Pretreated Sugarcane Bagasse Result in More Ecient Degradation by Streptomyces sp S2

Streptomyces genera plays important role in lignocellulose degradation. Many research founds Streptomyces has cellulolytic and ligninolytic enzymes that sucient to degrade lignocellulosic materials. However, minimum lignocellulosic material condition that can eciently degraded by Streptomyces sp. has not been fully understood. In this research, three pretreament conditions (physical, alkaline-hydrotermal, and hydrogen-peroxide chemical treatments) of sugarcane bagasse used as lignocellulosic material, to further degraded by Streptomyces sp. S2. Lignocellulose component measurement conclude that raw (physical treated only) bagasse wasn’t eciently degraded by Streptomyces sp S2. Hydrogen-peroxide was effective on reducing both syringil and guaiacyl lignin, meanwhile alkaline-hydrotermal pretreatment was very effective on reducing syringil lignin. This study suggest that hydrogen-peroxide pretreatment can be used in many type of lignocellulosic material, which can be further degraded by Streptomyces sp. S2. Alkaline-hydrotermal preteatment on the other hand is best suited to degrade lignocellulosic material that have high percentage of syringil lignin.


Introduction
Lignocellulose material consist of cellulose chained into hemicellulose, with lignin ll the gap between hemicellulose and cellulose bers. In recent years lignocellulose materials gained attention to be used as feedstock for bioethanol, biogas, bioplastics or simple sugar ( Lignin can be reduced using bleaching and/ or pretreatment. Bleaching used in paper industry, which reduces lignin and change lignocellulose color to brighter color (white). Bleaching methods use chemical that contain chlorine or not, such as H 2 O 2 , NaClO 2 , or ClO 2 . Pretreatment on the other hand focus on reducing size of lignocellulose particle and reducing lignin contents. Pretreatment can be divided into physical (milling, extrusion, ultrasonic radiation), physicochemical (ammonia ber explosion, supercritical CO 2 ). Chemical pretreatment can be done using acid or alkaline solution in high concentration or dilute, organosolv, ionic liquid or deep-eutectic-solvent (DES). Another type of pretreatment is biological pretreatment, that use whole organism (such as fungi or bacteria) or enzyme that can degrade lignin Streptomyces sp. is Gram positive actinobacteria that abundant in soil and some other extreme environtment such as deep sea, volcano and extreme cold environment like Artic. Most Streptomyces sp. are well known to produce secondary metabolites, such as antibacteria and anti-cancer, and also known to degrade substrate in dead-plants (Chater 2016;Sivalingam et al.2019). Streptomyces are substantial lignocellulose degrader alongside Clostridium, Flavobacteria, Pseudomonas and Xanthomonas for rst three days of lignicellulose degradation (Ma et al., 2020). High number of binding sites that called CebR enable Streptomyces to have high cellulolytic activities. The CebR sites in uence Streptomyces sp. to encode genes that associates for deconstruct plant biomass such as glycoside hydrolase, endoglucanase, cellobiohydrolase, β-glucosidase, mannanase, xylanase, chitinase and LPMO (lytic polysacharide monooxygenase) ( Book et al. 2016). Another nding conclude that ligninolytic Streptomyces sp. have high lignin degrading enzyme activities of lignin-peroxidase (LiP), aryl-alcohol oxidase (AAO), low laccase (Lac) and dye decoloring enzyme (DyP) activities, but don't have manganeseperoxidase (MnP) enzyme (Riyadi et al. 2020).
Many studies have found that Streptomyces sp. has su cient amount of cellulolytic and ligninolytic enzyme, therefore Streptomyces sp. potentially used to degrade lignocellulose material. However e ciency of Streptomyces sp degradation on different lignocellulose condition hasn't been fully understood. This study use three sugarcane bagasse condition as lignocellulose (control, alkalinehydrotermal pretreated, peroxide pretreated), then Streptomyces sp. degradation ability was tested on three bagasse conditon aforementioned above. In this study Streptomyces sp. S2 that was isolated from palm oil plantation, Jambi, Indonesia was used as biological pretreatment. Effect of Streptomyces sp S2 on sugarcane bagasse was evaluated its crude ber content, lignocellulose component, crystalinity using X-ray diffraction, its chemical contents using Fourier Transform Infrared and lastly 3D structure using Scanning Electron Microscope (SEM).

