Saccharification of chitin using solid-state culture of Aspergillus sp. S1-13 with shellfish waste as a substrate

JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 95,No.4,391-396.2003

Saccharification of Chitin Using Solid-State Culture of Aspergillus sp. S l-l 3 with Shellfish Waste as a Substrate NOPAKARN RATTANAKIT,’ SHIGEKAZU YANO,’ MAMORU WAKAYAMA,’ ABHINYA PLIKOMOL,’ AND TAKASHI TACHIKI’* Department of Bioscience and Biotechnology, Faculty of Science and Engineering, Ritsumeikan Universifv. Shiga 525-8577, Japan’ and Microbiologv Section, Department of Biology, Faculty of Science. Chiang Mai University, Chiang Mai 50200, Thailand’ Received 12 November 2002iAccepted 26 December 2002

Saccharification of chitin was performed in a suspension (mash) of a solid-state culture of chitinase-producing Aspergillus sp. Sl-13 with acid-treated shellfish waste as a substrate. The conditions for the saccharifying reaction and the solid-state cultivation were examined from the viewpoint of saccharification in the mash. Optimum cultivation conditions were defined: a solid-state medium consisting of 5 g of 10% lactic acid-treated crab shells (0.50-2.36 mm in size) and 3 ml of a basal medium (0.028% KH,PO,, 0.007% CaCl,.2H,O, and 0.025% MgSO,.7H,O) supplemented with 0.3% peptone was inoculated with 4 ml of spore suspension (1 x 10’spores/ml), and the water content of the medium was adjusted to 75%; static cultivation at 37°C for 7 d. When a culture obtained under the optimum conditions was suspended in 70 ml of 50 mM sodium phosphate-citrate buffer (pH 4.0) and incubated at 45’C for 11-13 d, 55 mM N-acetylglucosamine (GlcNAc) was formed in the solid-state culture mash, indicating that at least 33% of the initial chitin in the solid material was hydrolyzed. Through the experiments, the amounts of GlcNAc formed in the solid-state culture mash varied in a way similar to that of the water-extractable p-nitrophenyl p-D-N-acetylglucosaminide-hydrolyzing enzyme in the culture, but not to that of the colloidal chit&hydrolyzing enzyme. GlcNAc-assimilating lactic acid bacteria, which were inoculated into the mash after or at the start of the saccharification, formed lactic acid with decreasing GlcNAc. [Key words: solid-state cultivation, crab shellfish waste, chitin-saccharification, fermentation]

The waste in crustacean processing, which contains chitin as a carbohydrate component, is expected to be a renewable resource, and various investigations on its utilization have been carried out using microbial functions. The production of mono- and/or oligo-saccharides, which are usable as bioactive or fermentation substrates, is an example. The proposed process consists usually of two steps: the first is the chemical preparation of chitin from the waste, and the second is the hydrolysis of chitin with chitinolytic enzymes (chitinases) isolated from the culture filtrate of various microorganisms (l-4). The accumulation of saccharides in the culture medium by cultivating chitin-hydrolyzing microorganisms in a liquid medium containing the waste was also investigated (5). However, these methods might be impractical for local regions where the wastes occur because (i) for the chemical and biochemical processes, special facilities and equipment are required for the complicated techniques used, (ii) for preparing chitin, it is necessary to transport large amounts of bulky waste to the facilities, and (iii) highvolume effluents are generated due to the chemical treatment and recovery of product at low concentration in liquid

Aspergillus

sp. Sl-13, lactic acid

media. As a result, most of the waste is discarded through ocean dumping, incineration and land filling, causing secondary environmental pollution. On the other hand, conventional agrowastes have been utilized successfully to some extent in local regions in the production of useful substances (6-9), in which solid-state cultivation plays a role. Solid-state cultivation has the advantages of requiring relatively simple equipment and techniques, and generating little effluent with direct applicability of the products as enzyme sources or food amongst others. Thus, the solid-state cultivation might meet requirements for developing an alternative easy method for utilizing shellfish waste. We have reported, in a previous paper (lo), that shrimp shellfish waste was usable as a substrate for the solid-state cultivation of Aspergillus sp. Sl-13, and that the organism produced chitinases in the solid-state culture. The results indicated the possibility of utilizing shellfish waste, as well as various conventional agricultural materials, in solid-state cultivation. This paper deals with the saccharification of chitin using the solid-state culture of AspergilZus sp. S 1-13 on shellfish waste as a source of chitin and chitinase(s). It describes some properties of the saccharifying reaction in the suspen-

