J . Basic
,Microbial.
32 (1992) 2, 107 - 11 1
Departmenf of Botany and Microbiology, Faculty of Science, Alexandria University. Alexandria, Egypt)
Microbial degradation of shrimp-shell waste S. A. SARRV f Receiiied -75 Junc 1 9 9 l ; A ~ e p t e d 27 Septernher 1991)
A total of 40 strains of bacteria were isolated and tested for their potentiality to degrade chitin and utilize shrimp-shell waste for the production of chitinase. The activity ratio, percentage of weight loss and enzyme activity were determined for cultures exhibiting highest chitinolytic activities. The most active organisms were identified as Alcaligenrs denitrificans, Bacillus arnr’loliquejnrirns, B. inegoteriurn and B. subtilis. Thc potentiality of the first two organisms to degrade chitin was reported for the first time.
The processing plants of shell-fish such as shrimp and crab have difficulties coping with the disposal of the waste which contain chitin as a major constituent. The conversion of shell-fish chitin waste to single cell protein was investigated (REVAH-MOISEEV and CARROAD 1981). A waste bioconversion process has been proposed to utilize the extracellular chitinase system of Serrritia mareexens to hydrolyze the chitin fraction of the waste (CARROAD and TOM1978). Chitin degrading products can be used as feedstock chemicals, for the production of single cell protein and for animal or aquaculture feed (COSIOet al 1982). The current investigation is directed towards the isolation and selection of microorganisms capable of utilizing the shrimp-shell waste of the sea food packing industry for the production of chitinase. Materials and Methods Chitin source: A variety of “chitins” was used in the present work. Practical grade chitin (89%) from Crab shell (SIGMAC-3387). Swollen chitin was prepared by stirring 10 g of commerical chitin into 100 ml of 85% phosphoric acid in the cold for 48 h, and reprecipitated by pouring the gelatinous and REESE 1969). Waste chitin. is the shrimp-shell chitin mixture into excess of water (MONREAL waste collected from Torina Fish Company, Abu Kir, Alexandria. The waste was washed. dried and milled. Chemical analysis of shrimp-shell chitin waste: Chitin was determined in the waste using the modified WELINDER method reported by STELMOCK pr a / . (1985). The protein content was estimated by the microkjeldahl technique. Percentage of CaCO, was determined using a calcimeter of the type described by WRIGHT(1939). Element constituents were determined by flame photometric and atomic absorption techniques. lsolation of chitinolastic microorganisms: Soil samples were collected and serial dilutions were made and placed on chitin agar containing I % swollen chitin, 0.5% yeast extract and 2% agar. Plates were incubated at 30 “C and examined daily for the presence of active chitinolastic microorganisms. The isolates producing the largest zones of clearing were picked and obtained in pure cultures. The activity ratio is calculated as the ratio of the diameter of the clearing zone to the diameter of the colony. Determination of chitin decomposition (weight loss) with intact cells: The standard liquid culture medium contains in & / I ) : (NH,),SOJ, 1.0; MgSO, . 7H,O, 0.3; KH,PO,, 1.36 and yeast extract, 0.5. (CORROAD and TOM 1978). 50 aliquots were dispensed in 250 ml ERLENMEYER flasks. Each flask received 1 % chitinous solids (colloidal. commercial or shrimp-shell waste) as the sole carbon source. After sterilization, flasks were inoculated with the selected microorganisms and incubated on a rotary shaker at 30 -C for 7 days. Precipitates remaining after incubation were treated with 10% potassium X‘
108
S . A. SABRY
