preparation and use of media for protease-producing bacterial strains based on by-products from...

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Microbiological Research 163 (2008) 473—480

0944-5013/$ - sdoi:10.1016/j.

�CorrespondE-mail addr

www.elsevier.de/micres

Preparation and use of media for protease-producing bacterial strains based on by-productsfrom Cuttlefish (Sepia officinalis) and wastewatersfrom marine-products processing factories

Nabil Souissi�, Yosra Ellouz-Triki, Ali Bougatef,Monia Blibech, Moncef Nasri

Laboratoire de Genie Enzymatique et de Microbiologie, Ecole Nationale d’Ingenieurs de Sfax,Rte Soukra km 4.5, BP ‘‘W’’, 3038 Sfax, Tunisia

Received 5 May 2006; received in revised form 16 May 2006; accepted 7 July 2006

KEYWORDSCuttlefish;Cuttlefish powder;Protease;By-products;Sepia officinalis

ee front matter & 2006micres.2006.07.013

ing author. Tel.: +21622esses: Nabil.souissi@gm

SummaryCuttlefish powder (CFP) from Sepia officinalis by-products was prepared and tested asa fermentation substrate for microbial growth and protease production by severalspecies of bacteria: Bacillus licheniformis, Bacillus subtilis, Pseudomonas aeruginosa,Bacillus cereus BG1, and Vibrio parahaemolyticus. All microorganisms studied grewwell and produced protease activity when cultivated in medium containing only CFPindicating that the strains can obtain their carbon and nitrogen source requirementsdirectly from whole by-product proteins. Moreover, it was found that the addition tothe cuttlefish medium of diluted fishery wastewaters (FWW), generated by marine-products processing factories, enhanced the production of protease. Maximum activitywas obtained when cells were grown in cuttlefish media containing 5-times or 10-timesdiluted FWW. Five-times diluted FWW enhanced protease production by B. cereusBG1 and B. subtilis by 467% and 75% more than control media, respectively. Theenhancement could have been due to the high organic content or high salts in FWW.

As a result, cuttlefish by-products powder enriched with diluted FWW was foundto be a suitable growth media for protease-producing strains. This new process,which converts underutilized wastes (liquid and solid) into more marketable andacceptable forms, coupled with protease production, can be an alternative way tothe biological treatment of solid and liquid wastes generated by the cuttlefishprocessing industry.& 2006 Elsevier GmbH. All rights reserved.

Elsevier GmbH. All rights reserved.

950854; fax: +21674275595.ail.com, [email protected] (N. Souissi).

www.elsevier.de/micres

dx.doi.org/10.1016/j.micres.2006.07.013

mailto:[email protected],

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N. Souissi et al.474

(Coello et al., 2000; Ghorbel et al., 2005).
Introduction
The cuttlefish-processing industry generateslarge amounts of solid and liquid wastes, whichcan cause major problem to the environment.Wastewaters are characterized by high concentra-tions of nitrogenous ions, organic matter and saltconstituents (430 g L�1). Biological treatment ofthese saline wastewaters with conventional micro-organisms results in low chemical oxygen demand(COD) removal efficiency, because of the plasmo-lysis of the organisms (Woolard and Irvine, 1995;Kargi and Dinc-er, 1998). There are limited numbersof studies on biological treatment of saline waste-waters produced by a factory processing marineproducts. Vidal et al. (1997) studied the treatmentof wastewaters from a fishmeal factory by ananaerobic filter and reported that the reactorremoved up to 70% of the influent COD concentra-tions at organic loading rate of 9.5 and14.3 g COD L�1 day�1. Khannous et al. (2003) inves-tigated the influence of the organic loading rates,ranging from 250 to 1000mgCOD L�1 day�1 on CODremoval in an activated sludge reactor. The system,inoculated with a NaCl-acclimated culture, re-moved up to 98% and 88% of the influent CODconcentrations at an organic loading rate of 250and 1000mgCOD L�1 day�1, respectively.