* Corresponding author. e-mail: [email protected] phone: +81-(0)77-566-l 111 (ex. 8265) fax: +81-(0)77-561-2659 391

sion (mash)

of the solid-state culture, and the optimum solid-state cultivation conditions for obtaining effective saccharification. The formation of chitinase in the culture, which plays a major role in the saccharification, was also investigated. In addition, the results of preliminary work on the production of lactic acid (LA) by double fermentations in the mash are reported.

MATERIALS

AND METHODS

Microorganisms Aspergillus sp. Sl-13 was used for solidstate cultivation with shellfish waste (IO). Lactobucillus delbrueckii subsp. luctis IF0 3073 and a lactic acid bacterium isolate (strain T- 16) were used for LA fermentation combined with chitin hydrolysis. They were selected in a preliminary study on their assimilability of GlcNAc and productivity of LA (Rattanakit et al., Abstr. Proc. 14th Annu. Meet. Thai Sot. Biotechnol., p. 18, 2002). The amounts of GlcNAc consumed and LA formed by the organisms indicated that they carried out homo-fermentation. Their characteristics in LA fermentation with GlcNAc will be reported in a separate paper. Solid-state cultivation of Aspergillus sp. Sl-13 The conditions defined for the formation of a colloidal chitin-hydrolyzing enzyme, which were described in a previous paper (I 0), were used as the “initial conditions” for the present work with the shellfish waste preparation: 5 g of shellfish waste (native shells or acidtreated shells, see below) and 3 ml of basal medium (0.028% KH2P0,, 0.025% MgS0,.7Hz0 and 0.007% CaClZ.2H,0, pH 5.0) supplemented with 0.1% ammonium sulfate were sterilized, and then inoculated with 4 ml of a spore suspension (I x IO7spores/ml) of Aspergilfus sp. Sl-13 (initial water content of the medium, 58.3%); cultivation was at 37°C for several days with occasional mixing. Two series of cultivations were routinely performed. As described below, one series was used for saccharification, and the other for assaying the amounts of chitinase(s) in the culture. Saccharification in mash of solid-state culture One series of the solid-state culture was suspended in 70 ml of 50 mM sodium phosphate-citrate buffer (pH 4.0-5.5), and the suspension (solidstate culture mash) was incubated at 45°C statically with occasional mixing. During the incubation, aliquots of the mash were withdrawn periodically, and centrifuged (I 0,000 xg, 20 min). The supernatant was used for measuring reducing sugars. Extraction of chitinase from the solid-state culture Another series of the culture was used for extraction of chitinases according to the methods described in a previous paper (IO). Cultivation of lactic acid bacterium L. delbrueckii subsp. luctis or strain T-16 was cultured in 7 ml of an MRS medium (1 I) consisting of 5% GlcNAc, 1% beef extract, 0.5% peptone, 0.5% yeast extract, 0.5% sodium acetate, 0.2% diammonium hydrogen citrate, 0.1% tween 80, 0.2% K,HPO, and 0.02% MgS0,.7Hz0. After static cultivation at 37°C for 2d, the cells were collected, washed twice with 50mM sodium phosphatexitrate buffer (pH 4.0), and suspended in 7 ml of the same buffer. The cell suspension was used as an inoculum for LA fermentation in the mash. The activities of colloidal chitin- and p-nitrophenyl Assays p-D-N-acetylglucosaminide (p-NP-GlcNAc)-hydrolyzing enzymes were assayed according to the methods described previously (I 0). One unit of the enzyme activity was defined as the enzyme amount releasing I pmol of reducing sugar per min (for colloidal chitinhydrolyzing enzyme) or I pmol of p-nitrophenol per min (for p-NP-GlcNAc-hydrolyzing enzyme). The enzyme amount was expressed in U/glDS (IO): total units/g of initial dry substrate of solid-state medium. Reducing sugar (as GlcNAc) and p-nitrophenol were determined by the methods described previously (IO), and LA by the