hydroxidc for I h r to i y r e intact cells. a n d were washed. liltcred. and dried to determine weight loss of chitin. Enqme assay: The organisms were gronn i n shake flasks on the standard liquid medium previously mentioned with 2u/u of either commercial chitin or shrimp-shell waste for 48 hr. The medium w a b centrifuyed at 10.000 rpm for 15 min at 4 C and the supernatant was used as the crude enzyme extract. Enzyme assay was determined by incubating 3 ml of crude enzyme extract. added to 3 ml of 19'0 smollen chitin in citrate-PO, buffer ( ~ 2 0 pH , 6.6) at 40 C for 2 h in a shaking water bath (Hoon iind MEYERS1977). Following incubation. the tubes were placed in a water bath at 100 'C for 3 min to inactivate the chitinase. The solutions were centrifuged at 10.000 rpni at 4 'C for 15 min and er ul. (1955). Each treatment N-acetqlglucosaniine (NAGA) was measured using the method of REISSIC; uascarried out i n triplicate and the results obtained throughout this work were the arithmetic nicati. Results and discussion
Cotlipo.'itiorl #f the
II'LIStC'
The chemical composition of the solid waste from shrimp-shell processing is presented in Table 1 . The waste is mainly composed of chitin (21.4%), calcium carbonate (40%) and protein (27.994). Similar results were previously reported for shrimp and Crab shell waste (REVAH-MORISEEV and CARROAD 198 1). ScI~~c~/iorr 0f ' chitinolrtic
rllicr.oor.gclrli.~lll.s
A total of 40 strains were isolated from a variety of soil sources. The organisms were inoculated into agar plates with swollen chitin (SNEH1981) and incubated for 7 days for measuring the chitinolytic zones and determining the activity ratios. Based o n activity ratio. four were judged to have sufficiently active extracellular chitinase system lo be considered further. The four isolates were identified at the Micro Inc. Vermont, USA a s AIi,nligerz.r tleriitr.i/icuris. Bocillits trrri?~loliqir~fircreri.s, B. nirgtitrriurii and B. ,siih/ili.y. N o attempt was made to confirm the identify of all of the strains tested. In Table 2 are listed the activity ratios for the 4 most active chitinolytic organisms, with 4 microorganisms received from other researchers. The chitinolytic activity of Alcaligerirs derii/r.i/ic~nrisand Bcic~illrt.srrr~i~~loli~uc~fi~c.ieris has not been previously reported. Chitinase activity has been found in a wide variety of microorganisms including species of BLici/Iil.s and .Str.c)proiujws (CHIGALEICHIK 1976, BEWETTand HOOD1980. O'BRIENand DAVIS 1982, Z l K A K l S 1984, JANDA 1985. HARAv/ r i l . 1989).
(p),
Table I Chemical composition of shrimp-shell waste Constituents Moibturc contcnt Ash content Chitin C'rude protein Calcium carbonate Elements (as ppm) ca cu
c I1
Mn
NI
te Zn C ;1
'TO
to dry wt.
20
6 21 4 27.9 40
3 65 Zero 2.30 0 29 0.20 2 93 I .83 0 281
109
Microbial degradation of shrimp-shell waste Table 2 Chitinolytic activity of the tested strains Organism
Activity ratio*)
Alctiligenes deniirificutis Bucillrrs umjlaliquefuciens B. nieguterirrni B. .subtilis
2.8 2.4 2.4 I .8
Azorobacter chroococcurn Psmdomonus jlrrorescet~s sp. Acritiorii~~ces Streptor,1).ces sp.
2.0 I .o 0.8 1.6
*) Activity ratio is defined as thc ratio of the diameter
of the chitinolytic zone to the diameter of the colony (CHAN1970).
Atcaligenes derzitrificnns is seen to secrete the most active chitinase by this measure, followed by B. amyloliquefacicns and B. megaterium. Determinuion of Ir'eight loss For hydrolysis with intact cells, the microorganisms were cultured with 1% chitinous solids (colloidal, commerical and shrimp shells). The percentage weight losses of the chitin substrates in the culture flasks are listed in Table 3. The results indicate that on colloidal chitin Alcaligeries denitrficans affected the greatest weight loss while B. ~r~~~~lotiguejircier2.v and B. suhtitis yielded best results on shrimp-shell waste. Table 3 Percentage weight loss of different chitin substrates Organism
Percentage weight loss Colloidal chitin
Commercial chitin
Shrimp-shell waste
A lculigerres denitr@utis Baciflus aniy/oliquL.fLlcirns 5.niegateriiirii 5.subrilis
95.2 92.7 89.7 92.6
76.6 14.2 74.0 74.2
59.4 62.3 60.2 62.2
Arotobucter chroococcum Pseudornonus fluorescens Actinoniyces sp. S t r r p t o m y c e s sp.
