Isolation of Three Isolation of Three ThermophilicThermophilic Bacterial Strains (lipase, cellulose, Bacterial Strains (lipase, cellulose, and Amylase producers) From Hot and Amylase producers) From Hot

Springs in JordanSprings in Jordan

Amjad KhalilAmjad Khalil

Associate ProfessorAssociate Professor

Biotechnology Research GroupBiotechnology Research Group

King Fahad University of Petroleum King Fahad University of Petroleum and Mineralsand Minerals

Dhahran 31261, Saudi ArabiaDhahran 31261, Saudi Arabia

INTRODUCIONINTRODUCION

Microorganisms, specifically bacteria, Microorganisms, specifically bacteria, continue to be discovered living under continue to be discovered living under extreme environmental conditionsextreme environmental conditions previously thought to be unable to previously thought to be unable to sustain life.sustain life.

Bacteria have recently been found Bacteria have recently been found living in very acidic environment at pH living in very acidic environment at pH 0-1.0-1.

Other bacteria survive temperatures as Other bacteria survive temperatures as low as −20 °C and high salinity and low low as −20 °C and high salinity and low light conditions.light conditions.

Thermophilic bacteriaThermophilic bacteria

ThermophilicThermophilic microorganisms microorganisms are a group of extremophiles are a group of extremophiles whose optimal growth whose optimal growth temperature lies between 45-temperature lies between 45-80°C, while extremophiles whose 80°C, while extremophiles whose optimum is above 80°C are called optimum is above 80°C are called hyperthermophileshyperthermophiles

These extremophiles may These extremophiles may perform novel metabolic perform novel metabolic functions producing functions producing highly stable highly stable enzymes and proteinsenzymes and proteins capable of capable of significant geochemical functions significant geochemical functions in situ and potentially adaptable in situ and potentially adaptable to to industrial, biotechnological industrial, biotechnological and bioremediation applications.and bioremediation applications.

Thermostable EnzymesThermostable Enzymes

ThermophilicThermophilic and and thermotolerantthermotolerant microorganisms are potential sources of microorganisms are potential sources of suitable enzymes that are more suitable enzymes that are more thermostable as well as more resistant than thermostable as well as more resistant than their mesophilic counterparts to organic their mesophilic counterparts to organic solvents, detergents, low and high pH, and solvents, detergents, low and high pH, and other denaturing agents.other denaturing agents.

application of application of thermostablethermostable enzymesenzymes

Reduces the reaction timeReduces the reaction time and limits the probability of and limits the probability of microbial contaminationmicrobial contamination during the long operating time during the long operating time of the reactors which provide considerable energy. of the reactors which provide considerable energy.

They also cause They also cause inactivation of undesired enzymesinactivation of undesired enzymes originating, from (for example) food material, during the originating, from (for example) food material, during the industrial process. industrial process.

Moreover, small activities of thermostable enzymes at Moreover, small activities of thermostable enzymes at low temperatures allow terminating the reaction just by low temperatures allow terminating the reaction just by coolingcooling. .

Enhanced solubilityEnhanced solubility of the reagents and decreased of the reagents and decreased viscosity of the reaction environment. viscosity of the reaction environment.

It improves the mixing and pumpingIt improves the mixing and pumping when concentrated when concentrated substrate solutions are used. substrate solutions are used.

Examples of Examples of ThermostableThermostable enzymesenzymes

1- Amylases1- Amylases Applications of amylasesApplications of amylasesA- Liquefaction- A- Liquefaction- is a process of dispersion of insoluble starch granules in is a process of dispersion of insoluble starch granules in

aqueous solution followed by partial hydrolysis using aqueous solution followed by partial hydrolysis using thermostable amylasesthermostable amylases . .

B- Manufacturing of maltose- B- Manufacturing of maltose- Maltose is widely used as sweetener and also as Maltose is widely used as sweetener and also as

Intravenous sugar supplement.Intravenous sugar supplement. C- Manufacture of oligosaccharides mixtureC- Manufacture of oligosaccharides mixture It is used for preventing crystallization of sucrose in foods It is used for preventing crystallization of sucrose in foods

and keeping a certain level of hardness of the texture and keeping a certain level of hardness of the texture during storage.during storage.

D- Manufacture of maltotetraose syrup.D- Manufacture of maltotetraose syrup.E- Removal of starch sizer from textile .E- Removal of starch sizer from textile .

