Study of Glucosamine Production from Shrimp Shells by Fermentation Using Trichoderma harzianum

Shrimp shells are one of chitin sources in Indonesia which is potential to be converted into multifunctional glucosamine. This research was aimed to study the glucosamine production by fermentation using Trichoderma harzianum. Method used was experimental fermentation with pH treatment of 3-5 and fermentation duration of 10-20 days, and designed using Response Surface Methodology (RSM). Results showed that fermentation duration of 10-20 days has reached the optimum point of glucosamine production from shrimp shells using T. harzianum. The highest production of Dglucosamine in fermentation using T. harzianum occurred on initial pH of 5.41 and fermentation duration of 15 days (18,294.95 ppm), while the highest N-Acethyl-D-Glucosamine production occurred on initial pH of 3.00 and fermentation duration of 20 days (127,000.00 ppm).

they are widely used as coating material for cellulose fiber, nylon, cotton and wool [4].
Glucosamine is one of chitin derivatives that are widely used. Glucosamine production can be done physicochemically [7][8][9][10][11], enzymatically [12][13][14][15] and microbiologically [16]. To produce glucosamine chemically is relatively fast, but the chemical compounds used are dangerous for the environment, meanwhile to produce glucosamine enzymatically, the limitations are on the stability of enzyme and enzyme extraction cost that are quite expensive [17,18]. To produce monomer (likes glukosa, glucosamine) using microorganism is more advantageous because it is easier, more practical and the waste does not pollute the environment [19].
Glucosamine production using microorganisms is usually done by fermentation. There are many kinds of microorganisms, including bacteria and molds that can produce chitinolytic enzyme that can break down chitin into glucosamine. One of the microorganisms that can produce chitinase quite actively is Trichoderma. One of the strains, T. viride, produces chitinase enzyme with enzyme activity value of about 210.14 U.mg -1 [20].
Trichoderma have several strains and in this research, the characteristics of Trichoderma harzianum in fermenting chitin into glucosamine with different pH condition and fermentation duration using RSM method, was studied. or random variables applied in this fermentation experiment were pH (X1 = 3, 4, 5) and fermentation duration (X2 = 10, 15, 20 days). The determination of fermentation duration was based on report [21,22] that optimum fermentation duration of Trichoderma was 15 days, whereas the pH of fermentation was based on optimum pH for T. harzianum growth [23], i.e. pH of 5.6. The experimental matrix was derived by Software Design Expert, as can be observed on Table 1. The fixed factor was nutrition composition inside the fermentation media (KH2PO4 and MgSO4) and fermentation temperature, i.e. about 30°C (room temperature). Therefore, the treatment applied in this research was combination from initial pH column and duration column, i.e. 21 treatments (run column).

Preparation of Chitin from Shrimp Shells
Chitin isolation from Vannamei shrimp shells was performed by demineralization step using HCl and deproteination using NaOH [24]. First, Vannamei shrimp shells were washed and dried under sunlight until dry and then milled using Buhrmill and sieved using 60 mesh sieve to obtain shrimp shells powder.
Shrimp shells powder was then added with 1.5 M HCl with ratio of 1:15 (w/v) between sample and solvent. The mixture was then heated at temperature of 70-80°C for 4 hours under agitation of 50 rpm, and was filtered afterwards. The solid obtained was washed with aquadest to remove the remaining HCl. Then, the solid was dried in the oven with temperature of 70°C for 24 hours and cooled inside the desiccator, to obtain low mineral shrimp shells powder.
Demineralized shrimp shells powder was put into beaker glass and added with 3.5% NaOH solution with ratio of 1:10 (w/v). The mixture was then heated at temperature of 65-70°C for 4 hours under agitation of 50 rpm. The mixture was filtered to obtain chitin solids and washed with aquadest until neutral pH is obtained.