72.6 79.2 69.0 85.20
55.4 58.8 48.3 68.6
44.3 44.3 42.6 56.3
Enzyme activity The values of chitinase activity varied according to the substrate and the bacterial species as well. Bacillus anz~~lotiqiiclfacietis growing on shrimp shell waste as substrate supported the highest activity. This might be attributed to the presence of small amounts of chitin hydrolysate (NAGA) which induces the production of chitinase as previously reported (MONREALand REESE1969, YOUNGet a1 1985). The presence of protein in the waste enhance the bacterial growth and brings about increased enzyme productivity (Table 4).
S. A . SABRY
110 Table 4 Chitinase activity of the tested organisms grown on chitin SUbStrdteS Chitinase activity U/ml
Organism
.4 lculigenes denirrificms Bucillus uni~loliquefrrciens B. niegureriuni B. sirhiilis
Commercial chitin
Shrimp-shell waste
2.7 3.4 2.2 4.3
1.9 3.9 3.6 1.7
Effect qf incuhution period on cnzjme production
To evaluate the chitinase productivity by cultures of different ages, the cultures were allowed to grow under shaked condition for 84 hours. The crude enzyme was obtained by centrifugation at time intervals and the enzyme activity was estimates. The resdlts presented in Table 5 show that maximal activities were maintained for 36 and 60 hours old cultures of B. anzjVoliqirqfuciens and B. megaterium. respectively. Table 5 Chitinase activity of B. u~ii~~loliqirqfacieris und B. meggareriutii grown on I % shell fish waste under different incubation periods Organism
Chitinase activity (Ujml) Hours incubation 6
B. ani~loliquefuciens B. tneguterium
12
-
-
-
-
18
0.5 0.5
24 3.0 1.5
36 4.8 3.4
48 3.9 3.4
60 3.9 3.8
72 2.5 2.2
84 1.2 1.2
The results of this study reveale the potentiality of the two newly reported chitinoclastic organisms named Alcaligenes detiitrficuns and Bacillus amyloliquqfacierzs to utilize shrimp shell waste for the production of chitinase enzyme. Further studies concerning optimization of enzyme productivity are under investigation.
References BENSETT,C. B. and HOOD,M . A , , 1980. Effects of cultural conditions on the production of chitinase by a strain of Bucilhr~inegu~eriuin.Developments in Industrial Microbiology, 21. 357. CARROAD, P. A. and TOM,R. A , , 1978. Bioconversion of shell-fish chitin wastes. Process conception and selection of microorganisms. J. Food Sci., 43. 1158. CHAN,J. G., 1970. The occurrence, taxonomy and activity of chitinolastic bacteria from sediment, water and fauna of Puget Sound. Ph. D. thesis, University of Washington. CHIGALEICHIK, A . G., 1976. Chitinase of Bacillus rhuringiensis. Mikrobiologiya, 45, 966. COSIO.I. G., FISHER.R. A. and CARROAD. P. A.. 1982. Bioconversion of shell fish chitin waste pretreatment, enzyme production, process design and economic analysis. J . Food. Sci.. 47, 901.
