Leya Thomas et al., Enzymatic Saccharification of Rice Straw Cellulose…..
September 1, 2010 Leave a comment
Leya Thomas*, Jiby.K.Kurien** and V.V.N.Kishore, Enzymatic Saccharification of Rice Straw Cellulose and its Subsequent Fermentation to Bioethanol, (Ed.) S.Jisha, B.Hari & T.K.Remesan, Proc. Nat. Sem. on Env. Biotech. Chall. and Oppor., ENVIROTECH-2008, P.G. Dept. of Zoology, S.N.C. Natiika, pp 89-101
(*School of environmental studies, CUSAT, Cochin, **The Energy and Resource Institute, New Delhi, e.mail:layam31@gmail.com)
Abstract
Objective of the study is enzymatic hydrolysis of rice straw cellulose and its subsequent fermentation to ethanol. Five cellulolytic strains namely C1,C2,C3,C5,C8 could be successfully isolated from different sources such as compost piles, rice straw, soil samples etc. and the performance has been compared with cellulose enzyme (Carezyme 1000 L, Sigma Aldrich). Solid state Fermentation (SSF) was performed for the Enzymatic saccharification of cellulose to glucose. Rice straw was undergone a two stage pre-treatment (acid and alkali pre-treatment) in order to remove lignin and hemicellulose so that the strains could readily conduct the enzymatic saccharification process. The presence of glucose by the conversion of cellulose was checked by DNS method and the percentage of conversion calculated. The crude extract which contains glucose was further used for fermentation to ethanol using yeast strains. The culture C2 had a saccharification efficiency of 24.27 % were as carezyme 1000 L Sigma Aldrich Had only 3.45% efficiency.
By altering the various parameters cellulase production can be enhanced. The maximum cellulase production was obtained at an incubation period 48 Hours, pH 5.0, Temperature 500C, Nitrogen source:Yeast extract for culture C2, NH4SO4 for culture C1and a moisture content of 10 %. Maximum conversion efficiency for cellulose to glucose was 81.4%.The Next step was the optimization of yeast growth conditions. The optimum growth rate was obtained at a pH of 4.5, Incubation period of 120 Hours and substrate concentration 20 g/L. The reducing sugar concentration of the crude extract obtained by solid state fermentation was adjusted to a substrate concentration of 2%. It was then inoculated with yeast strains TERI βCR and IITD followed by fermentation.
Introduction
Lignocelluloses have been recognized as an abundant and cheap raw material for fuel ethanol production. It contains both cellulose and hemicellulose as source of hexose’s and pentose sugars respectively. Several economic evaluations do indicate that efficient utilization of hexose’s is an extremely important factor for lignocelluloses to ethanol conversion process. In this context enzymatic saccharification of rice straw cellulose in its least expensive way deserves prior attention.
A variety of environmental problems now affect our entire world. The biggest environmental problem facing the world today is probably global warming. About 33% of U.S carbon dioxide emissions come from the burning of gasoline in internal-combustion engines of cars and light trucks (US Emissions Inventory 2006). Bioethanol (ethanol from biomass) is an attractive, sustainable energy source to fuel transportation it is an effective tool for reducing air toxics that come from the transportation sector. Ethanol can also protect our land and water against the effects of large fuel spills or leaks. Based on the premise that fuel bioethanol can contribute to a cleaner environment and with the implementation of environmental protection laws in many countries, demand for this fuel is increasing. Efficient ethanol production processes and cheap substrates are needed.
Current ethanol production processes using crops such as sugar cane and corn are well established; however, utilization of a cheaper substrate such as lignocellulose could make bioethanol more competitive with fossil fuel. The processing and utilization of this substrate is complex, differing in many aspects from crop-based ethanol production. One important requirement is an efficient microorganism able to ferment a variety of sugars (pentoses, and hexose’s) as well as to tolerate stress conditions. While there are ongoing efforts to further enhance their properties, improvement of the fermentation process is just one of several factors that needs to be fully optimized and integrated to generate a competitive lignocellulose ethanol. As an alternative fuel, not only does bioethanol reduce carbon dioxide, it also helps reduce our dependence on oil significantly. This is a major advantage, since oil is a finite resource for which global demand is expected to eventually exceed supply.
Materials and Methods Isolation of cellulolytic organisms from the natural environment. The sources selected were soil samples from different locations of Haryana, decaying wood, leachates, compost piles, Cow dung slurry.
