05. Sipriyadi.cdr Vol.14, No.1, March 2020, p 34-43 DOI: 10.5454/mi.14.1.5 Optimization of Xylanase Production by Streptomyces costaricanus 45I-3 Using Various Substrates through Submerged Fermentation 1,2 3 4 SIPRIYADI , ARIS TRI WAHYUDI , MAGGY THENAWIDJAYA SUHARTONO , AND ANJA 3* MERYANDINI 1 Microbiology Study Program, Department of Biology, Faculty of Mathematics and Natural Sciences, Institut Pertanian Bogor, Kampus IPB Dramaga, Bogor 16680, West Java, Indonesia; 2 Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Bengkulu, Kampus UNIB Kandang Limun, 38371, Bengkulu, Indonesia; 3 Department of Biology, Faculty of Mathematics and Natural Sciences; 4 Department of Food Science and Technology, Faculty of Agricultural and Technology, Institut Pertanian Bogor, Kampus IPB Dramaga, Bogor 16680, West Java, Indonesia. Xylanase is an important hydrolytic enzymes with many application in several industries, but to obtain enzyme derived products is not easy. Thus, the optimization of efficient xylanases production is a great interest for enzyme application. This study aims to determine the type of substrate, medium composition, and optimum conditions of xylanase production by S. costaricanus 45I-3. Determination of substrate type was done by growing the tested bacteria on birchwood xylan, beechwood xylan, oat spelled xylan, corn cobs xylan, and tobacco xylan substrate, meanwhile the determination of medium composition and enzyme production were done by measuring xylanase activity at various substrate concentration and replacing the carbon, nitrogen, phosphate and surfactants source. The results showed that the highest enzymatic index (EI) produced from corn cob xylan substrate at 3.60 meanwhile the second highest was beechwood xylan substrate at 2.87 EI, however this substrate is purer, thus this substrate was selected and used as xylan sources for further optimization measurement. The best xylanase activity -1 (2.29 U mL ) obtained on eighth day after inoculation on rotary incubator at 120 rpm in 28 ºC. Arabinose as the -1 source of carbon generate the highest activity at 3.161 U mL meanwhile the most preferred source of phosphate is -1 Na HPO (2.37 U mL ). Both source of nitrogen i.e. nitrogen ammonium sulphate (NH ) SO and yeast extract 2 4 4 2 4 -1 were able to produce xylanase at 2.57 and 2.36 U mL . The addition of surfactant in production medium showed -1 -1 addition of SDS surfactant (0.146 U mL ) and Tween 80 (0.438 U mL ) showed a negative response by decreasing the activity. The conclusion showed that the xylanase activity was increased after optimization at various C, N, and P sources, and the use of nitrogen source (NH4) SO ), become a more economical alternative to replacing a 2 4 nitrogen source yeast extract so it can lower the production costs of xylanase enzyme. Key words : fermentation, Streptomyces costaricanus 45I-3, substrate variation Xilanase merupakan enzim hidrolisis penting dengan banyak aplikasi di berbagai industri, namun untuk mendapatkan produk turunan enzim tidaklah mudah. Dengan demikian, optimalisasi produksi xilanase yang efisien merupakan perhatian besar untuk aplikasi enzim. Penelitian ini bertujuan untuk mengetahui jenis substrat, komposisi medium, dan kondisi optimum produksi xilanase oleh S. costaricanus 45I-3. Penentuan jenis substrat dilakukan dengan cara menumbuhkan bakteri uji pada media xilan birchwood, xilan beechwood, xilan oatspelt, xilan tongkol jagung, dan xilan tembakau, sedangkan penentuan komposisi media dan produksi enzim dilakukan dengan mengukur aktivitas xilanase pada berbagai konsentrasi substrat. dan mengganti sumber karbon, sumber nitrogen, sumber fosfat dan surfaktan. Hasil penelitian menunjukkan bahwa indeks enzimatik (EI) tertinggi dihasilkan dari substrat xilan tongkol jagung sebesar 3,60 sedangkan tertinggi kedua adalah substrat xilan beechwood sebesar 2,87 EI. Substrat ini lebih murni sehingga dipilih dan digunakan sebagai sumber xilan untuk -1 optimalisasi lebih lanjut. Aktivitas xilanase terbaik (2,29 U mL ) diperoleh pada hari kedelapan