The solid wastes, which may represent 35% of theoriginal material, constitute an important source ofprotein. Thus, it would be very advantageous ifsuch by-products could be converted into afermentation substrate for microbial growth pro-duction of bioproducts. Indeed, growth substratesconstitute a major cost in the production ofmicrobial cells and bioproducts by the fermenta-tion industry. In most instances, the growthmedium accounts for approximately 40% of theproduction cost of industrial enzymes.

Organic nitrogen substrates, such as peptones,are widely used in many biological and biotechno-logical applications, such as microbial biomassproduction (Singh et al., 1995), and metabolitesbiosynthesis including enzymes (Haltrich et al.,1994; Rapp, 1995). At present, peptones areobtained from casein, soy protein, gelatin andmeat (Reed Hamer and West, 1994; Reissbrodt etal., 1995). Due to its favourable amino acid balanceand high protein content, fish materials represent apotential source of industrial peptones.

Until now, fish protein hydrolysates (FPH) havebeen investigated only to a minor extent. Particu-larly, FPH have been reported as growth substratesfor bacteria (Gildberg et al., 1989; Dufosse et al.,1997). Several reports show also that FPH might beused as nitrogen source for metabolite production

However, no work has been reported on the useof cuttlefish flour or protein hydrolysates fromcuttlefish by-products as fermentation substratefor microbial production of bioproducts.

The aims of the present study are to preparepowder from cuttlefish by-products and examinethe suitability of this substrate as a microbialgrowth medium for protease production by pro-tease-producing strains.

Materials and methods

Materials

Cuttlefish by-products were obtained from mar-ine processing industry. The wastewaters used wereeffluents generated by marine-products processingfactories treating cuttlefish, octopus and shrimps.Sewage was collected from the SOCEPA Co. Waste-waters were kept at 4 1C in a refrigerator to avoiddecomposition. Before being used, effluents werediluted with distilled water. The wastewaters werecharacterized in terms of total and soluble COD,total organic carbon (TOC), total Kjeldahl nitrogen(TKN), organic matter, volatile solids (ash-free dryweight), total suspended solids (TSS), ammoniumnitrogen and chloride.

Microorganisms

Bacillus subtilis was provided by the ‘‘Centre deBiotechnologie de Sfax-Tunisia’’. The followingstrains were isolated in our laboratory: Pseudomo-nas aeruginosa an alkaliphilic bacterium isolatedfrom tannery wastewater; Bacillus cereus BG1,Bacillus licheniformis and Vibrio parahaemolyticuswere isolated from an activated sludge reactortreating fishing industry wastewaters.

Cultivation and media

Inocula were routinely grown in Luria–Bertani(LB) broth medium (g L�1): peptone, 10.0; yeastextract, 5.0; NaCl, 5.0 (Miller, 1972). Media wereautoclaved at 120 1C for 20min. The cuttlefishmedium (CF) used for growth and protease produc-tion was composed only of cuttlefish by-productspowder (10 g L�1). The CF medium was prepared indistilled water or in different concentrations offishery wastewater (FWW). The medium for pro-tease production (M1) by B. cereus BG1 wascomposed of (g L�1): maltose 10 (as carbon source);cuttlefish powder (CFP) 10 (as nitrogen source).

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Preparation and use of media for protease-producing bacterial strains 475

Artificial sea water (ASW): NaCl 400mM, CaCl220mM, MgSO4 100mM and KCl 20mM (Krieg andHolt, 1984).

Cultivations were performed on a rotatory shaker(200 rpm) at 37 1C, in 250mL Erlenmeyer flasks witha working volume of 25mL. The cultures werecentrifuged and the cell-free supernatants wereused for estimation of proteolytic activity. Allexperiments were carried out in duplicates andrepeated at least twice.

