Bổ sung cellulase trong quá trình nghiền đã phát huy hiệu quả bởi endoglucanase tham gia

vào quá trình phân cắt các cầu nối trong mạch cellulose, làm giảm năng lượng nghiền và
tiêu hao hóa chất, làm giảm năng lượng nghiền và tiêu hao hóa chất (làm giảm nhanh chiều
dài của chuỗi và làm tăng nhẹ số lượng các nhóm khử tự do)

There is an increasing demand for cellulases in the market for various applications, among which the
bioconversion of lignocellulosic biomass for ethanol production is the major one. Improvements in the
titers as well as specific activities of cellulases are highly desired for its use in bioethanol production as
well as in other applications. This review deals with developments in bioprocess technologies, solid-state
and submerged fermentation as well as on the strategies adopted for improving cellulase production or
properties

The development of new cost-efective bioprocesses for the production of cellulolytic enzymes is needed
in order to ensure that the conversion of biomass becomes economically viable.

Today we’re going to determine whether a novel sequential solid-state and submerged fermentation
method could be validated for different strains of the Trichoderma genus.

Introduction:

Figure 6.3: Preview to the process of producing ethanol from lignocellulosic biomass.

Different cultivation method have been used in study for cellulase production by T.reesei, such as:

- Submerged fermentation:
 Involves production of enzymes by microorganisms in a liquid nitrient media
- Solid state fermentation:
Cultivation of microorganism on a solid substrate
Solid-state fermentation uses a solid substrate to grow microorganisms while
submerged fermentation uses a liquid medium to grow microorganisms. Thus,
this is the key difference between solid state fermentation and submerged
fermentation. Solid-state fermentation takes place under low moisture level
while submerged fermentation takes place under high water content.

Solid State Fermentation Submerged Fermentation

Definition SSF is a type of fermentation Involves production of
that occurs by the enzymes by microorganisms
microorganisms grown on a in a liquid nitrient media
solid subtrate
Cultivation of On a solid subtrate In a liquid medium
microorganismms
Microbial cells Grow adhered to the solid Microbial cells evenly
surface distribute throughout the
medium
Culture medium Not free-flowing Always free-flowing
Water content of the medium 40-80% More thhan 95%
Nutrient distribution Nutrient concentration is not Nutrients are evenly
even distributed
Inoculum Ratio High Low
Different cultivation methods have been used for cellulase production by T. reesei, such as
submerged fermentation (SmF) and solid-state fermentation (SSF) . Each of these cultivation systems has its own
advantages and disadvantages associated with the environmental and operational conditions. Nevertheless, SSF
more closely resembles the natural environment of the fungus, because T. reesei grows naturally on solid cellulosic
material. However, industrial-scale production of enzymes by SSF still faces technical limitations. On the other
hand, SmF is the most widely used cultivation method for industrial enzyme production, because the operational
techniques and the control of environmental factors (such as temperature and pH) are well established

Method: Nguyên liệu và phương pháp

SSF is normally multistep processes involving the following steps:

Nói: Pre-treatment of raw materials for substrates whether through mechanical, chemical or
biochemical process to improve the absorption of bound nutrients as well as to minimize the
dimensions of the components, e.g., pulverizing straw or shredding vegetable material to enhance
the physical aspects to the method.

Schematic representation of a SSF process.

 Figure: Schematic of solid state fermenter for conversion of
lignocellulosic biomass to enzymes
1. The first step is Pre-treatment of subtrate raw materials either by:
a) Mechanical (Milling or grinding)
b) Chemical (Alkali or acid treatment)
c) Biochemical processing (Enzymatic)
d) Physicochemical (Steam or Liquid hot water)
e) Hydrolysis of primarily polymeric substates, e.g: polysacccharides and proteins
 The purpose of pre-treatment of subtrates is to to enhance the availability of the bound
nutrients and also to reduce the size of the components
2. Sterilization of the substrates
3. Inoculation of the substrate with the microorganism of interest
4. Fermentation of pre-treated substrates by the microbes
5. Separaton and purification of the end products
- Mostly, water is add so as to dissolve the desired product along with other metabolites in the
water
- The liquid obtained after the seperation of solids is futher subjected to product recovery
methods including its purification