setelah inokulasi pada inkubator bergoyang 120 rpm dan suhu 28 ºC. Arabinosa sebagai sumber karbon menghasilkan aktivitas -1 -1 tertinggi yaitu 3,161 U mL sedangkan sumber fosfat yang paling baik adalah Na HPO (2,37 U mL ). Kedua 2 4 sumber nitrogen yaitu amonium sulfat (NH )2SO dan ekstrak khamir mampu menghasilkan xilanase pada 2,57 4 4 -1 -1 -1 dan 2,36 U mL . Penambahan surfaktan pada media produksi SDS (0,146 U mL ) dan Tween 80 (0,438 U mL ) menunjukkan respon negatif dengan penurunan aktivitas. Kesimpulan menunjukkan bahwa peningkatan aktivitas xilanase setelah dilakukan optimasi pada berbagai sumber C, N, dan P, serta penggunaan sumber nitrogen (NH )2SO ), menjadi alternatif yang lebih ekonomis untuk menggantikan ekstrak ragi sumber nitrogen sehingga 4 4 dapat menurunkan biaya produksi enzim xilanase. Kata kunci : fermentasi, Streptomyces costaricanus 45I-3, variasi substrat MICROBIOLOGY INDONESIA Available online at http://jurnal.permi.or.id/index.php/mionline ISSN 1978-3477, eISSN 2087-8575 *Corresponding author: Phone: +62-251-8625481; Fax: +62- 251-8625708; Email: ameryandini@yahoo.com Xylan is the main component of hemicellulose. The main chain is composed by β-xilopironosa units with β-1,4-glycosidic bond with a subsidiary chain in Volume 14, 2020 Microbiol Indones 35 the form of glucopyranosyl, 4-O-methyl-D- glucopyranosyl, α-L-arabinofuranosyl, acetyl, or furulil and p-coumaril (Kulkarni et al. 1999; Li et al. 2000) Endo-β-1,4-xylanase (EC 3.2.1.8) is an enzyme which is important to hydrolyze xylan perfectly thus it can produce an usable product such as xylose, and xylobiose like xylooligosaccharides (Bernier et al. 1983; Chakrit et al. 2006). � Various microbes were reported able to produce xylanase. Bacillus from bacteria, Trichoderma and Aspergillus from fungi, and Streptomyces from actinomycetes were known to be a potential microbes producing xylanase. Those microbes are known to have varied and widespread ecological niches (Collins et al. 2005). Bacteria from Indonesia such as Bacillus licheniformis strain 15 produces endo-β-1,4-xylanase which grown on banana peels substrate (Helianti et al. 2007). Meryandini et al. (2007) reported that the isolate of Streptomyces costaricanus 45I-3 isolated from peat swamps soil in Kalimantan, Indonesia, has an optimum activity at pH 5.0 in 50 °C. However this study only used a single substrate i.e. oat spelt xylan and did not conduct the activity measurement of various carbon, phosphate and nitrogen sources also the effects of surfactants to produce an optimum conditions in producing extracellular xylanase. � The main nutrients for microorganisms growth were carbon sources, nitrogen and mineral components especially phosphate. The media formulation for the growth and fermentation S. costaricum is an important stage at designing an experiments in the work scale. Papagianni (2004) reported that the carbon sources, nitrogen sources and the fermentation time have a significant role in determining the enzyme production level in a culture. Therefore, it is important to optimize the conditions to produce enzyme inexpensively using the available nutrient sources abundantly. Response surface method has been used as successful statistical tools for the optimization of media composition in the fermentation process for enzyme production (Dobrev et al. 2007). Therefore the aim of this study was to optimize the nutritional conditions in producing xylanase using S. costaricanus 45I-3 by conducting a series of research stages and data analysis. MATERIALS AND METHODS Bacterial Strain. The bacteria used in this study was S. costaricanus 45I-3 isolated from peat swamp forest in Kalimantan, Indonesia, a collection of Dr. Yulin Lestari (staff of Department of Biology, Faculty of Mathematic and Natural Sciences, Bogor Agricultural University, Indonesia). These isolate was cultured on International Streptomyces Project No. 2 (ISP2), supplemented with antibiotics nalidixic acid (1 -1 -1 mg mL ) and cyclohexamide (5 mg mL ), and incubated for 8 days at room temperature (27 °C), and this isolate was used as stock culture for the further assays. Morphological Characteristic of S. costaricanus 45I-3 in Various Growth Media. The cultivation of S. costaricanus 45I-3 was conducted using various solid media, which aims to see the growth and morphological characteristics in media used; nutrient agar (NA), potato dextrose agar (PDA), ISP, yeast soluble starch agar (YSA), International Streptomyces Project No. 4 (ISP4), and beechwood xylan agar medium. Each media (25 mL) were sterilized at 121 °C for 15 min. After sterilization, a loopful of S. costaricanus 45I- was streaked on media, then incubated at 27 °C for 8 days. The bacterial cells that showed the best growth will be used for further study. The Measurement of β-Xylanase Activity of S. costaricanus 45I-3 Based on Clear Zone. A total volume of 250 mL fermentation medium (g/L) which consist of yeast extract 1.25 g, KH PO 0.25 g, 0.05 g 2 4 MgSO ∙7H O) and 0.8% (w/v) substrate (beechwood 4 2 xylan) were added to 250 mL distilled water. After the medium mixed perfectly the sterilization was performed on 121 °C for 15 min. After sterilization, the medium then inoculated by 1% (v/v) bacterial culture and incubated at 27 °C for 8 days with 120 rpm agitation. Furthermore, the culture was centrifuged at 8000 rpm for 15 min to remove bacterial cells. The supernatant then was precipitated using 80% acetone solvent in accordance with Meryandini et al. (2007). Xylan degradation activity assay was conducted using two layers agar method with modifications based on Chen et al. (2004). The base layer of agar medium consists of 50 mM acetate buffer (sodium acetate + acetic acid) pH 6.0 and 1.7% agar. Meanwhile the upper layer consists of 0.8% (w/v) xylan substrate (beechwood, birchwood, oat spelled, corn cobs, and tobacco) and 1.7% (w/v) agar in 50 mM acetate buffer pH 5.0. A total of 3, 5, 7, and 10 mL precipitated xylanase were dripped on each media with different substrates and dried subsequently at 27 °C for 15 min, then incubated at 37 °C for 4 days. After the incubation process the media were stained using 1% congo red for 15 min and rinsed using 1 mM NaCl. The hydrolysis of β-xylanase indicated by the clear zone (halo) formed with an orange margin around (Carder, 1986). This EI= Diameter of hydrolysis zone Diameter of colony halo was measured for subsequent calculation of the enzymatic index (EI) using the equation. The Effect of Xylanase Production using Various Concentration of Xylan. In this study a beechwood xylan was used as a carbon source. To determine the minimum concentration with the best activity in producing β-xylanase, a varying concentration of xylan were used in fermentation media. Beechwood xylan was added to each fermentation medium with concentrations 0.2, 0.4, 0.6, 0.8, and 1.0% (w/v) respectively, meanwhile the 0% (w/v) medium (without the addition of beechwood xylan) used as a control. The Effect of Various Carbon, Nitrogen, Phosphate, and Surfactant Sources towards β- Xylanase Produced by S. costaricanus 45I-3. Observation of various nutrients addition i.e. carbon, nitrogen, phosphate and surfactant sources, towards β- xylanase production was done by replacing the resource of various nutrients with a variety of other sources. The production of β-xylanase with the addition of major carbon source in the form of beechwood xylan 0.8% (w/v) with various other carbon sources, such as glucose, galactose, maltose, xylose, fructose, sucrose, arabinose and corn cob xylan with 0.3% (w/v) concentration, respectively. Meanwhile, the nitrogen source used were malt extract, peptone, tryptone, yeast extract, ammonium sulfate (NH )2 SO , and ammonium persulfate (NH )2 S O 4 4 4 2 8 respectively 0.3% (w/v). The effect of various phosphate sources addition was done by replacing the main source of phosphate KH PO with other 2 4 phosphate sources, i.e. Na HPO , NaH PO , K HPO 2 4 2 4 2 4 and AlPO respectively 0.03% (w/v). Measurement of 4 xylanase activity with 0.03% (w/v) surfactants addition was performed by adding Tween 80, SDS (sodium