Isolation of Cellulase producing organisms from soil

 Aim

To isolate cellulose producing organisms (cellulolytic) from the soil

 Principle 

Cellulose is a predominant component of higher plants and most abundant biopolymer and polysaccharide found on earth. It forms the main constituent of cell wall of plants, most algae and some fungi. Cellulose is a linear homologous polymeric chain consisting of D-glucose residues that make up to 10,000 glucose residues, linked by β-1,4 glycosidic bonds. 

Though cellulose is plentiful, it is not utilized completely due to the fact of being insoluble. It is the food for ruminant mammals and termites which have bacteria that secrete cellulose digesting enzymes-cellulases. A cellulosic enzyme system consists of three major components: endo-ß-glucanase (CMCase) exo-ß-glucanase (cellobiohydrolase) and ß-glucosidase (cellobiase). The co-operative action of these three enzymes result in the complete hydrolysis of cellulose to glucose.

 The organisms which produces cellulase are known as cellulolytic organisms. Soil contains numerous microorganisms which produce cellulase. Eg. Cellulomonas, Clostridium, Pseudomonas and Trichoderma, Aspergillus etc. Fungi are the main cellulase-producing microorganisms, though a few bacteria and actinomycetes have also been recently reported to produce cellulase. 

Cellulase production can be detected using agar containing cellulose. Cellulose agar is a medium where if we grow microorganisms which produce cellulase, a clear zone develops around the growth, which indicates cellulolysis.

 Cellulose is the most abundant and renewable feedstock for energy source and can be used for the production of alternative fuels by the conversion of lignocellulosic biomass to biofuel. It has a wide range of applications such as food and beverage, pulp and paper, textile, animal feed, detergent and agriculture. 

Materials Required

 Soil sample, Petriplates, Carboxymethyl cellulose agar,  Tubes for serial dilution, L- rod

 

Procedure

1.      1g fertile soil was dissolved in 100 ml sterile distilled water to obtain 10^-2 dilution. It was then serially diluted to obtain dilutions till 10^-6.

2.    Aliquots of I ml from 10^-4, 10^-5, 10^-6 dilutions were transferred into Carboxymethyl cellulose plates and spread plate performed.

3.      Inoculated Carboxymethyl cellulose plates were then incubated at 37⁰C for 2-5 days.

4.      After incubation, the plates were flooded with Gram’s Iodine and observed

 

Result

Presence of colourless zones or clear halo was detected around cellulolytic colonies on the plates, which indicated cellulose hydrolysis.

 

Media Composition (Don't write in record sheet)

 Carboxymethyl Cellulose Agar (pH 7.0)

Carboxymethyl Cellulose 10 g

NaCl 0.1 g

Yeast extract 0.4 g

NH 4 H 2 PO 4 0.1 g

MgSO 4 7H 2 O 0.2 g

Agar 15 g

Distilled water 1000 ml

Hepatitis A, B, C, D, E and G

 

Hepatitis cannot be differentiated by symptoms because of similar symptoms. Hepatitis is a common viral infection with a variety of strains , some acute and some chronic in nature.

 

Table showing the Differences between Hepatitis A, B, C, D and E
Different types of Viral Hepatitis
	Hepatitis A	Hepatitis B	Hepatitis C		Hepatitis D	Hepatitis E
Aetiology (cause)	Hepatitis A Virus	Hepatitis B Virus	Hepatitis C Virus		Hepatitis D Virus	Hepatitis E Virus
Structure	Single stranded RNA virus	Single Stranded DNA virus	Single Stranded RNA virus		Single Stranded RNA virus	Single Stranded RNA virus
Presence of Envelope	Non-enveloped virus	Enveloped virus	Enveloped virus		Enveloped virus	Non-enveloped virus
Viral Family	Picorna virus	Hepadna virus	Flavi virus		No Viral Family but known as Delta virus	Hepevirus
Incubation Period	2 to 7 weeks	6 weeks to 6 months	2 to 23 weeks		2 to 8 weeks	3 to 9 weeks
Mode of Transmission	Eating contaminated food and drinks (Feco-oral spread)	Mostly by blood and sexual contact		By blood and sexual contact	Coinfection or superinfection with Hepatitis B	Feco-orally spread
Mother to Child	May not be transmitted to baby in pregnancy	Mother can transmit to baby during birth		Mother can transmit to baby during birth	May not be transmitted to baby in pregnancy	May not be transmitted to baby in pregnancy
Treatment and Drugs	No drugs; only treated by supporting the body to fight the infection	Antiretroviral drugs (e.g. Lamivudine)		Ledipasvir and Sofosbuvir	Lonafarnib and pegylated interferon	No treatment, only supportive management
Severity and Complications	Does not cause Liver cancer	Can cause Liver cancer		Can cause Liver cancer	Does not cause Liver cancer on its own but worsen it in Hepatitis B infection	Does not cause liver cancer. It has bad outcome in Pregnant women
Vaccines availability	There is a vaccine	There is vaccine available		There is no vaccine	Vaccination against Hepatitis B is protective	There is vaccine