2-2- LipasesLipases A- production of carbohydrate esters of fatty A- production of carbohydrate esters of fatty

acids.acids. B- food and flavor making.B- food and flavor making. C- catalysis of several unnatural reactions C- catalysis of several unnatural reactions

such such

as esterification and transesterification. as esterification and transesterification. D- synthesis of fatty esters as cosmetic or D- synthesis of fatty esters as cosmetic or

surfactants. surfactants.

3-3- CellulasesCellulases Cellulose is the most abundant organic compound in Cellulose is the most abundant organic compound in

the world and it is a kind of renewable energy that the world and it is a kind of renewable energy that human beings can easily utilize.human beings can easily utilize.

Applications of cellulasesApplications of cellulases A- BiopulpingA- Biopulping B- Cellulases and Textiles :Cellulases are used to B- Cellulases and Textiles :Cellulases are used to

finish fabrics by degrading surface fibres to prevent finish fabrics by degrading surface fibres to prevent colour fading , give jeans a stonewashed effect, or colour fading , give jeans a stonewashed effect, or help in the tanning of leathers.help in the tanning of leathers.

C- Cellulases and Biofuels: C- Cellulases and Biofuels: In the States, the production of fuel alcohol from In the States, the production of fuel alcohol from

corn starch reached about 3.41 billion gallons in corn starch reached about 3.41 billion gallons in 20042004

AbstractAbstract

Three strains of Three strains of thermophilicthermophilic bacteria bacteria were isolated from three different hot were isolated from three different hot springs in Jordan. springs in Jordan.

These strains were designated These strains were designated 1- AM8- isolated from Ma’een, 1- AM8- isolated from Ma’een, 2- AZ22 - isolated from Zara and 2- AZ22 - isolated from Zara and 3- AN133 - isolated from North Shona3- AN133 - isolated from North Shona

Cells were Gram stain-positive, strictly Cells were Gram stain-positive, strictly aerobic, grew optimally at pH 7.5–8.5 aerobic, grew optimally at pH 7.5–8.5 and temperature 55–60 and temperature 55–60 00C and C and tolerated maximally 10% (w/v) NaCl. tolerated maximally 10% (w/v) NaCl.

The three isolates were lipase, The three isolates were lipase, cellulose, and amylase producers with cellulose, and amylase producers with variable degrees of enzymatic activity.variable degrees of enzymatic activity.

They grew on various complex substrates such as They grew on various complex substrates such as yeast extract, carbohydrates, and organic acids, yeast extract, carbohydrates, and organic acids, which included starch, D-galactose, glutamate, which included starch, D-galactose, glutamate, fumarate and acetate. fumarate and acetate.

The The DNA G+CDNA G+C content of the three strains were content of the three strains were as follows:as follows:

1- AM8 (49 mol%) 1- AM8 (49 mol%) 2- AZ22 ( 48mole%) 2- AZ22 ( 48mole%) 3- AN133 (49 mol%).3- AN133 (49 mol%). Microscopic observations showed that AM8 is long Microscopic observations showed that AM8 is long

thick rod and spore former, AZ22 very small short thick rod and spore former, AZ22 very small short rod, and ANS 133 rod and spore former. rod, and ANS 133 rod and spore former.

Materials and methodsMaterials and methods

Sampling and Enrichment:Sampling and Enrichment: Water samples were collected from thermal hot springs Water samples were collected from thermal hot springs

located along the Jordan Rift Valley during summer season, located along the Jordan Rift Valley during summer season, using 500 ml sterile thermal glass containers which keep using 500 ml sterile thermal glass containers which keep the temperature of the water samples constant Ref 10 the temperature of the water samples constant Ref 10 (Khalil (Khalil et al.,et al., 1998).Five ml from each water sample were 1998).Five ml from each water sample were inoculated to 500 ml Elrelemenary flask containing 250 ml inoculated to 500 ml Elrelemenary flask containing 250 ml TT media (ATCC medium 697). The inoculated flasks were TT media (ATCC medium 697). The inoculated flasks were incubated at 55 0C with shaking at 300 rpm for 48 hrs. Ten incubated at 55 0C with shaking at 300 rpm for 48 hrs. Ten ml of each cultured flask were centrifuged and bacterial ml of each cultured flask were centrifuged and bacterial pelle were preaded on TT agar media, then incubated at 55 pelle were preaded on TT agar media, then incubated at 55 0C in a bench top incubator for 24-48hrs. For the urpose of 0C in a bench top incubator for 24-48hrs. For the urpose of etting pure cu;tur, distinctive colonies from TT agar plates etting pure cu;tur, distinctive colonies from TT agar plates were picked up and transferred to a fresh TT agar plates, were picked up and transferred to a fresh TT agar plates, then incubated at 55 0C in abench top incubator for 24-48 then incubated at 55 0C in abench top incubator for 24-48 hrs.hrs.