dodecyl sulfate), and DMSO (dimethyl sulfoxide) respectively. Xylanase Activity Assay. The released reducing sugars were measured using DNS method (Miller, 1959). The DNS method was conducted by reacting 0.5 mL of 0.8% beechwood xylan substrate with 0.5 mL xylanase in acetate buffer (0.05 M, pH 5.0) and incubated at room temperature for 30 min. The reaction stopped by adding 1.5 mL of 3.5 dinitrosalicylic acid and heated for 15 min at 100 ºC. The solution then allowed to cool down and the absorbance value was measured using a spectrophotometer at 540 nm. Xylanase activity was calculated by using xylose standard curve. One unit of enzyme activity is defined as the amount of enzyme required to produce 1 mol xylose sugar per minute in standard conditions. RESULTS Morphological Characteristic of S. costaricanus 45I-3. S. costaricanus 45I-3 bacterial culture showed several morphological characteristics when grown on a various growing medium as shown in Figure 1. The S. costaricanus 45I-3 colony was round-shaped hyphae, undulate margin with mealy surface, the substrate mycelia are yellow, and the aerial mycelia are yellowish white with a bright yellow pigmentation on beechwood xylan medium, meanwhile on the other media (YMA, YSA, NA, PDA and ISP4) the aerial mycelia are brown with a yellowish white to brownish gray substrate mycelium (Table 1). Bacterial strains isolated from peat swamp forest in Kalimantan have flexous hyphae but has a spiral type on ISP4 medium. The spores have a smooth morphological characteristic. S. costaricanus 45I-3 was able to grow well in six different media as shown in Figure 1, but the most rapid and abundant growth were in beechwood xylan and YMA medium, the growth on those media nd was started on 2 day after incubation, the growth begins with substrate hyphae formation and followed Table 1 Morphological characteristic of S. costaricanus 45I-3 on various media Isolate Code Growth Media Color Pigmentation on Media Hyphae Type Spore Morphology Aerial Mycelia Substrate Mycelia 45I-3 YMA Grey Brownish grey None Flexous Smooth YSA Grey Brownish grey None Flexous Smooth Beechwood xylan Yellowish white Yellow Bright yellow Flexous Smooth NA White Yellowish white None Flexous Smooth PDA Grey Yellowish grey None Flexous Smooth ISP4 Grey Greyish white None Spiral Smooth 36 SIPRIYADI ET AL. Microbiol Indones with aerial hyphae covering the majority of growth media which followed by sporulation process. On the other hand, the four other media showed a slower growth and the aerial hyphae which formed was thinner and did not cover the overall surface of growth media. Xylanase Activity on Various Xylan Sources. This measurement aimed to determine which xylan source preferred by tested bacteria that could be seen its EI value. The highest EI value (3.6) was obtained in the solid culture which was added with corn cob xylan, while the lowest in tobacco xylan-enriched medium with a mere of 2.08 EI value. In addition, three other substrates i.e. birchwood xylan, beechwood xylan and oat spelt xylan produces an adjacent EI values respectively at 2.80, 2.87 and 2.77 (Fig. 2). Optimal Incubation Time in Producing Xylanase. The maximum activity of xylanase was th obtained in 8 day after incubation. The optimum th activity of extracellular enzyme was generated on 8 -1 day i.e. at 2.29 U mL (Fig. 3). The Effect of Various Concentration of Substrate/Beechwood Xylan for Xylase Production. Every microorganism requires a certain concentration of carbon source for their growth. The results in Figure 4 shows that the addition of a carbon source ranging from 0-1% (w/v) beechwood xylan give a different effect on xylanase production. In general, it can be concluded that the addition of higher concentrations produce a greater xylanase. However, the provision of th th 0.8% (w/v) and 1.0% (w/v) carbon source at 7 and 8 day after incubation showed not significant result with a nearly same amount of xylanase production i.e. 2.29 -1 U mL . The Effect of Various Carbon Sources towards Xylanase Production. Figure 5 shows the