method of Barker and Summerson ( I? 1. Shrimp shells (f’ewem Shellfish waste and acid treatment monodon, black tiger) kindly supplied by Katokichi Bio, Kagawa. were dried and broken as described previously (IO; designated “native shrimp shells” in this paper). Dried and broken crab shells (Chionoecetes,juponicus, red snow crab) were a kind gift of Yaizu Suisan Kagaku Industry, Shizuoka (native crab shells). To remove calcium carbonate (and other salts), the native shells were soaked in 10% (or 15%) LA solution or ‘2N HCI (native shell:acid solution = I : 10, w/v) at room temperature for 3 d until no bubbles were formed. The acid-treated shells were washed well with water, and then dried at 50°C overnight (LA-treated or HCItreated shrimp shells, crab shells). Based on an experiment on LA concentration for the treatment, the 10% (or 15%) LA-treated crab shells were used in most of the experiments: the recovery of dry matter after the LA treatment was 50% (or 48%). The approximate chitin content was 22% (native shrimp and crab shells), 40% (I 0% LA-treated shrimp shells) and 47% (10% LA-treated crab shells), which was determined by the analytical method described previously (I 0). The water content was 446%. Chitin (crab shell) and p-NP-GlcNAc w’ere perReagents chased from Nacalai Tesque, Kyoto. Colloidal chitin was prepared according to the method described previously (10). Other chemicals were analytical grade commercial reagents.

RESULTS

AND DISCUSSlON

in solid-state culture mash of AsperThe organism was waste cultivated on solid-state media using several shellfish wastes, and the formation of reducing sugar in the culture mash was compared. Figure 1 indicates that, regardless of the type of shell, reducing sugar was formed in the mash with LAtreated shells whereas hardly any was formed in that with the native shells. Thin-layer chromatography (TLC) and HPLC analyses indicated that the soluble product in the mash was monosaccharide (GlcNAc : glucosamine = 10 : 1 or less), whereas oligosaccharide was hardly detected. We described the product as GlcNAc in this paper. GlcNAc formation was not observed (data not shown) in a mash which was heated in boiling water for 10 min before incubation for the saccharification indicating that GlcNAc was formed from residual shell chitin in the solid-state culture, and that chitinase(s) produced by Aspergillus sp. S 1- 13 catalyzed the hydrolysis (saccharification). In the mash with LA-treated shells, GlcNAc increased to a maximum level on day 7 of the incubation (about 10 mM in the mash with LA-treated shrimp shells, 12 mM with LAtreated crab shells), and then did not vary significantly to day I 1. As chitinases were active in the mash for 7-9 d of the incubation (100% p-NP-GlcNAc-hydrolyzing activity and 80% of the initial colloidal chitin-hydrolyzing activity were found), the stagnation of the saccharification later in the incubation period seemed to be due to an insufficiency of chitin in the mash. However, the addition of chitin (1%) or colloidal chitin (0.13%) to the mash did not increase GlcNAc, indicating that only the chitin in shells on which mycelia grew was hydrolyzable. The LA-treated crab shells were selected for further experiments considering the content of chitin, convenience of preparation, and amounts of GlcNAc formed. An LA concentration of 10% for the treatment was also decided based Saccharification

gillus sp. Sl-13 with shellfish

SACCHARIFICATlON OF CHITIN BY ASPERGILLUS Sl-13

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Saccharification period (d) FIG. 1. Saccharification in solid-state culture mash with various shellfish preparations. Aspergdlus sp. Sl-13 was cultivated on a solidstate medium with native shrimp shells, native crab shells, 15% LAtreated shrimp shells, 15% LA-treated crab shells, HCl-treated shrimp shells or HCI-treated crab shells for 11 d under the “initial conditions” described in Materials and Methods. Afier the cultivation, each culture was suspended in 70 ml of 50 mM sodium phosphate-citrate buffer (pH 5.5), and the mash was incubated at 45’C. Aliquots of the mash were withdrawn periodically, and analyzed for reducing sugar. The results with HCl-treated shells are not indicated in the figure because almost the same results were obtained as those with LA-treated shells. Symbols: open circles, LA-treated crab shells; open squares, LA-treated shrimp shells; closed circles, native crab shells; closed squares, native shrimp shells.