Preparation of cuttlefish by-productspowders

Waste materials, including guts and stomachs,were rinsed with fresh water to eliminate the darkink, which consists of a suspension of melaningranules in a viscous colourless medium, thenheated till boiling. The cooked by-products werepressed to remove water and cuttlefish oil. Theresulting pressed product was minced in a meatgrinder, then dried at 80 1C for 24–48 h. The driedby-products preparation was minced again toobtain a fine powder, and then stored in glassbottles at room temperature.

Biomass

Cell concentration was estimated by the deter-mination of colony forming units (cfu). All experi-ments were carried out in duplicate and repeatedat least twice.

Assay of proteolytic activity

Protease activity was measured by the method ofKembhavi et al. (1993) using casein as substrate. A0.5mL aliquot of the culture supernatant suitablydiluted, was mixed with 0.5mL 100mM glycine–

NaOH (pH 10.0) or Tris-HCl (pH 8.0) containing 1%casein, and incubated for 15min at 60 or 70 1C. Inthe case of crude extract from B. cereus BG1, assayof proteolytic activity was carried out in Tris-HCl(pH 8.0) containing 2mM CaCl2. The reaction wasstopped by addition of 0.5mL 20% trichloroaceticacid. The mixture was allowed to stand at roomtemperature for 15min and then centrifuged at13,000 rpm for 15min to remove the precipitate.The absorbance of the supernatant was measuredat 280 nm. A standard curve was generated usingsolutions of 0–50mg L�1 tyrosine. One unit ofprotease activity was defined as the amount ofenzyme required to liberate 1 mg of tyrosine in1min under the experimental conditions used.

Chemical composition

Dry weight of CFP was determined after heatingsamples at 105 1C to constant weight, and ashcontent determined after heating dried samples at600 1C for 24 h. Total nitrogen content was deter-mined using the Kjeldahl method. Crude proteinwas estimated by multiplying total nitrogen con-tent by the factor 6.25. Crude fat was determinedgravimetrically after Soxhlet extraction of driedsamples with diethyl ether.

Analytical method

TOC was determined by a Shimadzu Analyser,TOC-5000. Soluble and total COD were determinedaccording to methods described by Ross Knechtel(1978). Volatile suspended solid (VSS), TKN andammonium nitrogen NH4-N were determined ac-cording to Standard Methods for the Analysis ofWater and Wastewaters (APHA, 1989). TSS weredetermined by filtering effluent samples through apre-weighed fibre-glass membrane (0.4 mm); thefilter was then dried at 105 1C to constant weight.The ionic composition was measured by atomicabsorption spectrophotometer (Hitachi Z 6100).

Results and discussion

Preparation of cuttlefish substrates

A flow diagram for the cuttlefish by-productspowder preparation is shown in Fig. 1. The rawmaterial was cooked, pressed, minced, dried thenground. The material obtained was termed CFP.Table 1 shows the chemical composition of the CFP.The powder contained high protein content(70–75%) but low ash content (4–7%). The CFP hadrelatively high lipid content 7–10%, by weight. Theyield (weight of CFP/weight of raw material)obtained for the CFP was 8.5%.

Characteristics of FWW

The characteristics of the FWW, generated bymarine-products processing factories treating cut-tlefish, octopus and shrimp are given in Table 2,which lists the average values of three separateanalyses for each parameter. The wastewater wascharacterized by high salt concentrations, whichwere derived from saline ground water used forwashing and from the marine products processed.The soluble COD was very high 2665mg L�1. FWWcontains high amounts of total nitrogen and

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proteins, which are essential elements for micro-bial growth. However, ionic compounds concentra-tions were low.