effect of various carbon sources usage for xylanase produced by S. costaricanus 45I-3 isolates, then different treatments were done by adding various carbon sources (0.3%) i.e. glucose, galactose, maltose, xylose, fructose, sucrose, arabinose, and corn cob xylan. The addition of 0.3% arabinose and fructose as additional -1 carbon sources produced the highest (3.16 U mL ) and -1 lowest xylanase (0.45 U mL ), respectively. The Effect of Various Nitrogen Sources towards Xylanase Activity. Ammonium sulphate (NH ) SO 4 2 4 and yeast extract are the best nitrogen sources for -1 xylanase production i.e. 2.52 and 2.36 U mL individually compared to the other organic and inorganic nitrogen sources which used, such as malt extract, peptone, tryptone and ammonium persulfate (Fig. 6). The Effect of Various Phosphate Sources towards Xylanase Activity. The results obtained indicate that the best phosphate source for xylanase production was Na HPO . The addition of Na HPO can 2 4 2 4 -1 induce xylanase production up to 2.37 U mL . The Effect of Various Surfactants towards Xylanase Activity. The effect of surfactant on xylanase production was tested using 3 different surfactants sources including Tween 80, SDS, and DMSO. The results showed that both of 0.03% Tween 80 and SDS had a significant effect to decline of xylanase production. The addition of Tween 80 decreased -1 xylanase production to 0.44 U mL and SDS addition -1 also decreased xylanase production to 0.15 U mL . The addition of DMSO (dimethyl sulfoxide) did not affect xylanase production which was proven with the -1 xylanase production that still reached 2.46 U mL . DISCUSSION Xylan is one of the carbon sources used by microorganisms, particularly bacteria, to be reformed into a simpler molecular structure which can then be used as an energy source. Several carbon sources used in this study were birchwood xylan, beech wood xylan, oat spelled xylan, corncob xylan and tobacco xylan. This may occur because xylan from corn cobs, birchwood, beechwood and oats contains many growth factors needed, vitamins and proteins which can provide source of carbon and nitrogen for the test bacteria (Sasmitaloka et al. 2019; Revanker and Lele 2006), low lignin and silica (Battan et al. 2006). Based on the results, xylanase production peaked at -1 the eighth dayof incubation, accounted at 2.29 U mL . Several other research results showed that the optimal incubation time to produce xylanase depends on the type of bacteria used regarding with the time of bacterial growth. Gram-negative and gram-positive bacteria, which are non-actinomycetes, tend to have a shorter incubation time compared to actinomycetes group. Several studies reported that Bacillus subtilis cho40 merely produced xylanase during 4 days of incubation time (Khandeparker et al. 2011). Another study revealed the optimal extracellular xylanase production time, carried out by Li et al. (2010), of actinomycetes th isolate, Streptomyces rameus L2001, was on the 7 day. Kavya and Padmavathi (2009) reported that the xylanase produced by Aspergillus niger was at 6.11 U -1 th mL on the 6 day after incubation. As can be observed th from curve, bacterial cells started to decrease on the 8 day which was the peak of exponential period. In this phase, bacterial cells grow dramatically and require Volume 14, 2020 Microbiol Indones 37 Fig 1 Morphological characteristic of Streptomyces costaricanus 45I-3 on various substrates. A1 (beechwood xylan), A2 (yeast solube startch), A3 (YMA), A4 (ISP4), A5 (PDA) and A6 (NA). B1 (morphology of aerial hyphae), B2 (crossed hyphae). C (morphological spore and hyphae) figure taken using Scanning electron microscope (SEM). Fig 2 The effect of different substrates on xylanase production by Streptomyces costaricanus 45I-3 after 4 days o incubation at 28 C. -1 Fig 3 Cell biomass of S. costaricanus 45I-3 (mg mL ) ( ) and S. costaricanus 45I-3 xylanase activity ( ) o on 0.8% beechwood xylan medium at 120 rpm in room temperature (27 C). 