Effect of Carbon and Nitrogen Sources on the Growth and Production

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15(1), 45-50 (1992)

Effect of Carbon and Nitrogen Sources on the Growth and Productionof Cellulase Enzymes of a Newly Isolated Aspergillus sp.

CHOW-CHIN TONG * and K RAJENDRADepartment of Biochemistry and Microbiology

Universiti Pertanian Malaysia43400 UPM Serdang, Selangor Darul Ehsan Malaysia

ABSTRAKiltrat kultur yang didapati dari Aspergillus sp. yang baru dipencil menunjukkan aktiviti yang baik terhadap

kertas turas sebagai substrat. Suhu dan pH optimum bagi pertubuhan organisma ini masing-masing adalah pada3'?C dan 6.5. Walau bagaimanapun, suhu dan pH optimum bagi aktiviti enzim selulase masing-masing adalah44C dan 4.5 untuk masa pengeraman 48 jam. Penghasilan enzim selulase dalam medium cair mencapai tahapmaksimum pada hari ke 15 daripada keseluruhan tempoh pengeraman sepanjang 21 hari. Dari beberapa sumberkarbon dan nitrogen yang diuji, karboksimetil-selulosa (kelikatan medium) dan (NH)2 Fe(S))2' 6H20 merupakansumber terbaik bagi penghasilan enzim manakala serbuk selulosa dan NH/103 merupakan sumber yang terbaikbagi penghasilan miselia.

ABSTRACTThe culture filtrate obtained from a newly isolated Aspergillus sp. exhibited good activities against the filter paperas substrate. The optimum temperature and pH values for growth were 3'?C and 6.5, respectively. However, theoptimum temperature and pH for the activities of the cellulase enzymes were recorded as 44'C and 4.5, respectively.Production of the enzymes in liquid medium reached its maximum level (9 units/ml) at the 15th day of incubationwith an incubation period of up to 21 days. From the various carbon and nitrogen sources tested, carboxymethyl-cellulose (medium viscosity) and (NH) 2 Fe(SO) 2' 6H20 were found to be the best for cellulase enzyme production,whereas cellulose powder and NH/103 enhanced mycelial growth.

INTRODUCTIONThe production and utilization of cellulolytic enzymes is a topic of great interest in the worldsearching for renewable resources. The lack of aneconomical process for saccharification of wastecellulose by microbial enzymes is one of themajor problems yet to be solved by fermentationtechnology. Reduction of costs will involve theproper selection of strains yielding high levels ofthe enzymes. Thus, the search for new and different cellulose degrading microorganisms has increased and various new species have been isolatedand characterized as regards to their capacities toproduce cellulases (Tanaka et al. 1980; Tong et al.1980; Hudson et al. 1990)

In an attempt to isolate cellulose-degradingfungi from various natural habitats, samples fromcompost heaps, decomposed wood and rubbishdumping grounds were collected. Of the forty-* Author to whom all correspondence should be sent.

eight isolates tested, the most active organismidentified was Apergillus sp. obtained from compost heaps of oil palm waste. Our preliminarystudies indicated that this newly isolated specieshad the ability to degrade quickly a wide variety ofcellulosic materials. In this paper, the effect ofcarbon and nitrogen sources on the growth andcellulase production by this organism is reported.

MATERIALS AND METHODSWhatman o. 1 filter paper and Whatman chromatography paper were obtained from WhatmanLtd. Palm-press-fibres were kindly supplied by apalm oil factory in Bukit R ajah, Klang.Carboxymethyl-cellulose and cellulose powderwere purchased from Sigma Chemical Company,St. Louis USA. Potato dextrose agar was a productof Difco Laboratories, Detroit USA. Xylan fromoat-spelt was obtained from Fluka, Switzerlandand peptone was purchased f rom Topley House,England.