Sugarcane Bagasse Preparation
Bagasse used in this research derived from the sugarcane juice seller in Bogor. Total of 40 kg (wet weight) sugarcane bagasse was cut, washed until clean then further sun-dried for 3 days and/or accelerated using oven until water content lower than 9%. All dried bagasse then milled using Wiley Mill, ltered through 40 mesh sieve. Bagasse that pass-through the sieve used for further treatment.
Alkaline-hydrotermal pretreatment Every 100 g prepared sugarcane was treated using 1.5L sodium hydroxide (NaOH) 1M concentration (solid: liquid ratio 1:15) in 2L Erlenmeyer. Erlenmeyer was closed using plastic and rubber bands, before put into autoclave for 30 minutes at 121 o C (Lemoes et al. 2018 modi ed temperature used). All of alkaline-autoclaved pretreated bagasse was neutralised using aquadest until the neutralised pH solution is near or same with the aquadest used (pH 5.5). Neutralised sugarcane bagasse further ltered using lter paper before dried using oven between temperature 60 80 o C.

Hydrogen peroxide pretreatment
Every 100 g sieved sugarcane bagasse was treated with total volume of 1L 6% (w/v) H 2 O 2 , 1% (w/v) trisodium citrate dihydrate, 1% NaOH (w/v) and 92% (w/v) aquadest in 2L Erlenmeyer. Erlenmeyer was closed using plastics that holed using skewers, for gas release that formed during pretreatment and tightened using rubber bands. Peroxide treatment was done in waterbath at 60 o C for 90 minutes (Yan et al., 2019). Sugarcane bagasse then further neutralised using aquadest until the solution pH near or same with the aquadest used (pH 5.5). Neutralised bagasse was ltered through lter paper before dried using oven until water content lower than 9%. Degradation using Streptomyces sp. S2 On the 14th day of incubation, the liquid medium containing Streptomyces sp. S2 was inoculated into 500 ml Erlenmeyer that contains 15 g of previously sterilized (autoclaved 125 o C, 40 minutes) sugarcane bagasse from previous pretreatment (control, alkaline-hydrotermal treated, peroxide treated). Erlenmeyer that contains solution of bagasse and Streptomyces then further incubated for another 4 days (total incubation 18 days in liquid ISP 4 solution). On 4th days of degradation, enzyme reaction was stopped by ltered the bagasse through lter paper then put the bagasse into freezer. Each pretreatment (control,  506 Å). Background noise was separated from selected XRD pattern, then X-ray pattern was corrected and normalized using computer. Crystalinity index was calculated using formula: (I cr / (I cr + I a )) x 100%, where I cr is area of crystaline meanwhile I a is area of amorph.
Functional Group Analysis Fourier-transform analysis was done using Spectrum Two, Perkin Elmer (USA) on absorbance between 400-4000 cm −1 and spectrum area between 4 cm −1 and 16 scans. Wave intensity was based on percent of transmitance which calculated and observed later.