38 SIPRIYADI ET AL. Microbiol Indones Fig 6 The effect of various phosphate sources towards xylanase produces by S. costaricanus 45I-3 after incubated for 8 days. Fig 4 Screening of ISB64 isolate-producing cellulase thermophilic bacteria from the Ie Seuum hot spring, Aceh Besar. (a) Clear zone, (b) Bacterial colony, and (c) CMC media flooded with 1% congo red. Fig 5 The effect of various carbon sources towards xylanase produced by S. costaricanus 45I-3 after incubated for 8 days. Volume 14, 2020 Microbiol Indones 39 Fig 7 The effect of various phosphate sources towards xylanase produced by S. costaricanus 45I-3 after incubated for 8 days. Fig 8 The effect of various surfactant towards xylanase produced by S. costaricanus 45I-3 after incubated for 8 days. more energy source for survival than in other periods. Therefore, production of xylanase was excreted abundantly in order to gain more energy in terms of degrading media-contained xylan. Moreover, xylan concentration strongly related to enzyme production and for this reason, this study also monitored the effect of several concentrations of xylan to xylanase activity. Based on the results presented in Figure 4, the concentration used in the next optimization test was 0.8% (w / v). This result is in accordance with Guha et al. (2013) which states that the best concentration to produce xylanase is to provide a carbon source (xylan) from 0.25 to 1.0% (w / v). Lawrence et al. (2015) reported that the xylan concentration variation of 0.5-1% (w / v) added to the fermentation medium did not have a significant effect on xylanase production at concentration more than 1%. The administration of 0.5- 3.0% molasses as a supplement for B. subtilis and B. megaterium can reduce xylanase production in line with the increase in the concentration of molasses given. This probably due to the nutrients contained in molasses produce catabolite suppressants in xylanase production (Irfan et al. 2016). Application of several additional carbon sources also shows an influence on xylanase activity. Figure 5 shows the addition of fructose to the beech wood xylan production media significantly decreased the xylanase production, it was because of the xylanase produced in beech wood xylan media is 2.11 U / mL without the addition of fructose. Additional fructose can inhibit xylanase production, since fructose as a simple sugar will be used first as a carbon source by S. costaricanus 45I-3 isolates or bacterial cell growth. As supposed by Guan et al. (2016), simple sugars, including fructose, 40 SIPRIYADI ET AL. Microbiol Indones have no remarkable effect with xylanase production since, perhaps, those sugars could prevent synthesis of enzymes. This is also supported by study conducted by Ajijolakewu et al. (2016) who suggested that there were repressive effects produced by simple carbohydrate molecules in producing xylanase. Indeed, those were only utilized for growth. On the other hand, there is breakthrough discovering that S. thermocoprophilus TC12W was able to produce 1204.8 U/g of xylanase by using alkaline pretreated empty fruit bunch (APEFB) as a carbon source, which was the first report utilizing that (Sinjaroonsak et al. 2019). Giving different nitrogen sources turned out to have various effects on xylanase activity, indicating that yeast extract had the effect of being the best nitrogen source on xylanase activity. Bhardwaj et al. (2019) stated in their review that nitrogen is one of the most important elements for metabolisms, including enzymatic activity. A study by Zuhri et al. (2013) showed that ammonium sulfate is the best nitrogen source for the cultivation of Bacillus sp. M123 for the production of alkaline proteases. Addition of 1% casein played role in both the highest xylanase activity and xylanse production, 1.78 -1 -1 U g and 3.69 U mgG , respectively (Tai et al. 2019). Sudan and Bajaj (2007) reported that adding 0.3% ammonium sulfate to the production medium was able to make Aspergillus niveus RS2 produce 15 U/mL xylanase after 5 days of incubation which was the second best after yeast extract. The role of nitrogen, especially ammonium