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Source ofMicroorganismVarious isolates of fungi (48 isolates) were obt ained f rom soil samples collected around thevicinity ofKuala Lumpur. These samples includedcompost , wood chips, decomposed tree trunksand leaves. The most active cellulolytic organismfrom these isolates was identified to be anAspergillus sp. isolated from the oil palm compostheap. It was then selec ted for the followingstudies.BuffersThe citrate-phosphate buffer from pH 3.0 to 8.0was prepared using 0.1 M-citric acid and 0.2 M-dibasic sodium phosphate. The buffer 0.2M tris/HCI was used for pH 8.0 and 9.0.Preparation of Culture FiltrateAspergillus sp. was subcultured on PDA and grownat 37C fo r 48 h. Three agar-mycelium discs (8.0mm in diameter) were transferred to a Wheatonmedical flask (C-16, 500 ml) containing 60 ml ofFergus (1969) medium with 3.3 g filter paper ascarbon source. Mter 15 days of incubation at theabove mentioned temperature, the liquid mediumwas filtered by suction through a Whatman GF/Cfil ter to remove hyphal fragments and any isolublecellulose residues. The clear culture f il trate obtained was s to red in deep freeze, fo r later use ifnecessary.

The inoculum was taken from the culturegrown on PDA for 48 h at 37C. An agar-myceliumdisc (8.0 mm in diameter) was cut from theperimeter of the colony with a sterile cork borer,inverted and placed at the centre of a 9 cmdiameter petri dish containing agar medium.

For seeding liquid media, three agar-myceliumdiscs (8.0 mm in diameter) were placed into eachmedical flask containing 60 ml of Fergus (1969)medium.Maintenance of Stock CultureThe organism was cultured onto PDA slopes andincubated for two days at its optimum temperatureand stored at 4C for not more than three monthsprior to further subculturing.Determination of Opitmum Temperature for GrowthThe optimum growth temperature on solid agarwas ascertained by measuring the d iameters ofcolony on PDA in 9 cm diameter petri dishescontaining approximately 30 ml of agar medium.Each agar plate was inoculated with one agar-

mycelium disc as described earlier and incubatedfor 48 h at the following temperatures, that is,20C, 28C, 37C, 44C, 50C and 60C. To red uce desiccation of the agar medium at hightemperatures, a beaker of distilled water was placedin each incubator.Determination of Optimum pH for GrowthThe optimum pH on solid agar was determinedby measuring the diameters of colony on PDAmedium in a 9 cm petri dish. Medium with different pH, that is, 4.5, 5.5, 6.5, 7.5, 9.0 were eachinoculated with an agar-mycelium disc (8.0 mm indiameter) and incubated for 4S h a t the opt imumtemperature fo r growth.Effect of Incubation Period on the Production ofEnzymesMedical flasks containing 60 ml of Fergus mediumwere inoculated and incubated at 37C for different times. The contents of two flasks from eachculture were harvested at intervals of two to threedays up to 21 days by suction through a WhatmanGf/C filter. The culture filtrates were then assayed for cellulase activity on filter paper determined by estimating the reducing sugars formedusing the Nelson-Somogyi method (Nelson 1944;Somogyi 1952) as described below.Determination ofOptimum Temperature and pH for theProduction of Cellulase EnzymesThe opt imum tempera tu re and pH fo r the production of cellulase enzyme in liquid mediumwere determined using a range of temperatures(28C, 37C, 44C, 50C, 60C, 70C and SOC)and pH (3.0, 4.5, 5.5, 6.5, 7.5, 8.0 and 9.0) with anincubation period of 15 days. The culture filtratewas then assayed for cellulase activities as describedbelow.Effect ofDifferent Carbon Sources on Mycelial Growthand Cellulase Enzyme ProductionTo test the effects of different types of carbohydrate as the sole carbon source on the mycelialgrowth and cellulase enzyme product ion, t endifferent carbon sources were used and incorporated separately into the Fergus (1969) mediumincubated at optimum conditions.

A rough estimation of the amount of mycelialgrowth was done by making visual observation ofthe mycelium in the medical flasks whereas theamount of cellulase enzymes produced was determined by the method described below.

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EFFECT OF CARBON AND NITROGEN SOURCES ON THE GROwrH & PRODUCTION OF CELLULASE ENZYMES

Effect ojDifferent Nitrogen Sources on Mycelial Growthand Production oj Cellulase EnzymesThe effect of nine nitrogenous compounds onthe mycelial growth and enzyme production inFergus (1969) medium was determined underoptimum conditions. The concentration of eachni trogen source in the medium was 0.1% (wIv).In all cases, the pH of the medium was adjustedto 6.5. Upon incubation for 15 days, the dryweight of mycelia was recorded and the amountof cellulase enzymes produced was estimated bythe method described below.