Microbial degradation of shrimp-shell waste
111
HARA,s.,YAMAMURA, Y., FUGII, Y., MEGA,T. and IKENAKA, T., 1989. Purification and characterization of chitinase produced by Streptoinyces eryrlrraeus. J. Biochem., 105, 484. HOOD, M. A. and MEYER,S. P.. 1977. Microbiological and chitinolastic activities associated with Penaeus sefi/erus. J. Ocean. SOC.Japan., 33, 235. JANDA.J . M., 1985. Biochemical and exoenzyrnatic properties of Aerornonos species. Diagn. Microbiol. Infect. Dis., 3, 223. MONREAL, J. and REESE,E. T.,1969. Thechitinase of Serratia marcescens. Can. J. Microbiol., 15,689. OBRIEN,M. and DAVIS,G. H. G., 1982. Enzymatic profile of Pseudotnonas maltophilia. J. clin. Microbiol., 16, 417. REISSIG, A. R., STROMINGER, R . S. and LELOIR, V. F., 1955. A modified colorometric method for the estimation of N-acetylamino sugars. J. Biol. Chern., 217, 959. S. and CARROAD, P. A., 1981. Conversion of the enzymatic hydrolysate of shell fish REVAH-MOISEEV, waste chitin to single-cell protein. Biotechnol. Bioeng., 23, 1067. SNEH,B., 1981. Use of rhizosphere chitinolytic bacteria for control of Fusariuni orysporum f. sp. clinnthi in carnation. Phytopath. Z., 100, 251. STELMOCK, R. L., HUSBY,F. M. and BRUNDAGE. A. L., 1985. Application of Van Soest Acid detergent fiber method for analysis of shell fish chitin. Dairy Sci., 68, 1501. WRIGHT,C. H., 1939. Soil Analysis. Physical and Charnical Methods, Thomas Murly. London. YOUNG,M. E., BELL.R. L. and CARROAD, P. A., 1985. Kinetics ofchitinase production, 11, Relationship between bacterial growth, chitin hydrolysis and enzyme synthesis. Biotechnol. Bioeng., 27, 776. ZIKAKIS. J . P. (ed.), 1984. Chitin, Chitosan and Related Enzymes. Academic Press, Inc. New York. Mailing address: Dr. S. A. SABRY.Department of Botany and Microbiology, Faculty of Science. Alexandria University, Moharram Bey, Alexandria, Egypt
,Microbial.
32 (1992) 2, 107 - 11 1
Departmenf of Botany and Microbiology, Faculty of Science, Alexandria University. Alexandria, Egypt)
Microbial degradation of shrimp-shell waste S. A. SARRV f Receiiied -75 Junc 1 9 9 l ; A ~ e p t e d 27 Septernher 1991)
A total of 40 strains of bacteria were isolated and tested for their potentiality to degrade chitin and utilize shrimp-shell waste for the production of chitinase. The activity ratio, percentage of weight loss and enzyme activity were determined for cultures exhibiting highest chitinolytic activities. The most active organisms were identified as Alcaligenrs denitrificans, Bacillus arnr’loliquejnrirns, B. inegoteriurn and B. subtilis. Thc potentiality of the first two organisms to degrade chitin was reported for the first time.