sulfate, from xylanase excretion was found from Anoxybacillus kamchatkensis strain NASTPD13 (Yadav et al., 2018). Ravindran et al. (2019) reported the highest xylanase (6495.6 IU/g of dry SCW) produced by A. niger was successfully resulted using media supplemented with 0.2g/g of yeast extract as nitrogen source. All nitrogen sources added to the production media showed that all of them were able to promote cell biomass growth and xylanase production except for ammonium persulfate (NH )2S O which 4 2 8 inhibited cell biomass growth and xylanase production. This is presumably because (NH )2 S O is toxic so that 4 2 8 it inhibits the growth of S. costaricanus 45I-3 cells and also inhibits xylanase production. The effect of different inorganic phosphate resources towards xylanase production has been tested and presented in Figure 7, shows that S. costaricanus 45I-3 isolates was able utilizing other phosphate sources i.e. NaH PO , KH PO , and AlPO to produce xylanase, 2 4 2 4 4 it is seen from the xylanase production which were -1 relatively stable at 2.17 to 2.34 U mL . A slightly different results in the addition of phosphate sources was K HPO which only capable inducing xylanase 2 4 -1 production at 1.69 U mL . A similar result were reported by Mandal (2015) which stated that Na HPO is the best 2 4 phosphate sources to Bacillus cereus BSA1 for xylanase production, with the production of xylanase at 5.53 U -1 mL . The phosphate salts with a certain concentration can encourage organism growth and stimulates the synthesis of extracellular enzymes in production medium Chellapandi and Jani (2008). Microbes using phosphate source as an ingredient synthesis of nucleic acids and phospholipids on the cell wall.The addition of other biosurfactant such as 0.03% sodium dodecyl sulfate (SDS) decrease xylanase activity by 13 fold -1 (0.15 U mL ) compared with the production media -1 without surfactant addition i.e. 2.11 U mL (Figure 8). Silva et al. (2015) reported that the addition of SDS on production media decreasing xylanase activity by 7.75 -1 fold (12.9 U mL ) which is lower than controls (100 U -1 mL ). SDS is a strong denaturant, the usage of SDS at certain concentrations can interfere enzyme activity function and lipid solubility (Wamack et al. 1983) As conclusion, from the results obtained it can be concluded that S. costraricanus 45I-3 was able to grow on a variety of media, but the best growth medium was medium containing xylan (beechwood xylan) which was produced a yellow aerial hyphae, smooth-shaped spores and produces bright yellow pigmentation. The th usage of 0.8% beechwood xylan substrate on 8 day after incubation was able to produce the highest xylanase production. The best fermentation conditions were on the addition of arabinose carbon source, and nitrogen source i.e. ammonium sulfate (NH )2 SO and 4 4 yeast extract. The most preferred source of phosphate was Na HPO . The addition of surfactants such as SDS 2 4 and Tween-80 gave negative effects because it decreased the level of xylanase production while the addition of DMSO did not decrease the activity. ACKNOWLEDGMENTS This research was funded by the Directorate of Research and Community Service, Directorate General for Strengthening Research and Development, Ministry of Research, Technology and Higher Education. In accordance with the Letter of Assignment Agreement Implementation Research Program Number: 044 / SP2H / LT / DRPM. Sipriyadi. Volume 14, 2020 Microbiol Indones 41 REFERENCES Ajijolakewu KA, Leh CP, Abdullah WNW, Lee CK. 2016. Assessment of the effect of easily-metabolised carbon supplements on xylanase production by newly isolated Trichoderma asperellum USM SD4 cultivated on oil palm empty fruit bunches. BioResources. 11(1):9611- 9627. Battan B, Sharma J, Kuhad RC. 2006. High-level xylanase production by alkaliphilic Bacillus pumilus ASH under solid-state fermentation. World J Microbiol Biotechnol. 22(12):1281–1287. doi:10.1007/s11274-006-9173-x. Bhardwaj N, Kumar B, Verma P. 2019. 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