Degradation of the filter paper in the Fergus(1969) liquid medium began after three days ofincubation at 37C. By the 15th day, the integrityof the f il te r paper was lost completely. The optimum tempera ture for growth on PDA was 37Cand the colony reached its maximum size of 9.0cm in diameter upon incubation for 48 h. Nogrowth was observed at 20C and 60C. At 37C,the opt imum pH value for growth was 6.5 with anaverage diameter of about 8.1 cm for an incubation period of 48 h.

2500 15Incubation period (Day)

10 1 ------------- --,

Effect oj Temperature on Cellulase Activity towardsFilter PaperIn general, cellulases characteristically have highoptimum temperatures compared with other enzyme systems. The cellulases described in thiswork have an opt imum temperature of 44C (Fig.2). At 60C, the activity decreased to about 60%of the maximum activity.

At temperatures higher than 70C, thecellulase activity was denatured almost completely.At lower temperatures, a sharp decrease in theactivity was observed at temperatures below 37C,

Effect ojIncubation Period on the Production ojCellulaseEnzymesFig. 1 demonstrates the product ion of cellulaseenzymes over a period of 21 days as measured bythe ability of the culture fil trate to degrade filterpaper. Initially, the activity was low and fluctuateduntil th e 12th day, after which the activity increased sharply up to the 15th day. By this time,the integrity of the filter paper in the medium waslost almost completely and a thin slur ry wasformed. Futher incubation resulted in a declinein the activity.

Fig. 1: Effect of incubation petiod on the production ofcellulase activities. Point represents an average ofduplicates.

Assay oj Cellulolytic ActivityAn indication of total cellulolytic activity was obtained by the determination of filter paper degrading activity. The standard reaction mixturecontains 20 mg of filter paper (Whatman No.1),0.5 ml of citrate-phosphate buffer, pH 5.0, 0.5 mlof enzyme solution (culture filtrate) of appropriatedilution (i f necessary) and one drop (l01l1) oftoluene. After incubation at 44C for three days,0.5 ml of the reaction mixture was withdrawn andassayed for reducing sugars using the methodmentioned above.

An absolute def init ion of a unit o f cellulaseactivity is difficult. There is l it tle to be gained byexpr es sing the activity in terms of glucoseequivalents, since glucose is not the only productof the enzyme reaction (Shepherd et al. 1988). Aunit of cellulase activity is defined as that amountof enzyme that produces an increase in absorbanceof 0.10 at 560 nm under the conditions defined.

change in absorbance of 0.10 is equivalent to 30Ilg of glucose under the conditions given, andthus cellulase preparations with units quoted I I Iglucose equivalents can be compared.

Determination oj Reducing SugarsThe number of reducing sugar groups created byhydrolysis of the cellulosic substrates was measuredspectrophometrically by using the Nelson-Somogyiprocedure (Nelson 1944; Somogyi 1952).

RESULTS AND DISCUSSIONorty eight fungal isolates were tested for their

cellulolytic activity towards filter paper as carbonIt was found tha t of all the fungi tested,

one showed a high cellulase activity towards filteraper and other cellulose substrates. This active

cellulolytic fungus was identified as an Aspergillus

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CHOW-CHIN TONG AND K RAJENDRA

120r--------------------,

and at room temperature (28C), the cellulaseactivity was only about 20% of the maximum.

pH

120

100

aD60

40>

:.!20

0 0 4 6 10

80

20

100

60

Fig. 3: pH ojJtimun of cellulase activities. Point j'epresentsan average of duplicates.000000

oL-__-L - .L.-__ - ' - _ ~ : : _ . . . . . . . . .- -_ . JoTefTIlerature (OC)