The processing plants of shell-fish such as shrimp and crab have difficulties coping with the disposal of the waste which contain chitin as a major constituent. The conversion of shell-fish chitin waste to single cell protein was investigated (REVAH-MOISEEV and CARROAD 1981). A waste bioconversion process has been proposed to utilize the extracellular chitinase system of Serrritia mareexens to hydrolyze the chitin fraction of the waste (CARROAD and TOM1978). Chitin degrading products can be used as feedstock chemicals, for the production of single cell protein and for animal or aquaculture feed (COSIOet al 1982). The current investigation is directed towards the isolation and selection of microorganisms capable of utilizing the shrimp-shell waste of the sea food packing industry for the production of chitinase. Materials and Methods Chitin source: A variety of “chitins” was used in the present work. Practical grade chitin (89%) from Crab shell (SIGMAC-3387). Swollen chitin was prepared by stirring 10 g of commerical chitin into 100 ml of 85% phosphoric acid in the cold for 48 h, and reprecipitated by pouring the gelatinous and REESE 1969). Waste chitin. is the shrimp-shell chitin mixture into excess of water (MONREAL waste collected from Torina Fish Company, Abu Kir, Alexandria. The waste was washed. dried and milled. Chemical analysis of shrimp-shell chitin waste: Chitin was determined in the waste using the modified WELINDER method reported by STELMOCK pr a / . (1985). The protein content was estimated by the microkjeldahl technique. Percentage of CaCO, was determined using a calcimeter of the type described by WRIGHT(1939). Element constituents were determined by flame photometric and atomic absorption techniques. lsolation of chitinolastic microorganisms: Soil samples were collected and serial dilutions were made and placed on chitin agar containing I % swollen chitin, 0.5% yeast extract and 2% agar. Plates were incubated at 30 “C and examined daily for the presence of active chitinolastic microorganisms. The isolates producing the largest zones of clearing were picked and obtained in pure cultures. The activity ratio is calculated as the ratio of the diameter of the clearing zone to the diameter of the colony. Determination of chitin decomposition (weight loss) with intact cells: The standard liquid culture medium contains in & / I ) : (NH,),SOJ, 1.0; MgSO, . 7H,O, 0.3; KH,PO,, 1.36 and yeast extract, 0.5. (CORROAD and TOM 1978). 50 aliquots were dispensed in 250 ml ERLENMEYER flasks. Each flask received 1 % chitinous solids (colloidal. commercial or shrimp-shell waste) as the sole carbon source. After sterilization, flasks were inoculated with the selected microorganisms and incubated on a rotary shaker at 30 -C for 7 days. Precipitates remaining after incubation were treated with 10% potassium X‘
108
S . A. SABRY
hydroxidc for I h r to i y r e intact cells. a n d were washed. liltcred. and dried to determine weight loss of chitin. Enqme assay: The organisms were gronn i n shake flasks on the standard liquid medium previously mentioned with 2u/u of either commercial chitin or shrimp-shell waste for 48 hr. The medium w a b centrifuyed at 10.000 rpm for 15 min at 4 C and the supernatant was used as the crude enzyme extract. Enzyme assay was determined by incubating 3 ml of crude enzyme extract. added to 3 ml of 19'0 smollen chitin in citrate-PO, buffer ( ~ 2 0 pH , 6.6) at 40 C for 2 h in a shaking water bath (Hoon iind MEYERS1977). Following incubation. the tubes were placed in a water bath at 100 'C for 3 min to inactivate the chitinase. The solutions were centrifuged at 10.000 rpni at 4 'C for 15 min and er ul. (1955). Each treatment N-acetqlglucosaniine (NAGA) was measured using the method of REISSIC; uascarried out i n triplicate and the results obtained throughout this work were the arithmetic nicati. Results and discussion
Cotlipo.'itiorl #f the
II'LIStC'
The chemical composition of the solid waste from shrimp-shell processing is presented in Table 1 . The waste is mainly composed of chitin (21.4%), calcium carbonate (40%) and protein (27.994). Similar results were previously reported for shrimp and Crab shell waste (REVAH-MORISEEV and CARROAD 198 1). ScI~~c~/iorr 0f ' chitinolrtic
rllicr.oor.gclrli.~lll.s
A total of 40 strains were isolated from a variety of soil sources. The organisms were inoculated into agar plates with swollen chitin (SNEH1981) and incubated for 7 days for measuring the chitinolytic zones and determining the activity ratios. Based o n activity ratio. four were judged to have sufficiently active extracellular chitinase system lo be considered further. The four isolates were identified at the Micro Inc. Vermont, USA a s AIi,nligerz.r tleriitr.i/icuris. Bocillits trrri?~loliqir~fircreri.s, B. nirgtitrriurii and B. ,siih/ili.y. N o attempt was made to confirm the identify of all of the strains tested. In Table 2 are listed the activity ratios for the 4 most active chitinolytic organisms, with 4 microorganisms received from other researchers. The chitinolytic activity of Alcaligerirs derii/r.i/ic~nrisand Bcic~illrt.srrr~i~~loli~uc~fi~c.ieris has not been previously reported. Chitinase activity has been found in a wide variety of microorganisms including species of BLici/Iil.s and .Str.c)proiujws (CHIGALEICHIK 1976, BEWETTand HOOD1980. O'BRIENand DAVIS 1982, Z l K A K l S 1984, JANDA 1985. HARAv/ r i l . 1989).