Isolation of Fungi: The samples were mixed with sterile distilled water and a series of dilutions were made. From the dilutions, 0.5ml volumes were pipetted in to the isolation media. For checking the bacterial growth 500 mg/L of penicillin was also added. After 3-7days of incubation at 50oC on a shaker, a patch of a yellow pigmented material appeared at the liquid air interface on the filter paper. As soon as the pigmented material appeared, a portion of filter paper was transferred with a sterile wire and inoculated in to fresh medium. This process was repeated several times to enrich the aerobic and thermophilic cellulose-utilizing organisms. The filter paper from the enriched culture was removed, macerated in a small amount of sterile water, and streaked on to plates containing filter paper agar (a plate of nutrient agar covered with filter paper). Representatives of various colonies which developed on each of these media were picked and inoculated in to fresh media in to test tubes. Tubes showing visual degradation of filter paper were selected and alternatively transferred to liquid and solid media for enrichment and isolation of cellulolytic organisms.
Media (Composition in g\L)
Filter paper 50mg
KH2PO4 1.0g
(NH4)2SO4 0.543g
KCl 0.5g
MgSO4.7H2O 0.2g
CaCl2 0.1g
Thiamine hydrochloride 0.001g
Distilled water 1000mL
pH 5.5
Confirmation of Cellulolytic fungi
a) Visual determination of cellulolytic activity: The isolated fungus were grown on cornmeal agar and water agar containing filter paper, and checked for clearing zones. The visual determination was used as a preliminary screening test for the cellulolytic organisms.
b) Colorimetric determination of cellulolytic activity: The filter paper medium, in which the fungus was grown, was checked for the presence of reducing sugar by DNS method.
Pretreatment of rice straw (acid and alkali pretreatment): The rice straw was treated with 1% of H2SO4 and kept at 15 psi for 1 hour at 120oC in an autoclave. The contents of the flask were filtered using a nylon mesh. The acid hydrolysate obtained after filtering was discarded and the acid treated rice straw was washed well until a neutral pH was reached. The next step is alkali treatment. The previously treated rice straw was taken in a conical flask. It was treated with 15% of NaOH and 2.5% H2O2 and kept at 15 psi for 1 hour at 120oC in an autoclave. The contents filtered using a nylon mesh and the hydrolysate discarded. The resulting rice straw was washed until neutralized. This was done inorder to make it lignin free so that the microbes can act quickly. Adaptation of the isolated strains to the pretreated rice straw: The isolated strains were inoculated on the pretreated rice straw which were varied based on pH, moisture, temperature, nutrients other than carbon source were supplied the strains which survived these conditions were taken and the parameters optimized.
Solid State Fermentation (SSF)
Optimization of Solid State Fermentation (SSF) conditions: Substrates used were rice straw [pretreated], which is cost effective and supplies the nutrients and anchorage to the microbial culture growing in it. However, some of the nutrients may be available in sub-optimal concentrations, or even absent in the substrates. In such cases, it would become necessary to supplement them externally with these. Various parameters such as incubation period, pH, temperature, and moisture content and nitrogen source were optimized. The SSF was carried out in 100ml Erlenmeyer flasks with 5g of carbon substrate the moisture content was 10%, pH 5. The spore inoculums [0.2ml] in the range of 107 spores was used tween 80 [1-2] drops was added to give uniform suspension of spores. The flasks were incubated at 50oC cellulase production was calculated by the amount of cellulose converted to glucose in a time period of 24, 48, 72 etc.
Enzyme Extraction (crude extract): After the growth has taken place 20ml of 0.05mMol sodium acetate buffer was added in to each. The flasks were then kept in a microbial shaker at 200rpm for 1 hour for the release of enzyme. The suspension was centrifuged and filtered. The supernatant was taken for cellulase and reducing sugar assay.
Reagents
- Buffer stock solution: (Sodium Acetate buffer, 1 M, pH 4.5)
- Extraction/Dilution buffer: [Sodium acetate, 25 mM, pH 4.5 containing sodium azide (0.02 %)]
Cellulase Estimation: For the estimation of cellulase 2 types of tests are conducted, Indirect test and Direct test
Indirect test: It is done by checking the amount of glucose produced in the crude extract Method: DNS test Instrument Used: Spectrophotometer
Procedure
- Standard glucose solution of concentration 50mg/mL is made.