Result And Discussion
Lignocellulosic component of sugarcane bagasse Crude ber consist of insoluble and soluble ber in which contain cellulose, hemicelulose, lignin and some amount of dextrin, β-glucan, mucilages, pectin, inulin, also oligosaccharides/ oligofructose (Slavin et al. 2009). Raw sugarcane bagasse used in this research have 31.90% crude ber from total dry weight. Streptomyces sp. S2 alone ineffectively digest crude ber in native sugarcane bagasse used, shown by not statisticaly different crude ber amount compared to control. On the other side both alkalinehydrotermal and hydrogen peroxide treatment does affect bagasse crude ber, increased percentage of ber by 19.96% and 10.95% respectively. Streptomyces sp. S2 increase amount of crude ber by 5% after bagasse treated using alkaline-hydrotermal which is not statistically signi cant with alkaline-hydrotermal treated only. Decreased amount of crude ber by 4.6% occur after peroxide bagasse further treated with Streptomyces sp S2, is statistically signi cant to bagasse that treated using peroxide only (Table 1).
Raw sugarcane bagasse that used consist of 31.83% hemicellulose, 46.65% cellulose and 9.90% lignin. Streptomyces sp. S2 degradation on raw bagasse wasn't effective, just slightly reduce cellulose to 44.14% and slightly increase hemicellulose and lignin to 32.28% and 10.78% respectively. Both alkalinehydrotermal and hydrogen peroxide pretreatment reduce hemicellulose by 19.37% and 10.45% respectively. Cellulose content on the other hand increased by 31.56% and 4.99% after alkalinehydrotermal and hydrogen peroxide pretreatment, respectively. Lignin concentration was reduced signi cantly by 4.08% after alkaline treatment, meanwhile peroxide treatment increase lignin signi cantly by 6.34%.
Increase in cellulose content and reduced lignin content after bagasse treated with alkaline was similar to Contrary to the alkaline-hydrotermal pretreatment, further degradation of alkaline pretreated bagasse using Streptomyces sp.S2 was reduce cellulose content signi cantly by 4.86% from 78.21-73.35%. Its hemicellulose and lignin content on the other hand only slightly increased. Streptomyces sp. S2 degradation to peroxide pretreated bagasse furthermore increase celulose content by 1.52-53.16%, also increase hemicellulose signi cantly by 3.87-25.25%. Lignin after peroxide bagasse degraded using Streptomyces sp. S2 was reduced signi cantly compared to peroxide only treated bagasse by 6.48-9.76%, but not signi cant compared to control (raw) bagasse (Table 1). This is consistent and similar to Xu et al (2017) ndings that use Cupriavidus basilensis to further degrade acid pretreated rice straw. Crystalinity index analysis Raw sugarcane bagasse (control) used have crystallinity around 45%, means about 45% of cellulose content are distributed in well-ordered crystal lattice and the rest 54.66% are distributed unevenly (amorph). Crystallinity after alkaline-hydrotermal pretreatment was reduced to 35.44% even the cellulose content increased indicates wider amorph area and loosened cellulose bonds. Peroxide pretreatment meanwhile increasing crystallinity of raw sugarcane bagasse by 5-50%, indicates increase in cellulose purity or more crystalline area.
Increase in crystalinity also mean wider speci c surface area, that accesible to bacteria and can be further digested (Xu et al. 2017). Result after Streptomyces treatment indicates that well-ordered cellulose are harder to digest by enzymes, by looking at amount of reduced crystalinity of raw bagasse ( 9% after Streptomyces treatment only) was lower compared to 11% reduction after hydrogen peroxide bagasse (H 2 O 2 ) treated by Streptomyces. Slight crystalinity increase after alkaline-hydrotermal (NaOH) bagasse treated with Streptomyces (from 35.44-36.95%) might indicate increase in cellulose purity after Streptomyces treatment (Figure 1 and Table 2).

Functional Group Analysis
The FTIR peak between 3500 and 3000 cm −1 in control was higher than in alkaline and peroxide treated, indicates the hydrogen bonds in cellulose was cut after both pretreatment. Both control-Streptomyces and NaOH-Streptomyces have higher peak on 3300 cm −1 after treated with Streptomyces sp S2 compared to its previous treatment, indicates that bonds between cellulose is stronger despite the cellulose content was lower compared to its previous treatment. On the other hand cellulose bond was weaker after hydrogen-peroxide bagasse was further treated using Streptomyces sp. S2, indicated from lower peak number 3300 cm −1 on peroxide-Streptomyces bagasse compared to hydrogen-peroxide only. Simple carbonic compound just found in control con rmed with peak number 1726 cm −1 , which more likely to be ester (Nandiyanto et al.2019). This ester bonds was decreased after all pretreatment.
Present bands of raw bagasse around 1300-1200 cm -1 with higher bands around 1300 compared to around 1200, indicates that sugarcane bagasse have higher S (syringyl) compared to (G) guaiacyl lignin (Watkins et al.2015). This con rms Miyamoto et al.(2018) results that sugarcane bagasse have higher syringyl lignin. Peak around 1300 was reduced after alkaline-hydrotermal pretreatment, meanwhile peak around 1200 wasn't. This indicates that alkaline-hydrotermal pretreatment attack mainly on syringil lignin, and not too effective on reduce guaiacyl lignin. Syringyl and guaiacyl bonds remains unchanged after alkaline pretreated bagasse further treated using Streptomyces sp. S2. Both peak around 1300-1200 was reduced after hydrogen-peroxide treatment, although the drop on 1300 peak not as much as the alkaline-hydrotermal treatment. This indicates weaker both syringyl and guaiacyl bonds despite high amount of lignin concentration remains after hydrogen-peroxide pretreatment. Both peak around 1300-1200 cm was even lower after peroxide bagasse treated with Streptomyces sp. S2, resulted in weaker syringyl and guaiacyl bonds ( gure 2). This weaker bonds contribute to signi cant lignin content drop after peroxide bagasse treated with Streptomyces sp. S2, although not signi cant compared to control (table 1) Bands around 1200-800 shows especially, but not limited to hemicelulose, because this bands stacked with cellulose and lignin identi er bands (Gogna and Goacher 2018