Comparative study of protease production bydifferent species

Five bacterial strains secreting neutral or alka-line proteases were tested. Proteases, one of themost important group of industrial enzymes, arewidely used in leather processing, the detergentindustry, production of protein hydrolysates, foodindustries, recovery of silver from used X-ray films,etc. (Kelly and Fogarty, 1976; Kalisz, 1988). Usinginexpensive protein sources may reduce consider-ably the cost of protease production. Table 3 listssome characteristics of proteases produced bystrains tested in this study. In order to test ifcuttlefish preparation alone promotes biomass

Table 1. Chemical composition of CFP (g/100 g product)

Water Protein (N� 6.25) Lipids Ash

4–8 70–75 7–10 4–7

By-products

Cuttlefish (Sepia officinalis)

Dark ink

Water, Lipids

Diluted FWW

Thermal treatment at boilingtemperature, 10–15min

Washing

Pressing

Drying 24-48 h at 80˚C

Microbial growth media

Grinding

Grinding

Cuttlefish by-products powder

Figure 1. Flow diagram for the preparation of cuttlefishby-products powder (CFP) and microbial growth media.

protease synthesis experiments were first con-ducted in media containing only cuttlefish by-products powder as the sole organic carbon,nitrogen, phosphorus and salts sources. In addition,we investigated the effects of different concentra-tions of FWW on the biomass and protease produc-tion. FWW was diluted in distilled water.

Evidence of protease production by B. cereusBG1

Growth and protease synthesis in medium M1containing different concentrations of FWW aregiven in Table 4. Protease synthesis in control M1medium, which contains only maltose as carbonsource and CFP as nitrogen source, was lower thanthose obtained in M1 medium enriched withdifferent concentrations of FWW. Protease produc-tion increased significantly with increasing dilutionof FWW while biomass did not vary significantly.Highest enzyme production was achieved with five-times diluted FWW and protease production was

Ca Mg Na K

0.6–0.7 0.2–0.3 0.25–0.35 0.06–0.07

Table 2. Average composition of the wastewater fromfish processing factory

Parameters

pH 7.85Total COD (mg L�1) 3245Soluble COD (mg L�1) 2665BOD5 (mg L�1) 1654TOC (mg L�1) 1657VSS (g L�1) 0.23TSS (g L�1) 5.7TKN (mg L�1) 1289NH4-N (mg L�1) 1152Proteins (mg L�1) 856COD/BOD 1.6Cl (g L�1) 28P (mg L�1) 36.25Na (g L�1) 5.30Ca (mg L�1) 242.50Fe (mg L�1) 3.68Zn (mg L�1) 1.17Pb (mg L�1) 0.01K (mg L�1) 160Mg (mg L�1) 0.40Mn (mg L�1) 0.19Cu (mg L�1) 0.11

Average values of three samples.

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Table 3. Some characteristics of proteases produced by the strains studied

Strains Type of protease Optimum pH Optimum Tre (1C)

Bacillus cereus BG1 Metalloprotease 8.0 60Bacillus subtilis Serine protease 8.0–10.0 60Pseudomonas aeruginosa Metalloprotease 8.0 60Bacillus licheniformis Serine protease 10.0 70Vibrio parahaemolyticus Serine protease 10.0 60

Table 4. Growth and protease production by B. cereusBG1 in M1 medium supplemented by different concentra-tions of FWW

Medium Protease(UmL�1)

UFC(� 107)

Final pH

M1+DW 487 58 8.7M1+normal FWW 756 83 8.2M1+two-times diluted FWW 1461 79 8.45M1+five-times diluted FWW 2771 95 8.5M1+10-times diluted FWW 1592 101 8.6

Cultivations were performed for 48 h at 37 1C. Values are meansof three independent experiments. Standard deviations were72.5% (based on three replicates).DW: distilled water; FWW: fishery wastewater.

Table 5. Growth and protease production by B. subtilisin CF medium supplemented by different concentrationsof FWW


UFC(� 107)

Final pH

CF+DW 178 13 8.5CF+normal FWW 0 7 8.0CF+two-times diluted FWW 185 13 8.26CF+five-times diluted FWW 309 25 8.4CF+10-times diluted FWW 392 31 8.45

Cultivations were performed for 16 h at 37 1C. Values are meansof three independent experiments. Standard deviations were72.5% (based on three replicates).


about 5.7-times greater than the control medium.However, with undiluted FWW there was only a 55%increase in enzyme synthesis.