SmF is also a multistep process involving the following steps:

a. 5g bã mía khô (UB hoặc PB)
b. Inoculum:  ecoli from the  freezer and thawing it it's used to inoculate 
1mL spore suspension ( huyền phù bào tử) 10 7 spore/mL transferred to Erlenmeyer flask
100 mL of nutrient medium
 Fermenter: a broth tank for out final product the bioreactor is equipped with a water jacket
arroud the vessel to regulate temperature and integrated sensors  to monitor key environmental factors
including  
Culture conditions:  the bioreactor is set up for its fermentation cycle: The
incubation was carried out for 48 h at 30 °C with stirring at 200 rpm
Media sterilization: sterilization is 121 degrees celsius  for 30 minutes
c. mix the media in the vessel the agitator  is turned on and the ingredients are added
d. The next process step is Recovery
- Cells are separated from the fermentation broth
- The filtered broth contains the desired product
- The broth is further subjected to down streaming processes including purificatioon process
obtained the final product
Quy trình phục hồi sử dụng nhiều công cụ khác nhau như máy ly tâm
để cô lập sản phẩm
1. Preparation of liquid media. No pre-treatment is required in this case
2. Sterilization of the media
3. Inoculation of the media with the microorganism of interest
4. Fermentation of media by the microbes
5. Separation and purification of end products
- Cells are separated from the fermentation broth
- The filtered broth contains the desired product
- The broth is further subjected to down streaming processes including purificatioon process
obtained the final product
 - O2 and CO2
SF: the pre-culture was initiated as solid-state fermentation (SSF)
Solid substrate (5g of dry sugarcane bagasse (UB or PB)) -----( add 107 spores/g of dry bagasse )
SmF
1. Fermentation may be carried out as
batch or continuous
2. Medium is added in large vessel
3. Surface area to volume height ratio is less
4. 5-10% of inoculums is added
5. Inoculum is usually in liquid form
6. Product used are usually high as
compared to input cost
7. Lesser space is required
8. Less contamination
9. If a batch get contaminated there is a loss
of entire batch
10. Entire fermentation media is utilized by
microorganism for growth and product
fermentation
11. Aeration and agitation of system is
possible by use of spaarger and impeller
12. Power consumption is high
13. Controlling parameters like temperature,
pH is easy
14. Foaming occurs
15. Automation and use of computer is easy
16. Less Labor required
Subtrate used
SmF
Soluble sugar
Liquid media
Fruit and vegetable juices
Waste water
pyrolysed sugarcane bagasse (PB)
UB = untreated bagasse
SSF
1. Fermentation may be carried out as
batch
2. Medium is add in flat vessel or trays
3. Surface area to volume height ratio very
high
4. Less inoculum is added
5. Inoculum is usually sprayed on surface of
medium
6. Product yield is comparatively less
7. More space is required
8. More contamination
9. If a tray gets contaminated then there is
a loss of only tray but not the batch
10. There is wastage of fermentation media
11. Aeration is usually carried out by passing
sterile air and no agitation
12. Power consumption is less
13. Controlling parameters like temperature,
pH is difficult
14. Foaming doesn’t occurs
15.
SSF
Wheat bran
Rice and wheat straw
Fruit and vegetable waste
Bagasses
Coconut coir
Synthethic media
 Egase activity (IU/L)
Assay pH Temperature SmF-UB SF-UB SmF-PB SF-PB
1 3 (-1) 30 (-1) 80.6 58.6 146.5 229.5
2 6 (+1) 30 (-1) 19.5 87.9 339.4 498.2
3 3 (-1) 80 (+1) 9.8 26.9 87.9 109.9
4 6 (+1) 80 (+1) 14.7 24.4 173.4 173.4
5 2.5 (-1.41) 55 (0) 7.3 9.8 4.9 4.9
6 6.5 (+1.41) 55 (0) 85.5 239.3 705.7 1,051.1
7 4.5 (0) 20 (-1.41) 24.4 67.8 255.6 258.9
8 4.5 (0) 90 (+1.41) 4.9 49.0 124.8 143.3
9 4.5 (0) 55 (0) 102.6 268.7 664.5 1,245.4
10 4.5 (0) 55 (0) 100.1 232.1 635.1 1,160.0
11 4.5 (0) 55 (0) 102.6 224.8 647.4 1,050.1
12 4.8 50 136.8 259.1 635.3 1,018.3