Morphological characteristic
Scanning electron photograph of physical-treated (control) sugarcane bagasse show regular and neatly bundled ber ( Figure 3A) with little to no difference of depth ( Figure 3A2). Outer layer of alkalinehydrotermal (NaOH) treated bagasse become unevenly folded, formed fabric-like folding and a big hole between the ber ( gure 3B). Higher magni cation shows the folding in alkaline-hydrotermal treated bagasse are multi-layered (consist of several bundle of ber, gure 3B2). This result was similar to wheat straw treated with 4% NaOH (Qi et al. 2018), despite different concentration and lignocellulose type of ber used. Peroxide pretreated bagasse shown to have rough edges, shallow holes scattered around outer layer of ber bundles and the bundles also have different depth in several area ( gure 3C), compared to control that doesn't have different height of ber bundles. Higher magni cation show more detail of shallow holes formed around the bagasse ber and rough edges formed around the bundles are also multi-layered ( gure 3C2).
Streptomyces sp. degradation creates folding in control bagasse, some deep holes also formed around the bagasse. Bagasse after alkaline and Streptomyces treatment have smooth web-like bers on its outer layer, some deep holes formed also can be seen (red arrows, gure 3E). Smooth layer of bagasse that exposed after alkaline-Streptomyces treatment might indicate lignin on outer surface of bagasse have completely removed (red arrows, gure 3D). Most outer area of bagasse after peroxide -Streptomyces have been completely removed, exposing another layer of hemicellulose and cellulose bundles. Deep holes that are formed by Streptomyces sp.S2 activities still can be seen (red arrows, gure 3F). Removal of lignin on outer layer of both alkaline and peroxide bagasse by Streptomyces sp. S2 is likely through bacterial digging mechanism, when Streptomyces sp S2 form holes and dig out the lignin. This mechanism similar to Cupriavidus basilensis that used to degrade rice straw (Xu et al. 2017) Conclusion Physical treated only sugarcane (control) cannot be further degraded Streptomyces sp. S2 degradation because have high lignin concentration with strong syringil and guaiacyl bonds, cellulose in high ordered crystal lattice, and neatly ordered ber bundle as observed from lignocellulose content measurement, Fourier transform infrared, X-ray diffraction and Scanning electron microscope. Streptomyces sp. S2 can further degrade hydrogen-peroxide pretreated bagasse. Lower lignin percentage, weaker syringyl and guaiacyl lignin were found on peroxide-Streptomyces bagasse compared to hydrogen-peroxide only treated bagasse. Peroxide-Streptomyces treated bagasse also have higher cellulose and hemicellulose content compared to hydrogen-peroxide treated bagasse. Alkaline-hydrotermal pretreatment can signi cantly increase cellulose content, lowering both hemicellulose and lignin, specially syringil lignin. Streptomyces sp. S2 however, cannot further degrade alkaline treated bagasse, despite more smooth bagasse layer after its degradation. Alkaline-Streptomyces bagasse have lower amount of cellulosse, similar syringil and guaiacyl lignin bonds, similar amount of lignin and hemicellulose compared to alkaline-hydrotermal only treated bagasse.

Declarations
Ethics approval and consent to participate Not applicable Consent for publication Not applicable Availibility of data and Materials All data generated or analysed during this study are included in this published article