In a previous paper, Ghorbel-Frikha et al. (2005)reported that the production of protease by B.cereus BG1 is absolutely calcium-dependent sinceno other divalent cations were able to induceenzyme synthesis. No protease activity was de-tected when the strain was grown in mediumlacking CaCl2, although such a medium yielded ahigher concentration of biomass. The fact thatprotease was produced even in control mediumwithout salts indicated that cuttlefish by-productspowder and FWW supplied the strain with calcium.The results obtained clearly indicated that CFP maybe an excellent nitrogen source for the growth andproduction of protease by B. cereus BG1.

Evidence of protease production byB. subtilis

Preliminary experiments showed that proteaseproduction by B. subtilis in YT medium (peptonefrom casein, 10 g L�1; yeast extract, 1 g L�1; NaCl,3 g L�1; K2HPO4, 1 g L�1; MgSO4 � 7H2O, 1 g L�1;MnSO4 � 2H2O, 0.1 g L�1; CaCl2 � 2H2O, 0.3 g L�1 andglucose, 2 g L�1) was about 300 UmL�1. Growth andprotease production by B. subtilis in CF mediumcontaining different concentrations of FWW are

shown in Table 5. Protease activity was notdetected in the culture medium containing normalFWW. This could be due to the inhibition of thegrowth of the strain by compounds present at highconcentrations in FWW. However, the production ofprotease by B. subtilis was enhanced with dilutedFWW. With 10-times diluted FWW there was a 120%increase in the protease production compared tothe control medium, which contains only CFP.

Evidence of protease production byV. parahaemolyticus

V. parahaemolyticus grew well and producedhigh protease activity in control medium containingonly cuttlefish substrates (1607UmL�1) (Table 6).The effects of various concentrations of FWW onthe growth and protease synthesis showed thatenzyme production increased with increasing dilu-tion of FWW, although final biomass was notsignificantly different. Protease production in CFmedium containing 10-times diluted FWW wasabout 155% of that obtained with control medium.However, protease production was considerablyaffected when the strain was cultivated in thepresence of undiluted FWW.

In a second experiment, and in order to explainthe enhancement of protease synthesis by dilutedFWW, the strain was grown in CF medium supple-mented with ASW. As shown in Table 6, the addition

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Table 6. Growth and protease production by V. para-haemolyticus in CF medium supplemented by differentconcentrations of FWW


UFC(� 107)

FinalpH

CF+DW 1607 11 8.76CF+normal FWW 196 9 8.13CF+two-times diluted FWW 1658 11 8.4CF+five-times diluted FWW 1883 12 8.6CF+10-times diluted FWW 2487 16 8.67CF+ASW 2196 11 8.5

Cultivations were performed for 24 h at 37 1C. Values are meansof three independent experiments. Standard deviations were72.5% (based on three replicates).ASW: artificial sea water.

Table 7. Growth and protease production byB. licheniformis in CF medium supplemented by differentconcentrations of FWW


UFC(� 107)

FinalpH

CF+DW 407 80 8.6CF+normal FWW 138 20 7.9CF+two-times diluted FWW 821 70 8.3CF+five-times diluted FWW 785 85 8.5CF+10-times diluted FWW 821 80 8.6


200

300

400

500

600

700

Prot

ease

act

ivity

(U

mL

-1)


of ASW caused a 36.6% increase in activity(2196UmL�1) over the control (1607UmL�1), andthe enzyme activity was slightly higher than thatobtained with five-times diluted FWW(1883UmL�1). From these results, it seems likelythat FWW supplies the strain with some elementsfor growth and protease production since theaddition of ASW enhanced enzyme synthesis.

0

100

CF CF+ASW CF+KCl CF+CaCl2 CF+NaCl

Culture media

Figure 2. Effects of addition of artificial sea water (ASW)and some inorganic salts to the cattlefish culture mediumon protease production by B. licheniformis. CF: cuttlefishmedium containing only 10 g L�1 CFP. Values are means ofthree independent experiments. Standard deviationswere 72.5% (based on three replicates).