on the characteristics of the saccharification: the final amounts of GlcNAc in the mash with the 2.5% or 5% LAtreated shells were as low as those with the native shells, and the amounts with the 10% LA-treated shells increased up to almost the same level as those with the 15% LAtreated shells. The mash with 10% LA-treated shells gave a maximum yield in a shorter period than that with the 15% LA-treated shells. Conditions for saccharification Incubation temperature The degree of saccharification at 4°C was very low, and 2.7 mM GlcNAc was formed in 11 d of incubation. At 37”C, Aspergillus sp. Sl-13 again grew vigorously, and GlcNAc was not detected. A satisfactory degree of saccharification was observed when the mash was incubated at 45°C as shown in Fig. 1, and the amounts of GlcNAc formed decreased at higher incubation temperatures (10 mM at 50°C; 7 mM at 60°C). The optimum temperature for the growth of the organism was 40°C in liquid medium with glucose as the carbon source, and 37-40°C on an agar plate containing native shrimp shells (10). The growth was repressed at 45°C. The unsatisfactory saccharification at 37°C might be caused by GlcNAc assimilation by the growing organism, and the temperature of 45°C was favorable due to the stable action of chitinase and repressed consumption of GlcNAc by the organism. Among the mashes at different initial pH ofthe mash pHs, the mash at pH4 gave the maximum amount of GlcNAc: 90%, loo%, 82%, 60%, 54% and 45% for the ini-

393

tial pHs of 3, 4, 5, 6, 7 and 8, respectively. During the saccharification, the pH of the mash usually increased about one pH value from the initial value. However, precise pH control throughout the saccharification might be unnecessary because the amounts of GlcNAc formed in the mash, the initial pH of which was maintained at pH 4.0, 4.5 and 5.0 with 6 N HCl, did not vary markedly (respectively, lOO%, 95% and 90%). Actually, the amounts of GlcNAc formed were almost the same in two mashes with an initial pH of 4.0: one was controlled to maintain the pH at 4.0, and the other was not. A solid-state culture ground Grinding of the culture with a mortar before saccharification was expected to facilitate efficient saccharification due to the release of chitinase(s). However, the grinding did not increase the amount of GlcNAc formed although there was a slight acceleration of the formation. Improvement of condition of solid-state cultivation In a previous study (lo), the conditions for solid-state cultivation with native shrimp shells were defined with the formation of a water-extractable colloidal chitin-hydrolyzing enzyme as a factor indicating the growth of Aspergillus sp. S 1- 13 : the enzyme was assumed to reflect efficient degradation of chitin for assimilation by the organism. However, the conditions are not always favorable for saccharification when LA-treated crab shells are used, suggesting the need for valuation of the conditions. Cultivation period Solid-state cultures with different cultivation periods were assayed for GlcNAc formation in the mash. The saccharification in each mash proceeded as outlined in Fig. 1, and the final concentrations of GlcNAc formed are indicated together with the amounts of chitinase in the water-extract of the culture (Fig. 2). The GlcNAc formation varied according to the cultivation period of the culture (Fig. 2), and the mash of the 5-d culture gave the maximum (about 20 mM). Unexpectedly, the variations in the amount of GlcNAc formed were not similar to those observed for the water-extractable colloidal chitin-hydrolyzing enzyme, but to those observed for the p-NP-GlcNAc-hydrolyzing enzyme, which might play a secondary role in chitin degradation. Particle size oJ‘crab shells The LA-treated crab shells were divided into four groups according to their size, and each group was used for preparation of the solid-state medium. As shown in Table 1, the mash of the cultures with crab shells of particle size 0.50-1.39 and 1.40-2.36 mm gave high concentrations of GlcNAc. The distribution of these particle sizes in the LA-treated shells was, respectively, 40% and 47%, and the two sizes were combined (i.e., size 0.50-2.36 mm) for further study. Particles of less than 0.5 mm were not suitable for use in the solid-state medium: mycelial growth was limited in the muddy medium. Water content of the medium Aspergilka sp. S 1- 13 was cultivated on solid-state media of different water contents, and the mash of the culture with 75% water content formed the highest amount of GlcNAc (Fig. 3). The amount of water-extractable p-NP-GlcNAc-hydrolyzing enzyme formed varied similarly to that of GlcNAc formed. The amount of colloidal chitin-hydrolyzing enzyme