Hepatitis cannot be differentiated by symptoms because all Hepatitides have similar symptoms.

Hepatitis is a common viral infection in the hospital population, especially with liver failure patients. There are a variety of strains of hepatitis, some acute and some chronic in nature.

Hepatitis A

Cause:  Fecal-oral route of transmission, meaning that a person ingests food or water that has been contaminated by human waste containing the Hepatitis A virus. It is often spread among household members via the passage of oral secretions (kissing) or stool (poor hand hygiene).

Length of Infection: Hepatitis A does not cause a long-term infection. Hepatitis A can be prevented with a vaccine.

Hepatitis B

Cause: Hepatitis B is a blood borne infection, meaning it is spread by direct contact with infected blood or bodily fluids.  Specific examples of transmission include sharing dirty needles when injecting IV drugs, having sexual contact with an infected individual, or passing infection from an infected mother to her unborn child.

Length of Infection: This form of hepatitis causes chronic liver damage in a small percentage of infected individuals; however, many people recover from this within six months. Hepatitis B can cause cirrhosis, liver failure, and liver cancer.  There is also a vaccine available to prevent Hepatitis B.  

Hepatitis C

Cause: Hepatitis C is spread similarly to Hepatitis B.  It is most commonly spread by sharing dirty needles for IV drug use or coming into contact with infected blood.

Length of Infection: The majority of people infected with Hepatitis C have this as a life-long infection. It can lead to cirrhosis. There is no vaccine for this form of Hepatitis.  Many people with this infection have no symptoms until their cirrhosis is very advanced.  Hepatitis C can also cause liver failure and liver cancer. 

Hepatitis D, Hepatitis E, And Hepatitis G

Hepatitis D virus (HDV), also known as the delta virus or agent. It is a small virus that requires concomitant infection with HBV to survive. HDV cannot survive on its own because it requires a protein that the HBV makes (the envelope protein, also called surface antigen) to enable it to infect liver cells. The ways in which HDV is spread are by shared needles among drug abusers, contaminated blood, and by sexual contact; essentially the same ways as HBV.

Individuals who already have chronic HBV infection can acquire HDV infection at the same time as they acquire the HBV infection, or at a later time. Those with chronic hepatitis due to HBV and HDV develop cirrhosis (severe liver scarring) rapidly. Moreover, the combination of HDV and HBV virus infection is very difficult to treat.

Hepatitis E virus (HEV) is similar to HAV in terms of disease, and mainly occurs in Asia where it is transmitted by contaminated water.

Hepatitis G virus (HGV, also termed GBV-C) was recently discovered and resembles HCV, but more closely, the flaviviruses; the virus and its effects are under investigation, and its role in causing disease in humans is unclear.

Symptoms Of Hepatitis

The symptoms of hepatitis are similar for all three types:

· Fever

· Nausea and vomiting

· Loss of appetite

· Flu-like symptoms

· Fatigue and weakness

· Lack of nutrition

· Aching in the abdomen

· Pale or clay-colored stool

· Dark urine

· Yellowing of skin or eyes

Treatment

For acute hepatitis, the main treatment is symptom relief.  Special consideration should be given to any medications that a patient takes to see the effect it may have on an impaired liver.

Chronic hepatitis (B and C) is managed mainly with medications to eradicate the virus. Patients should avoid alcohol and cigarettes which can aggravate the liver.