Evidence of protease production byB. licheniformis

Growth and protease production by B. licheni-formis is given in Table 7. The addition of normalFWW reduced considerably the biomass and enzymeproduction compared to the control medium whichcontained only CFP. This inhibitory effect may bedue to the high COD of FWW and/or the presence oftoxic compounds at high concentrations. However,the addition of diluted FWW enhanced proteasesynthesis. With 10-times diluted FWW there wereabout 100% and 495% increases in protease produc-tion compared to the control medium and to thatcontaining undiluted FWW, respectively.

Additional experiments were conducted to elu-cidate the effects of FWW on protease production.In the first experiment, the strain was grown in CFmedium supplemented with ASW. In contrast to theresults observed with V. parahaemolyticus, addi-tion of ASW to the control medium reduced enzymeproduction by 50% (Fig. 2). In a second experiment,the effects of addition of some inorganic saltspresent in ASW to the control medium wereexamined. Among the compounds investigated,KCl (20mM) enhanced enzyme production byapproximately 57% (Fig. 2). However, the additionof NaCl (400mM) or CaCl2 (20mM) to the controlmedium decreased enzyme synthesis by 36% and

73%, respectively. From these results, it seemslikely that reduction of enzyme activity could havebeen due to the high salts concentrations in FWW.

Evidence of protease production byP. aeruginosa

When cultivated in a medium containing onlyCFP, P. aeruginosa produced about the sameamount of protease (1680UmL�1) as that obtainedwith CFP prepared in 5- and 10-fold diluted FWW(Table 8). However, protease production wassignificantly low (about 160 UmL�1) with two-timesdiluted FWW although biomass formation in all runswas about the same. This indicated the presence ofsubstance(s) in FWW which may repress proteaseproduction. The results obtained clearly indicatedthat CFP alone promotes biomass and protease

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Table 8. Growth and protease production byP. aeruginosa in CF medium supplemented by differentconcentrations of FWW


UFC(� 108)

FinalpH

CF+DW 1680 20 8.6CF+two-times diluted FWW 160 16 7.6CF+five-times diluted FWW 1680 18 8.4CF+10-times diluted FWW 1694 22 8.5



synthesis, and does not need to be enriched by theaddition of salts.

Conclusion

This work is the first on the use of cuttlefish by-products as bacterial substrate for enzyme synth-esis. In this study, we reported that protease couldbe produced by protease-producing strains in mediacontaining only CFP. Cuttlefish powder is a complexsubstrate, notably it contains substances essentialto microbial media such as carbon and nitrogensources and minerals. The fact that protease wasproduced even when cells were grown in thepresence of undigested proteins as sole carbonand nitrogen source indicated that the basal levelof protease was sufficient to release inducingpeptides from undigested cuttlefish proteins. Addi-tion of undiluted FWW to CF medium reducedconsiderably both bacterial growth and proteaseproduction compared to control media. The in-hibitory effect may be due to the presence in FWWof unknown compounds at high concentrations.However, the production of the enzyme was greatlyincreased with the addition of diluted FWW to CFmedium. It may be due to the availability of theadequate amounts of organic and inorganic com-pounds to the bacteria for the production ofprotease.

Based on this study, it appears that cuttlefish by-product could be converted into a fermentationsubstrate for protease-producing strains. In orderto better understand the enhancement or thereduction of protease synthesis by fishery waste-water (FWW), further research on this subjectshould also include the study of the effect ofvarious metallic ions on growth and proteasesynthesis. In addition, it seems that FWW maycontain other compounds that can inhibit or

stimulate protease production; other investigationsmust be done to elucidate such compounds.

Acknowledgements

This work was supplied by grants from ‘‘Ministerede la recherche Scientifique, de la Technologieet du Developpement des Competences, Tunisie’’.

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