- Glucose solution of known concentration (0.2mg/mL, 0.4mg/mL, 0.6mg/mL, 0.8mg/mL, 1mg/ml) was prepared.
- 3ml of DNS is added to1 mL of sample in all tubes and boiled for exactly 5 minutes. Cool immediately. Make up to 20mL using distilled water
- The OD is read at 540 nm wavelengths on a spectrophotometer after
adjusting it using the blank.
Direct test
Procedure: Modified Worthington, C.E
Optimization of Yeast growth conditions: In order to find out the optimum growth conditions the yeast cultures were inoculated in to the media with varied substrate (glucose) concentration and pH. The substrate concentrations used were 2g, 3g, 4g, 5g and 6g were as the pH were 4.5, 3.5 and 2.5. The best growth rate was found out from the growth curve using absorbance values from a spectrophotometer at O.D 600nm.
Identification of Fungus: One of the isolated fungus were identified as Aspergillus sps. by conventional methods and given for further identification.
Results
Table 1 Filter paper disintegration by cultures Vs time
Isolation and Screening of Cellulolytic Fungi
| CULTURE | 24h | 48h | 72h | 96h | 120h | 144h |
| C1 | – | – | – | + | + | + |
| C2 | – | – | – | – | – | + |
| C3 | – | – | – | + | + | + |
| C4 | – | – | – | – | – | – |
| C5 | – | – | + | + | + | + |
| C6 | – | – | + | + | + | + |
| C7 | – | – | + | + | + | + |
| C8 | – | – | + | + | + | + |
| C9 | – | – | – | – | – | – |
| C10 | – | – | – | – | – | – |
| C11 | – | – | – | – | – | – |
| C12 | – | – | – | – | – | – |
Table 2 Cultures grown on CMA Vs Time.
Confirmation of Cellulolytic Fungi
| CULTURE | 24h | 48h | 72h | 96h |
| C1 | – | + | + | + |
| C2 | – | + | + | + |
| C3 | – | – | + | + |
| C5 | – | – | + | + |
| C8 | – | – | + | + |
Table 3 Cultures grown on water agar (containing filter paper) Vs Time
| CULTURE | 24h | 48h
|
72h | 96h |
| C1 | – | – | + | + |
| C2 | – | + | + | + |
| C3 | – | – | + | + |
| C5 | – | – | + | + |
| C8 | – | – | + | + |
Table 4 Percentage composition of rice straw and treated rice straw
Pretreatment of Rice Straw (Acid and Alkali pretreatment)
| Sample
|
Cellulose (%) | Hemicellulose(%) | Lignin (%) | Ash (%) |
| Rice straw | 31.45 | 35 | 20.4 | 14.78 |
| Treated Rice straw | 76.6 | Nil | 11.2 | 1.15 |
Table 5 DNS test results of crude extract of various cultures
Optimization of Cellulase Production Conditions
| Culture | DNS test (crude extract) |
| C1 | + |
| C2 | + |
| C3 | – |
| C5 | – |
| C8 | – |
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Table 6 Sugar Concentration by Direct Method Direct Method (48 Hours)
| Culture | Reducing sugar concentration (g/L) | Saccharification efficiency
|
|||||
| Test (t) | Control (c) | (t)- (c) | Total | ||||
| C1 | 0.26 | 0.26 | 0 | 0 | 0 | ||
| C2 | 1.19 | 0.316 | 0.874 | 4.37 | 24.27 | ||
| C3 | 0.170 | 0.004 | 0.166 | 0.14 | 0 | ||
| C5 | 0.560 | 0.453 | 0.170 | 0.255 | 1.4 | ||
| C8 | 0.420 | 0.167 | 0.253 | 0.605 | 3.36 | ||
| Enzyme
(Carezyme 1000 L, Sigma Aldrich) |
0.124 | 0 | 0.124 | 0.622 | 3.45 | ||
Table7 Reducing sugar concentration of crude extract in percentage Reducing sugar concentration of crude extract (%)
| Hour | C2Y | C2 | C2U | C1 |
| 2 4 | 29.3 | 2.2 | 30.7 | 65.7 |
| 48 | 81.4 | 2.4 | 39.3 | 61.6 |
| 72 | 0 | 0 | 15 | 0 |
| 96 | 0 | 0 | 0 | 0 |
|
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