Fig. 2: Temperature optimun of cellulase activities. Pointj

Effect of pH on Cellulase Activity towa-rds Filter PaperBasically, fungal cellulases are stable from pH 3.0to 8.0 at 30C, active at pH 3.7 to 7.0 (Tong &Cole 1982). The optimal pH observed for cellulasesproduced by Aspergillus sp. occurred at pH 4.5(Fig. 3). At a lower pH of 3.0, there was a sharpdecrease in the cellulase activity to about 35% ofthe max.imum. At a pH higher than 4.5, a gradualdecrea e in th e activity was observed with only 9%of the maximum activity remaining at pH 8.0 and9.0. In some fungal species, such as T7'ichodermakoningii (Wood 1969) and Aspergillus sp. (Khatijahet al. 1983) double pH optimum has been reported.Effect ofDifferent Carbon Sources on the Mycelial Growthand Production of Cellulase EnzymesTo test the effect of different types of carbohydrate as the sole carbon source on mycelial growthand on the production of extracellur cellulolyticenzymes, culture was inoculated into Fergus (1969)medium and incubated for 15 days at 37C.

The results are shown in Table 1 and Fig. 4.There were some difficulties experienced inmeasuring the amoun t o f mycelial dry weight insome of the substrates used. This was due to thefact that some of the mycelia became adsorbedonto the subst ra te thereby making its separationdifficult. Therefore, a visual obervation of themycelial growth in the liquid medium was adopted.Cotton, cellobiose, fIlter paper and particularlycellulose powder were found to be good sourcesfor the mycelial growth, whereas xylan, citric acidand mannitol were less suitable for mycelialgrowth.

The amount of extracellular cellulolytic en-zymes in the crude mtrates was subsequently determined. It is interesting to note that there is nocorrelation between the amount ofmycelial growthand the amount of cellulase enzymes produced.Xylan and CMC which did not promote muchmycelial growth turned out to be good carbonsources in enhancing the production of cellulaseenzymes. Other carbon sources which yieldedgood cellulase activity included cellobiose andcellulose powder. This supported the fIndings ofReese and Mandels (1971) that cellulase enzymes

TABLE 1. Effect of different carbon sources onth e mycelial growth.

Carbon source Arbitrary scaleXylan +Citric acid +Mannitol ++

CMC ++Yeast +++Cotton +++

Cellobiose ++++Filter paper ++++

Cel lulo e powder ++++++++++

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EFFECT OF CARBOI AND NITROGE TSOURCES 01 THE GROWTH & PRODUCTION OF CELLULASE ENZYMES

XylanCitric acidMannitol

(MeYeast

(ottonCellobiose

Fi 1ter paperCellulose powder

production of cellulase enzymes in Fergus (1969)medium with filter paper as the carbon sourcewas determined.

As shown in Fig. 5, sodium nitrate, sodiumnitrite and ammonium nitrate were found to bethe best nitrogen sources for mycelial growth.However, fer rum ammonium sulphate and ammonium molybdate were no t sui table for thegrowth o f Aspergillus sp. Surprisingly, peptone didnot support good mycelial growth either.

o 3

IlltrO!iJellSOlCt5

Fig. 5: Effect of different nitrogen sources on the m)Jcelialgrowth. Al l values are average of duplicates.

1.2.8.6Mycelial weight (g)

0.4.2

PeptoneINHq 16FelSOq)2 . H2O INHql

Effect ojNine Different Nitrogen Sources on the MycelialGrowth and Production oj Cellulase EnzymesThe effect of nine different nitrogenous compounds on the mycelial growth of Aspergillus sp and

produced when cellulose was present as thesource in submerged cultures of fingalOther carbon sources which are easily

and have been shown to promotengal growth may not induce the production oflulolytic enzymes (Mandels and Reese 1965;viella, 1966). In addition, Rautela and King

(1965), as well as Fan et al. (1982) have showndifferent forms of cellulose vary from one

in their growth-supporting, enzyme-inand reacting capabilities. This may be due

o the dif ference in the microstructures amonge cellulose substrates (Wang 1982). The cellulaseobserved may no t be due to the actualof enzymes secreted in the cultures. This

is due to the fact that cellulases have an affinityr certain cellulose sources. The cellulase enzymes

then be ad so rbed onto the celluloseubstrate thereby reducing the amoun t o f detect

able cellulase enzymes in the culture filtrate(Halliwell 1961). The percentage of this adsorbedcellulolytic activity has been estimated to varyrom 0 to 40% depending on the species of theungus. Greaves (1971) reported that by successively washing and gently agitating the myceliumand cellulose several times, the cellulase enzymescan be released from cellulose. Similarly, certaincellulose substrates, for example, the non-absorben t cotton wool, have surfaces that are not suitable fo r the absorption of cellulolytic enzymesleaving a higher amount of enzymes detectable inthe filtrates.