394

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55

60

I 65

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70

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Water content (%)

Sohd-state cultivatton (d) FIG. 2. Saccharification in solid-state culture mash with different cultivation periods and the amounts of chitinase formed in the cultures. Aspergilh sp. Sl-13 was cultivated on solid-state media with 10% LA-treated crab shells at 37°C. At each cultivation time indicated in the figure, two cultures were treated as follows: one was suspended in 70 ml of 50 mM sodium phosphate-citrate buffer (pH 4.0), and incubated at 45°C for saccharitication; the other was used for the extraction of chitinases with water. The saccharification was analyzed as outlined in Fig. 1, and the final concentration of GlcNAc formed is indicated. The amount of chitinase in each culture was estimated as described in Materials and Methods. Symbols: open circles, GlcNAc; closed circles, p-NP-GlcNAc-hydrolyzing enzyme; squares, colloidal chitin-hydrolyzing enzyme.

FIG. 3. Saccharification in solid-state culture mash with different water contents and the amount of chitinase formed in the cultures. The weight of the basal medium and the spore suspension (3 and 4 ml=3 and 4g) are included when calculating the water content (assuming their specific gravity is I). To prepare solid-state medium with more than 7 ml of water, water was added to the medium to achieve the water content indicated in the figure. For the media with less than 7 ml of water, concentrated basal medium and/or spore suspension was used. Two media of the same water content were prepared with 10% LA-treated crab shells. After cultivation at 37°C for 7 d, one culture series was used for saccharitication, and the final concentration of GlcNAc formed is indicated in the figure. The other series was used for the extraction of chitinases. Symbols: open circles, GlcNAc; closed circles, p-NP-GlcNAc-hydrolyzing enzyme; squares, colloidal chitinhydrolyzing enzyme.

TABLE I. Saccharification in the solid-state culture mash with LA-treated crab shells of different particle size Size of particle (mm) CO.50 mm

0.50-I .39 mm

I .40-2.36 mm

Cultivation (d) 3 5 7 3 < 3 c

>2.36 mm

Control (not sieved)

GlcNAc formed (mM) 10.8 12.3 13.4 16.6 17.1 18.3 17.6 18.6 18.0 15.7 15.8 14.1 15.5 17.1 17.2

The 10% LA-treated crab shells were put through sieves and divided into four groups of different particle sizes as indicated in the table. Solid-state media were prepared with each group, and Aspergillus sp. S I - I3 was cultivated for 3, 5 and 7 d. The culture mash was incubated at 45°C until GlcNAc formation was completed as in Fig. 2. The tinal concentration of GlcNAc is indicated in the table.

increased up to the maximum in the culture with 70% water content, which was considerably different from the optimum water content of 58-65% with native shrimp shells (10). The present and previous (10) findings on the water content of the solid-state medium are noteworthy: a higher

=o0 00

u

0 05

0 10

0.15

020

Peptone (%)

025

030

0.35

h

FIG. 4. Saccharitication in solid-state culture mash, which was prepared with basal medium supplemented with various concentrations of peptone, and the amount of chitinase formed in the cultures. Aspergillus sp. Sl-I3 was cultivated on two series of solid-state media with 10% LA-treated crab shells, which were prepared using basal medium containing various concentrations of peptone. After the cultivation at 37°C for 1I d, one series was used for saccharitication, and the other for the extraction of chitinases. Symbols: open circles, GlcNAc; closed circles, p-NP-GlcNAc-hydrolyzing enzyme; squares, colloidal chitinhydrolyzing enzyme.

water content was required for the medium containing shellfish waste than for the medium containing conventional agrowastes (13, 14). This might be due to the water retaining ability of materials. The agrowastes initially contain a certain amount of water, suggesting that their structure facilitates water retention and penetration of hyphae (1.5, 16). Shellfish waste contains little water and might not have a structure similar to that of agrowaste. Supplementation of the basal medium The effect on

