1. Countercurrent Enzymatic Saccharification of Cellulosic
Biomass
Andres Beascochea Sagar Lonkar, Mark T. Holtzapple
Bioenvironmental Sciences, Department of Plant Pathology and Microbiology, Texas A&M University
References
The Countercurrent Saccharization method previously showed a
reduction in the required enzymatic load in order to produce the
two sugars glucose and xylose necessary to produce ethanol (1)
However, the industry currently uses a batch process and few
studies have examined protocol variations to improve this
process have been limited. We tested a Concurrent
Saccharization of Cellulosic Biomass method for enhanced
efficiency of conversion was conducted using a 16 multistage
transfer process with various enzymatic loads. Solids and
liquids were transferred counter currently every two days while
liquid samples were taken to determine sugar concentration at
each stage. The results showed a high percentage yield of
glucose and xylose production with low enzymatic loads of
Htec3 and CTec3 (1mg/g dry biomass)..
Abstract
Since last summer , two enzymatic loads have been used. The
first enzymatic load was 1mg per gram of dry biomass of CTec3:
which resulted in a 60% yield of glucose, 32% yield of xylose with
50% total sugar.. The second contained both CTec3 and HTec3
enzymes at 1mg/g dry biomass. The results were a 67% yield of
glucose, 41 % yield of xylose and 57% total sugar (Figure 3). Note
the sugar concentrations increase overtime.
More recently, experiments with a load of 2mg/g dry biomass is
being tested. The samples collected from the trials will be
analyzed soon.
Introduction
Method
Transfer procedures
The trials involved a sixteen centrifuge bottle train process. The experiment would be run
every two days, until a steady state was attained. This could take over 35 days or more
depending on the trials. Transfers consisted of moving the liquid phase upwards manually
while moving the solid phase downwards as the enzymatic hydrolysis occurred. Each bottle
was recorded for the mass of each phase both liquid and solid, as well as the total weight
of each bottle, the pH of the liquid phase, calculation of the transfer wet cake or what was
moved upwards in the train, transfer liquid, as well as the addition of fresh enzyme and
antibiotics after each trial run. Samples of the liquid phase were collected from each of the
sixteen bottles every time the trial was run. The samples were prepared and analyzed via
HPLC for concentration of glucose and xylose. The enzyme load was placed in bottle 4 train
rather than at the end bottle sixteen. This method is also tested, and has currently shown
an increase in overall sugar production.
Method is described in FIGURE 1 (1) below: (note this shows an 8 bottle
train, currently a 16 bottle train is used.)
Method
Even at a low enzymatic load concentration the yield percentage
of xylose and glucose increases over time. Glucose is next
fermented to produce ethanol, and the presence of xylose the
enhances ethanol production. Continuous Counter Current
Saccharification is showing a good yield of sugar production at
low enzymatic load concentrations.
Figure 1 shows the levels of glucose and xylose production
throughout the bottles can be seen in FIGURE 1. What is shown
is that as the liquid phase is allowed to move from bottle 4
upwards to eventually one, an increase in sugar production is
seen. This is important as the liquid from bottle one is the end
result for process. This also shows that the countercurrent
system is behaving as it should be.
Discussion
• The Continuous Countercurrent Saccharization method
produces a good yield of glucose/xylose at low enzymatic
loads.
• Work is continuing to determine the optimal enzymatic load
resulting in the highest glucose/xylose production at the
lowest cost.
• The countercurrent method shows promise of a more
efficient way to produce ethanol as opposed to the batch
fermentation process.
• Second-generation biofuel production is an interest of mine
and I am glad that I was involved in experiments to improve
its efficiency.
Conclusions
All gasolines contains some ethanol. Most gasoline carries 10% by volume
and is usually referred to as E10 gasoline. In 2014, around 13 billion
gallons of fuel ethanol were added to the motor gasoline in the United
States. (3) Therefore it is economically advantageous to develop more
efficient ways to produce ethanol to reduce cost. This can be achieved by
increasing efficiency in one of the most costly factors, the enzyme loads
used. The batch fermentation process is one of the most common
processes of producing ethanol. . Batch fermentation is a common process
for producing ethanol. This process produces ethanol quickly but
production quickly declines as ethanol accumulates. An alternative is the
Countercurrent Saccharization of cellulosic biomass.
My group studied the benefits of a process known as continuous
countercurrent saccharification as opposed to batch saccharification. We
focused on the effects of alterations in enzyme Loading using two
commercial enzymes, CTec3 and HTec3 (1). The project goal is to define an
optimal enzyme for this process as well as obtain data on the percentage
yield of production of glucose and xylose during the process. The Enzymes
themselves tend to be the most expensive part of the process. Much of it
is often used inefficiently in batch Saccharization ethanol production. The
reason for this is that batch saccharification need high enzyme loadings,
and left over active enzymes which are often discarded. (1) The
continuous countercurrent method can improve saccharification by
overcoming these disadvantages. By having the sugar-rich liquid phase
and the enzyme-rich solid phase contact counter currently it allows the
sugars to flow away from the less digestible mass which would decrease
product inhibition. Allowing the process to happen at a lower enzyme
load. (1)
Results
Figure 2.CTec3: 1 mg/g dry biomass
HTec3: 1mg/g dry biomass
Figure 3. Yield % of glucose, xylose, total sugar over 35 days
after addition of both CTec3 and HTec3 Enzyme
CTec3: 1 mg/g dry biomass
HTec3: 1mg/g dry biomass
Yield (%)
Glucose 67
Xylose 41
Total sugar 57
Acknowledgements
I thank Dr. M. Holtzapple for letting me participate in his lab.
Dr. Wilkinson for introducing me to Dr. Holtzapple. Sagar
Lonkar for training and explaining the processes and purpose
behind the lab as well providing data .
Sponsors for high impact experiences for BESC and the BESC
poster symposium include the Department of Plant
Pathology and Microbiology, the College of Agriculture and
Life Sciences, the Office of the Provost and Executive Vice
President for Academic Affairs.
•1. Zentay, Agustin N., and Mark T. Holtzapple. "Countercurrent Enzymatic Saccharification of
Cellulosic Biomass." 1-36.
• 2. Zentay, Agustin. "COUNTERCURRENT ENZYMATIC SACCHARIFICATION OF LIGNOCELLULOSIC
BIOMASS AND IMPROVEMENTS OVER BATCH OPERATION." 2014.
• 3. How much ethanol is in gasoline and how does it affect fuel economy? (2015, April 3). Retrieved
April 4, 2015, from http://www.eia.gov/tools/faqs/faq.cfm?id=27&t=10
FIGURE 1 (1)