Fermentation of Chitin from Shrimp Shells using T. harzianum
Fermentation of chitin using T. harzianum was performed using semi solid fermentation system. The substrate consists of 5 gram of chitin, 0.05 gram of KH2PO4, 0.25 gram of MgSO4, 0.05 gram of urea, 1 gram of glucose, and 25 ml of aquadest [25].
The substrate was then sterilized using autoclave with temperature of 121°C for 15 minutes. When the substrate has cooled down, pH was set according to the treatment using 0.1 M NaOH and/or 0.1 M HCl. Each substrate was added with 1 mL of T. harzianum spore and fermented at room temperature using shaker fermenter.

Chitinolytic Activity Test by Clear Zone Measurement
Chitinolytic activity test was done by observing the clear zone formed on agar media enriched with chitin, surrounding the growth of molds. Agar-chitin media was made from PDA added with 0.5% colloidal chitin (w/v media) [26,27] and acidified using 50% lactic acid for about 0.14% from total volume of media [28]. This media was then sterilized using autoclave with temperature of 121°C, pressure of 1 atm for 15 minutes. The media was then poured into Petri dish and let solidify. After the media has solidified, a well with 6 mm diameter was made, aseptically. Then, 20 μL of T. harzianum spores with density of 20.48×10 7 .mL -1 , 38.48×10 7 .mL -1 , 63.52×10 7 .mL -1 , 158.72×10 7 .mL -1 , were respectively added into the well (6.0 mm diameter), and incubated at temperature of 35°C for 48 hours.

Glucosamine Analysis using Spectrophotometry Method [29]
About 0.01 gram of fermented sample was dissolved uisng 1 mL of 0.25 M CH3COONa to obtain concentration of 10,000 mg.L -1 . This solution was then diluted gradually until concentration of 12 ppm and 100 ppm were obtained. Each sample (concentration of 12 ppm and 100 ppm) was reacted with same concentration of PITC. The absorbance of sample solution of 12 ppm concentration that has been added with PITC was determined using UV-Vis spectrophotometer at wavelength of 273 nm to obtain absorbance of D-Glucosamine compound. Meanwhile, the absorbance of sample solution of 100 ppm concentration that has been added with PITC was determined using UV-Vis spectrophotometer at wavelength of 584 nm to obtain absorbance of N-Acethyl-D-Glucosamine compound. These absorbance results were then plotted on standard curve of D-Glucosamine and N-Acethyl-D-Glucosamine.
Standard curve of D-Glucosamine and N-Acethyl-D-Glucosamine were prepared by dissolving 0.01 gram of standard D-Glucosamine or N-Acethyl-D-Glucosamine in 10 mL of 0.25 M CH3COONa and allowed to stand for 24 hours (to stabilize the solution). After that, standard D-Glucosamine or N-Acethyl-D-Glucosamine solution with concentration of 1,000 ppm as a stock solution was obtained. The stock solution was then diluted to obtain concentration of 3, 6, 9 and 12 ppm. About 10 mL of each solution was added with 10 mL of PITC solution, homogenized for 5 minutes and derivatives compound, i.e. phenylthiourea (PTH), was obtained. The absorbance of this compound can be determined according to the wavelength used. In this case, PITC solution was prepared by dissolving PITC using methanol that has the same concentration with glucosamine standard solution. Table 2 shows that chitin used has not fulfilled the commercial chitin standard in terms of protein content, although its moisture content, mineral content and deacetylation degree have fulfilled the commercial chitin standard. High protein content in chitin indicates that deproteination process was not enough. This might be caused by insufficient NaOH concentration and/or insufficient deproteination process. Effectivity of deproteination process depends on the concentration of basic solution and tempera-ture used [30]. Besides, protein content of chitin is related to its moisture content [30]. Lots of protein matrix that have been degraded cause hydrophilic properties of protein to bind water molecule becomes weaker [33]. As a result, the moisture content will become less [31]. It is shown by the moisture content of shrimp shells which is about 60-65% [32]. Chitinolytic Activity of T. harzianum Molds Chitinolytic activity of molds is defined as the ability of molds to degrade chitin into other simpler compounds, e.g. glucosamine by chitinase enzyme. This activity can be measured by clear zone formation around the wells, paper disc or colony [20,35,36]. In this research, chitinolytic activity obtained based on clear zone formation was used to determine the amount of spores added during chitin fermentation. The results of clear zone based on density of T. harzianum spores can be observed on Table 3. The size of clear zone formed shows the amount of N-acetylglucosamine formed from chitin breakdown by chitinase enzyme. The larger the clear zone, the higher amount of chitinase formed, which means the higher the chitinolytic activity [36]. Based on Table 3, it can be observed that the largest clear zone diameter for T. harzianum molds was obtained from spores with density of 20.48 x 10 7 spores.mL -1 , with clear zone of 6.52 ± 0.73 mm, and then decreased as the spore density was increased. The smallest clear zone occurred at density of 63. therefore there was no growth competition among the molds. Thus, T. harzianum can work optimally to degrade chitin during fermentation. Chitin degradation is done by chitinase enzyme that has different activity based on the types and stage of growth of microorganisms, and factors which influence the growth of microorganisms, such as pH, temperature and fermentation period [12,37,38]. Chitinase activity from Trichoderma sp. and T. viridae was 33.19 IU.mL -1 and 18.77 IU.mL -1 , respectively [20].