(p),
Table I Chemical composition of shrimp-shell waste Constituents Moibturc contcnt Ash content Chitin C'rude protein Calcium carbonate Elements (as ppm) ca cu
c I1
Mn
NI
te Zn C ;1
'TO
to dry wt.
20
6 21 4 27.9 40
3 65 Zero 2.30 0 29 0.20 2 93 I .83 0 281
109
Microbial degradation of shrimp-shell waste Table 2 Chitinolytic activity of the tested strains Organism
Activity ratio*)
Alctiligenes deniirificutis Bucillrrs umjlaliquefuciens B. nieguterirrni B. .subtilis
2.8 2.4 2.4 I .8
Azorobacter chroococcurn Psmdomonus jlrrorescet~s sp. Acritiorii~~ces Streptor,1).ces sp.
2.0 I .o 0.8 1.6
*) Activity ratio is defined as thc ratio of the diameter
of the chitinolytic zone to the diameter of the colony (CHAN1970).
Atcaligenes derzitrificnns is seen to secrete the most active chitinase by this measure, followed by B. amyloliquefacicns and B. megaterium. Determinuion of Ir'eight loss For hydrolysis with intact cells, the microorganisms were cultured with 1% chitinous solids (colloidal, commerical and shrimp shells). The percentage weight losses of the chitin substrates in the culture flasks are listed in Table 3. The results indicate that on colloidal chitin Alcaligeries denitrficans affected the greatest weight loss while B. ~r~~~~lotiguejircier2.v and B. suhtitis yielded best results on shrimp-shell waste. Table 3 Percentage weight loss of different chitin substrates Organism
Percentage weight loss Colloidal chitin
Commercial chitin
Shrimp-shell waste
A lculigerres denitr@utis Baciflus aniy/oliquL.fLlcirns 5.niegateriiirii 5.subrilis
95.2 92.7 89.7 92.6
76.6 14.2 74.0 74.2
59.4 62.3 60.2 62.2
Arotobucter chroococcum Pseudornonus fluorescens Actinoniyces sp. S t r r p t o m y c e s sp.
72.6 79.2 69.0 85.20
55.4 58.8 48.3 68.6
44.3 44.3 42.6 56.3
Enzyme activity The values of chitinase activity varied according to the substrate and the bacterial species as well. Bacillus anz~~lotiqiiclfacietis growing on shrimp shell waste as substrate supported the highest activity. This might be attributed to the presence of small amounts of chitin hydrolysate (NAGA) which induces the production of chitinase as previously reported (MONREALand REESE1969, YOUNGet a1 1985). The presence of protein in the waste enhance the bacterial growth and brings about increased enzyme productivity (Table 4).
S. A . SABRY
110 Table 4 Chitinase activity of the tested organisms grown on chitin SUbStrdteS Chitinase activity U/ml
Organism
.4 lculigenes denirrificms Bucillus uni~loliquefrrciens B. niegureriuni B. sirhiilis
Commercial chitin
Shrimp-shell waste
2.7 3.4 2.2 4.3
1.9 3.9 3.6 1.7
Effect qf incuhution period on cnzjme production
To evaluate the chitinase productivity by cultures of different ages, the cultures were allowed to grow under shaked condition for 84 hours. The crude enzyme was obtained by centrifugation at time intervals and the enzyme activity was estimates. The resdlts presented in Table 5 show that maximal activities were maintained for 36 and 60 hours old cultures of B. anzjVoliqirqfuciens and B. megaterium. respectively. Table 5 Chitinase activity of B. u~ii~~loliqirqfacieris und B. meggareriutii grown on I % shell fish waste under different incubation periods Organism
Chitinase activity (Ujml) Hours incubation 6
B. ani~loliquefuciens B. tneguterium
12
-
-
-
-
18
0.5 0.5
24 3.0 1.5
36 4.8 3.4
48 3.9 3.4
60 3.9 3.8
72 2.5 2.2
84 1.2 1.2
The results of this study reveale the potentiality of the two newly reported chitinoclastic organisms named Alcaligenes detiitrficuns and Bacillus amyloliquqfacierzs to utilize shrimp shell waste for the production of chitinase enzyme. Further studies concerning optimization of enzyme productivity are under investigation.
