various enzyme recycling strategies and their pros and cons
List of equations
• Equation 1: MeOH wt% by Karl-Fischer and Brix refractometry
68
LIST
List of appendices
• Appendix I
Novozymes Eversa® Transform MSDS (complete)
• Appendix II
Enzymatic Biodiesel Application Sheet (complete)
• Appendix III
NaOH determination method SOP (in preparation)
• Appendix IV
Laboratory settling test assay SOP (in preparation)
• Appendix V
Laboratory pre-treatment test assay SOP (in preparation)
• Appendix VI
Laboratory enzymatic biodiesel mini-batch assay SOP (in preparation)
• Appendix VII
Brix method for water/MeOH/glycerin SOP (in preparation)
69
Novozymes A/S
Krogshoejvej 36
2880 Bagsvaerd
Denmark
Tel. +45 4446 0000
visit www.novozymes.com
bioenergy@novozymes.com
Novozymes is the world leader in bioinnovation. Together with customers across a broad array of industries we
create tomorrow’s industrial biosolutions, improving our customers’ business and the use of our planet’s resources.
Read more at www.novozymes.com.
Main sales offices

For more office addressess, visit www.novozymes.com
Africa, Sub-Sahara Eastern Europe, Middle East Korea Russia & CIS countries
Sandton, South Africa & Northern Africa Seoul, South Korea Moscow, Russia
Tel. +27 11 444 8124 Dittingen, Switzerland Tel. +82 2 795 0882 Tel. +7 495 234 44 01
Tel. +41 61 765 6111
Australia & New Zealand Mexico, Central America South America
Sydney, Australia Indian Subcontinent & Caribbean Araucária, Paraná, Brazil
Tel. +61 2 9630 8466 Bangalore, India Mexico City, Mexico Tel. +55 413 641 10 00
Tel. +91 80 30593500 Tel. +52 555 318 96 80
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Laws, regulations, and/or third party rights may prevent customers from importing, using, processing, and/or reselling the products described herein in a given manner. Without separate, written
agreement between the customer and Novozymes to such effect, this document does not constitute a representation or warranty of any kind and is subject to change without further notice.
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THE

CHAPTER1. ENZYMATIC TRANSESTERIFICATION  7

CHAPTER 2. NOVOZYMES ENZYMATIC BIODIESEL PROCESS  17

CHAPTER 3. FEEDSTOCK FLEXIBILITY FOR ENZYMATIC BIODIESEL  33

CHAPTER 4. LABORATORY TOOLS FOR QUALITY ASSESSMENT AND CONTROL  41

CHAPTER 5. PROCESS OPERATION  53

CHAPTER 6. NOVOZYMES AS ENZYME SUPPLIER  63

Enzymatic biodiesel has been considered a promising alternative for the

This Enzymatic Biodiesel Handbook is intended for use as a practical

Here is a short summary of the key requirements for processing low-cost

• Robust pre-treatment and mineral acid neutralization

All the information provided herein is advisory and it is recommended to

CHAPTER1. ENZYMATIC TRANSESTERIFICATION

In order to reduce complexity, this document focuses on glycerides

Fig. 1: A: Oleic triacylglycerol (TG), B: Oleic diacylglycerol (DG), C: Oleic monoacylglycerol

(MG), D: Oleic free fatty acid (FFA)

In biodiesel terms, “transesterification” describes the reaction between

Fig. 2: Reaction scheme of transesterification of acylglycerides and FFA

Biodiesel production by transesterification of glycerides and fatty acids

Enzymatic technology enables biodiesel producers to circumvent these

Fig. 3. An enzyme is a molecule from a living organism consisting of a

The functionality of any enzyme is usually limited to one specific

Enzyme Enzyme-substrate complex Enzyme

Fig. 3: Lock-and-key model of enzymatic catalysis

Novozymes is world leader in production of industrial enzymes. In

Fig. 4 exemplifies the industrial production of enzymes.

Fig. 4: Step-by-step overview of enzyme production

Enzymes in biodiesel production

As biological molecules, most enzymes are water-soluble and generally

Water can be added to an enzymatic system as free water, with

In a biodiesel system, the hydrophilic nature of the enzyme causes it to

Fig. 5 shows a snapshot of the interactions between a suspended droplet

phases in an enzymatic biodiesel system

In a real-life application of enzymatic biodiesel production, a pseudo

(Note about Fig. 6: In real-life enzymatic biodiesel production, the feed

Taking advantage of this fact, Novozymes has developed strategies for

In full scale application, it has been shown

Further strengthening the overall process

Finally, compared to conventional biodiesel

Fig. 7: Comparison of enzymatic and chemical glycerin quality. Courtesy of Piedmont

Being biological molecules, enzymes are generally stable at ambient

The formulation of Eversa® Transform ensures a high stability and long

In case of skin contact, active enzymes can cause irritation. Therefore

Please see Novozymes MSDS sheet and enzymatic biodiesel application

CHAPTER 2. NOVOZYMES ENZYMATIC BIODIESEL PROCESS

The Novozymes enzymatic biodiesel process has been developed in

This close collaboration has resulted in an efficient and robust process

FAME after reaction

Fig. 8 Enzymatic biodiesel process

It is important to mention that Novozymes, as the enzyme supplier,

Alternatively, the engineering can be done internally but Novozymes

Novozymes’ engineering partners have extensive experience with the

Equipment and unit operations

Table 1 shows a list of the required equipment and unit operations

6 Enzyme reactor Good temperature control is required. Depending on the ambient

Gravimetric settling in the reactor is possible and offers a simple

A secondary separator can be beneficial to separate the heavy phase

Control parameters for feed oil

Table 2 shows a list of analyses to be used as control parameters for

Table 2 describes the minimum analysis required to approve a pre-treated

It is highly important to perform these initial feedstock analyses

Table 3 lists the key operating conditions for the enzymatic

The MeOH load is calculated based on the glyceride composition of the

CHAPTER1. ENZYMATIC TRANSESTERIFICATION 7

CHAPTER 2. NOVOZYMES ENZYMATIC BIODIESEL PROCESS 17

CHAPTER 3. FEEDSTOCK FLEXIBILITY FOR ENZYMATIC BIODIESEL 33

CHAPTER 4. LABORATORY TOOLS FOR QUALITY ASSESSMENT AND CONTROL 41

CHAPTER 5. PROCESS OPERATION 53

CHAPTER 6. NOVOZYMES AS ENZYME SUPPLIER 63