4: 1'-Ifect of diJJerent carbon SOU1"CeS on the production ofcellulase activities. All values are average of dupli-cates.

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CHOW-CHI TONG Aj\lD K. RAJENDRA

cellulase production as that achieved by ferrumammonium sulphate heptahydrate and glycine.This may be exp la ined by th e fact that ammonium ions were absorbed by the mycelium whichresulted in a lowering of th e pH that subsequentlyinhibited th e production of cellulase enzymes(Wang 1982).

REFERENCESFAN, L.T., Y.H. LEE & M.M. GHARPURAY. 1982. The

Nature of Lignocellulosics and Their Pre-treatment for Enzymic Hydrolysis. Adv. Biochern. Eng.23: 157-187.

FERGUS, C. L. 1969. The Cellulolytic Activity ofThermophilic Fungi an d Actinimycetes. Mycologia61: 120-129.

GREAVES, H. 1971. Effect of Substrate Availability onCellulolytic Enzyme Production by SelectedWood-rotting Microorganisms. Aust. J. Bioi. Sci.24(6): 1169-1182.

HALLIWELL, G. 1961. The Action of Cellulolytic Enzymes from jVJ.yrotheciurn verrucaria. Biochern. J.79: 185-192.

HUDSON, JA., H.W. MORGAN & R.M. DANIEL. 1990. ASurvey of Cellulolytic Anaerobic Thermophilesfrom Hot Springs. Syst. Appl. Microbial. 13: 72-76.

KHATIZAH, M., M. 1. YAZIZ & C.C. TONG. 1983. Degradation of Cellulose by Aspergillus sp.,Trichoderma koningii and Myriococcurn sp. Pertanika6: 8-16.

M DELS, M. & R.T. REESE. 1965. Inhibition of aCellulase. Ann. Rev. Phytopath 3: 85-102.

NELSON, N. 1944. A Photometric Adaptat ion for theSomogyi Method for the Determination of Glucose. J. Biol. Chern. 153: 375-380.

NOVIELLA, C. 1966. Pectolysis and Cellulolytic Activities of Sclerotiurn rofsii. Ann. Fac. Sci. Agron. Univ.Studio Napoli Portici 30: 461-474.

RAUTELA, C. & K W. KING. 1965. Significance of theCrystal Structure of Cellulose. Arch. Biochem.Biophys. 123(3): 599-601.

REESE, E.T. and M. MANDELS 1971. Degradation ofCellulose and its Derivatives. New York: John Wiley.

SHEPHERD, M.G., A.LJ. COLE & e.C. TONG. 1988.Cellulases of Thermoascus aumntiacus. Methods inEnzymology 160: 301-307.

SOMOGYI, M. 1952. Notes on Sugar Determination. J.Biol. Chern. 195: 19-23.

TA..t'JAKA, M., T. MORITA, M. TANIGUCHI, R. MATSUNO &T. KAMIKUBO, 1980. J. Ferment Tech. 58: 517.

TONG, e.C., A.LJ. COLE & M.G. SHEPHERD. 1980. Purification and Properties of the Cellulases fromthe Thermophilic Fungus, Thermoascusaumntiacus. Biochem. J. 191: 83-94.

TONG, C.C. &A.LJ. COLE 1982. Cellulase Productionby t he Thermophi li c Fungus, Thermoascusaumntiacus. Pe1tanika 5: 255-262.

WA..t'1G, C.W. 1982. Cellulolytic Enzymes of Volvariellavolvacea. p. 167-185. Hong-Kong: The ChineseUniversity Press.

WOOD, T.M. 1969. Cellulolytic Enzymes System ofTrichoderma koningii. Separation of ComponentsAttacking Native Cotton. BiocheJn. J. 109: 217-277.

(Received 29 Novernber 1991)

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Effect of Carbon and Nitrogen Sources on the Growth and Production

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