References BENSETT,C. B. and HOOD,M . A , , 1980. Effects of cultural conditions on the production of chitinase by a strain of Bucilhr~inegu~eriuin.Developments in Industrial Microbiology, 21. 357. CARROAD, P. A. and TOM,R. A , , 1978. Bioconversion of shell-fish chitin wastes. Process conception and selection of microorganisms. J. Food Sci., 43. 1158. CHAN,J. G., 1970. The occurrence, taxonomy and activity of chitinolastic bacteria from sediment, water and fauna of Puget Sound. Ph. D. thesis, University of Washington. CHIGALEICHIK, A . G., 1976. Chitinase of Bacillus rhuringiensis. Mikrobiologiya, 45, 966. COSIO.I. G., FISHER.R. A. and CARROAD. P. A.. 1982. Bioconversion of shell fish chitin waste pretreatment, enzyme production, process design and economic analysis. J . Food. Sci.. 47, 901.
Microbial degradation of shrimp-shell waste
111
HARA,s.,YAMAMURA, Y., FUGII, Y., MEGA,T. and IKENAKA, T., 1989. Purification and characterization of chitinase produced by Streptoinyces eryrlrraeus. J. Biochem., 105, 484. HOOD, M. A. and MEYER,S. P.. 1977. Microbiological and chitinolastic activities associated with Penaeus sefi/erus. J. Ocean. SOC.Japan., 33, 235. JANDA.J . M., 1985. Biochemical and exoenzyrnatic properties of Aerornonos species. Diagn. Microbiol. Infect. Dis., 3, 223. MONREAL, J. and REESE,E. T.,1969. Thechitinase of Serratia marcescens. Can. J. Microbiol., 15,689. OBRIEN,M. and DAVIS,G. H. G., 1982. Enzymatic profile of Pseudotnonas maltophilia. J. clin. Microbiol., 16, 417. REISSIG, A. R., STROMINGER, R . S. and LELOIR, V. F., 1955. A modified colorometric method for the estimation of N-acetylamino sugars. J. Biol. Chern., 217, 959. S. and CARROAD, P. A., 1981. Conversion of the enzymatic hydrolysate of shell fish REVAH-MOISEEV, waste chitin to single-cell protein. Biotechnol. Bioeng., 23, 1067. SNEH,B., 1981. Use of rhizosphere chitinolytic bacteria for control of Fusariuni orysporum f. sp. clinnthi in carnation. Phytopath. Z., 100, 251. STELMOCK, R. L., HUSBY,F. M. and BRUNDAGE. A. L., 1985. Application of Van Soest Acid detergent fiber method for analysis of shell fish chitin. Dairy Sci., 68, 1501. WRIGHT,C. H., 1939. Soil Analysis. Physical and Charnical Methods, Thomas Murly. London. YOUNG,M. E., BELL.R. L. and CARROAD, P. A., 1985. Kinetics ofchitinase production, 11, Relationship between bacterial growth, chitin hydrolysis and enzyme synthesis. Biotechnol. Bioeng., 27, 776. ZIKAKIS. J . P. (ed.), 1984. Chitin, Chitosan and Related Enzymes. Academic Press, Inc. New York. Mailing address: Dr. S. A. SABRY.Department of Botany and Microbiology, Faculty of Science. Alexandria University, Moharram Bey, Alexandria, Egypt