Detection of extra-cellular Detection of extra-cellular enzymes productionenzymes production

Amylase:Amylase: The ability of thermophilic isolates to produce amylase was tesed by culturing The ability of thermophilic isolates to produce amylase was tesed by culturing

each isolate on a minimal media composed of soluble starch(1%).each isolate on a minimal media composed of soluble starch(1%). Cellulase testCellulase test The bacterial isolates were streaked on TT media containing 1% CMC The bacterial isolates were streaked on TT media containing 1% CMC

(Carboxymethyl cellulose) and incubated at the optimum temperature for (Carboxymethyl cellulose) and incubated at the optimum temperature for each strain for 48 to 72 hours. each strain for 48 to 72 hours.

Congo red (1 mg/ml) were flooded on the agar plate for 2 minutes followed by Congo red (1 mg/ml) were flooded on the agar plate for 2 minutes followed by destining step using 1M NaCl, the clear zones indicated where the CMC has destining step using 1M NaCl, the clear zones indicated where the CMC has been hydrolysed which indicates the ability of the strain tested to produce been hydrolysed which indicates the ability of the strain tested to produce cellulase .cellulase .

Lipase testLipase test The bacterial isolates were streaked on TT media containing Rhodamine B The bacterial isolates were streaked on TT media containing Rhodamine B

and olive oil. Then the isolates were incubated at the optimum temperature and olive oil. Then the isolates were incubated at the optimum temperature for 48 to 96 hours. Plates were then exposed to the UV lamp at 350 nm.for 48 to 96 hours. Plates were then exposed to the UV lamp at 350 nm.

The strains which produce strong levels of lipases will glow brilliant pink –red The strains which produce strong levels of lipases will glow brilliant pink –red under UV lamp.under UV lamp.

DNA isolation and base DNA isolation and base composition:composition:

DNA was extracted using Qiagen DNA was extracted using Qiagen DNA kit (Qiagen Inc.) according to DNA kit (Qiagen Inc.) according to the manufacture’s instruction. the manufacture’s instruction.

The DNA G+C content was The DNA G+C content was determined by high-performance determined by high-performance liquid chromatography .liquid chromatography .

Sugar Utilization ExperimentSugar Utilization Experiment Sugar utilization experiment was Sugar utilization experiment was

carried out in TTV medium [TT carried out in TTV medium [TT supplemented with trace supplemented with trace elements and vitamins with the elements and vitamins with the addition of 1% (w/v) of yeast addition of 1% (w/v) of yeast extract, D-galactose, glutamate, extract, D-galactose, glutamate, fumarate and acetate.fumarate and acetate.

RESULTSRESULTS

Amylase testAmylase test

Isolate #Isolate # TemperatureTemperature

50 50 00CC 55 55 00CC 60 60 00CC

AM8AM8 +++++ +++++ +++++ +++++ ++++

AZ22AZ22 +++++ +++++ +++++ +++++ __

ANS133ANS133 +++++ +++++ +++++ +++++ ++++++++

Cellulase testCellulase test

Isolate #Isolate # TemperatureTemperature

50 50 00CC 55 55 00CC 60 60 00CC

AM8AM8 ++ ++ ++ ++ ++++++++

AZ22AZ22 -- ++ ++ ++++++

ANS133ANS133 ++++ +++ +++ ++++++++++

LipaseLipase

Isolate #Isolate # TemperatureTemperature

50 50 00CC 55 55 00CC 60 60 00CC

AM8AM8 ++ ++ ++ ++++++

AZ22AZ22 ++++ + ++ + ++++++

ANS133ANS133 __ ++++ ++++++++

AM8 is long thick rod and spore AM8 is long thick rod and spore former former

AZ22 very small short rodAZ22 very small short rod

ANS 133 rod and spore ANS 133 rod and spore formerformer

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