Glucosamine Production
Glucosamine that is obtained from chitin fermentation can be in form of D-glucosamine and N-Acethyl-D-Glucosamine. The correlation between pH and fermentation duration of shrimp shells chitin for glucosamine production according to RSM program can be observed on Figure 1. Three-dimensional RSM graph on Figure 1 shows the flat shape of correlation between pH and fermentation duration of shrimp shells chitin for glucosamine production, which means there was no optimum point. This phenomenon shows there was no significant effect of fermentation duration and pH on D-Glucosamine and N-acetyl-D-Glucosamine obtained. This might happen because the fermentation duration was too long; therefore the production of D-Glucosamine is already maximum since the first observation (day 10) and there was only slight difference in glucosamine content on the following observations. It is based on a previous research that stated the production of chitinase by fermentation using T. harzianum reached its highest amount after 72 hours (day 3) when T. harzianum mycelia starter was used and after 96 hours (day 4) when T. harzianum spore starter was used [39].
This statement is also supported by another research that stated chitinolytic activity of T. harzianum reached its optimum amount in between day 2 and day 3 [40]. No significant change of glucosamine production after it has reached its optimum point is also in accordance with a research [41] which stated after 172 hours, glucosamine fermentation using T. harzianum mold starter did not undergo significant increase. Because there was no optimum point for glucosamine production, the glucosamine production can be observed based on interaction between pH and fermentation duration, as can be seen on Figure 2. Figure 2 shows that the highest Dglucosamine amount obtained from pH treatment of 5.41 and fermentation duration of 15 days, which produces D-glucosamine content of 18,294.95 ppm. This amount of glucosamine is still higher compared to other research reports. Fermentation using chitinase from Pseudomonas sp. was optimum after 35 hours, which produces 0.164 mg.mL -1 164 ppm of glucosamine [14], using Actinomycetes ANL-4 starter produces highest glucosamine about 9,784.25 ppm on day 5 [29], using Aspergillus sp. BCRC 31742 produces highest glucosamine of 7,480 ppm [42].
The highest N-acetyl-D-Glucosamine production in fermentation of shrimp shells chitin using T. harzianum obtained from pH treatment of 5 and 10 days of fermentation, which produces Nacetyl-D-Glucosamine of 127,000.00 ppm. This amount of glucosamine is still higher compared to other research reports. Fermenta-tion using Aspergillus rogulosus 501 on day 10 obtained Nacetyl-D-Glucosamine of 2,228 ppm [43], fermentation using enzyme combination from T. harzianum and T. reesei was highest after 72 and 96 hours of incubation, which was about 4,040 ppm [39], fermentation using T. harzianum was

CONCLUSION
The fermentation duration applied on optimization of fermentation of shrimp shells chitin for glucosamine production using T. harzianum was too long. The highest production of D-Glucosamine from fermentation using T. harzianum occurred on initial pH of 5.41 and fermentation duration of 15 days, which reached about 18,294.95 ppm, whereas the highest production of N-Acethyl-D-Glucosamine occurred on initial pH of 3.00 and fermentation duration of 20 days, which reached about 127,000.00 ppm.