SACCHARIFICATION OF CHITIN BY ASPERGILLUS Sl-13

VOL.95,2003

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B

.y 40

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Incubation time (d) FIG. 5. Saccharification under the optimum conditions, and conversion of GlcNAc to lactic acid by double fermentation. Aspergillus sp. Sl-13 was cultivated under the optimum conditions: the medium consisted of 5 g of 10% LA treated-crab shells (0.50-2.36 mm in size), 3 ml of basal medium containing 0.3% peptone, 4 ml of the spore suspension (1 x 10’spores/ml); with the water content adjusted to 75%. After cultivation in two flasks at 37°C for 7 d, one culture was suspended in 70 ml of 50 mM sodium phosphate-citrate buffer (pH 4.0) and incubated at 45°C (A). On day I5 of incubation (indicated with the dotted line), the mash was inoculated with a lactic acid bacterium T-l 6 and further incubated. As a result of the addition of the inoculum (7 ml), the concentration of GlcNAc was diluted about I. 1 times. The other culture was suspended in 70 ml of the same buffer containing the T-16 inoculum, i.e., inoculation of the organism at the start of saccharification (B). The mash was incubated under the same conditions as in panel A. Symbols: open circles, GlcNAc; closed circles, lactic acid.

the saccharitication of the supplementation of the basal medium, which used for preparing the solid-state medium, was determined. Among the supplements tested (ammonium sulfate, yeast extract, peptone, molasses, malt extract and corn steep liquor, each 0.1% in the basal medium), peptone enhanced GlcNAc formation in the mash. Figure 4 indicates that the optimum concentration of peptone was 0.3% in the basal medium, and the variation in the degree of saccharification in accordance with the peptone concentration was similar to the variation of p-NP-GlcNAc-hydrolyzing enzyme as in former experiments. Ammonium sulfate had no effect, although it increased the amounts of colloidal chitin-hydrolyzing enzyme in the culture with native shrimp shells (10). The other supplements had a repressive effect on the saccharification. Saccharification under optimum conditions and its The left side of conversion to lactic acid fermentation the dotted line in Fig. 5A indicates the saccharification under optimum reaction conditions using the solid-state culture obtained under the optimum cultivation conditions: the conditions were defined on the basis of the above results, and are described in the legend to the figure. Approximately 55 mM GlcNAc was formed in 11 to 13 d of incubation, indicating that at least 33% of the initial chitin in the LAtreated crab shells in the solid-state medium was hydrolyzed. The right side of Fig. 5A shows that inoculation of a GlcNAc-assimilating lactic acid bacterium T- 16 to the mash on day 15 of incubation resulted in the formation of LA with a concomitant decrease in GlcNAc over a further 7 to 10 d. When strain T- 16 was inoculated into the mash at the start of saccharification (Fig. 5B), 130 mM LA was formed in 13 d of incubation. The low-level accumulation of GlcNAc in the mash was due to its simultaneous conversion to LA.

L. delbrueckii subsp. lactis also gave similar results (data not shown). The chemical changes outlined in Fig. 5 indicated that two types of double fermentation proceeded in the mash: double and double-parallel fermentations. It should be noted that the degree of saccharification in the LA-fermenting mash (Fig. 5B), which could be calculated roughly from the concentration of LA formed, was higher than that in the mash where a single saccharification reaction proceeded (left side of Fig. 5A). This study demonstrated that chitin in shellfish waste was hydrolyzed to GlcNAc when the waste was used in the solid-state culture of Aspergillus sp. Sl-13: the culture was a source of chitin and chitinases. The double fermentations resulting in the production of LA suggested the possibility of converting shellfish waste, similarly to conventional agricultural waste materials, to useful substances via saccharification. However, improvement of the saccharification efficiency is still necessary for practical application, and this could be achieved by characterization of the chitinases in particular with respect to enzyme species participating in the saccharification and their interaction with chitin in the shells. The results of such a characterization also might explain some observations in this study: the unexpected variation in the amount of chitinases and the enhancement of saccharilication in double fermentations.

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