The Potential of Microbial Symbionts Macrotermes gilvus Hagen Termite Gut as Degrading Agents of Cellulose in Bioethanol Production

Water hyacinth is a potential feedstock for bioethanol production because of their high cellulose. The microbial symbionts of the Macrotermes gilvus termite’s gut have a high endoglucanase enzyme activity. This research was aimed to analyze the pH, temperature and agitation effects towards cell density, endoglucanase enzyme activity and reducing sugar, and to determine the effective optimum condition that can produce maximum reducing sugar. This research used central composite design (CCD) with the total number of run was . The independent variables were including pH (5.9, 6.4, 7.0, 7.6, 8.0), temperature (30 C, 33 C, 37 C, 41 C, 44 C) and agitation (90 rpm,114 rpm, 150 rpm,185 rpm, 210 rpm), with six replications at central points. Parameters measured were cell density, endoglukanase enzyme activity and reducing sugar, thus analyzed by the statistical software package MINITAB 18.0. The Student’s t-test result showed the primary sequence influencing cell density as pH ˃ agitation ˃ temperature and towards endoglucanase enzyme activity and reducing sugar as pH ˃ temperature ˃ agitation, P ˂ 0.05. The maximum reducing sugar (60.13 ± 3.16 mmolL) was obtained at pH 6.95, temperature 37 C and agitation 150 rpm. The results of this research can be used to explore the more potential microbial symbionts of the Macrotermes gilvus Hagen termite’s gut.

This aims of this research were to analyze the pH, temperature, and agitation effects towards cell density value, endoglucanase enzyme activity, and reducing sugar and also to determine the effective optimum condition that can produce maximum reducing sugar.

METHODS Preparation and delignification of water hyacinth biomass
Fresh water hyacinth was collected from a Rawa Pening lake, Ambarawa, Semarang.It is washed to remove impurities.The leaves and stems (7 kg each) were dried separately.Drying is done for ± 7 d.The water hyacinth is chopped and ground using a grinding machine with a 0.6 mm sieve.After that, the applying of delignification as the method of Reales-Alfaro et al. (2013).Then, there was a Chesson (Datta 1981) assay step towards the delignification result filtrate and the last cellulose with amount of 42.24 ± 0.24% was obtained as bioethanol feedstock.

Preparation of the microbial symbionts of the Macrotermes gilvus Hagen termite's gut
Live termites were taken from Semarang State University mini forest.It were collected in a clean jar bottles (Subekti & Febriana 2016).After that one hundred Macrotermes gilvus caste worker termites was sterilized with 70% ethanol (v/v) (Tay et al., 2010).Termite guts were taken aseptically using micro-tweezers and suspended at 1 mL NaCl (0.85%, w/v) (Sharma et al., 2015).The suspension was further centrifuged at 95 g in 1 min (Tay et al., 2010).Then, 0.5 mL aliquots were cultured with 4.5 mL of liquid medium I (Sharma et al., 2015), followed by incubation at 30 0 C for 7 d.Then 1 mL result of spread plate culture on sterile solid medium (Dickerman & Starr 1951), followed by incubation at 30 0 C for 24 h.While microbial screening was performed by culturing the inoculum obtained from spread plate on 50 mL of the second liquid medium (Sharma et al., 2015 modified without CMC) and incubated at 30 0 C for 72 h.

Optimization SSF factors
Filtrates of 1.5 g were added in the er-

INTRODUCTION
Bioethanol is one of potential energy sources that can be developed as renewable fuel and eco-friendly.Current bioethanol development has switched using lignocellulosic biomass, such as water hyacinth (Eichhornia carssipes).It is because the abundant availability of water hyacinth and does not compete with edible and also it has a high cellulose yield (Das et al., 2016).
As a bioethanol feedstock, water hyacinth should be de-lignified to degrade and remove lignin and hemicellulose from cellulose.The next step is saccharification to depolimerate complex sugar into simple sugar.Delignification can dissolve more hemicellulose and increase the amount of cellulose that in the sample of delignification can produce cellulose (35,4%) and hemicellulose (19,6%) while in the sample of non-delignification can only produce cellulose (24,7%) and hemicellulose (32,2%) (Das et al., 2016).It shows that delignification is important, that is why the water hyacinth in this research was delignified with H 2 SO 4 (2%, v/v) under steam at a constant temperature (121 0 C) for 20 min as same as the research of Reales-Alfaro et al. (2013).
The de-polymerization of cellulose into glucose and hemicellulose into glucose, manose, and xilose need complex celullolytic enzyme (Sánchez et al., 2011).It becomes a consideration in developing bioethanol because 40-60% from the total etanol production cost for commercial enzyme purchases (Dhillon et al., 2012).Thus, many researchers tried to reduce the production cost, as a substituting commercial enzyme by microbial symbionts of the Macrotermes gilvus Hagen termite's gut.Macrotermes gilvus Hagen is higher termite which identified have two cellulolytic microbial of termite gut symbionts that have high cellulotic activities, Bacillus megaterium and Paracoccus yeei (Ferbiyanto et al., 2016) On other hand, one of the effort to reduce the production cost by combining sacarification and fermentation steps into single step (simultaneous saccharification and fermentation (SSF)).SSF is more economical comparing with separate hydrolysis and fermentation (SHF) because the time needed to produce reducing sugar is shorter yet still can increase hidrolysis rates thus requiring lower enzyme loadings and also having low rate of contaminating risk (Sudiyani et al., 2014;Scully & Orlygsson 2015).Thus, many factors of SSF, such as pH, temperature and agitation, are urgently necessary to be optimized by central composite design (CCD) to increase enzyme production (Deka et al., 2013).lenmeyer and added 80 mL 0.05 M buffer pH KH 2 PO 4 -NaOH (5.9, 6.4, 7.0, 7.6, 8.0) and 20 mL microbes (30%, v/v) (the microbial symbionts of the Macrotermes gilvus Hagen termite's gut-Saccharomyces cerevisiae, ratio 1:1), those were shaked with shaker for 24 h at temperature (30 0 C, 33 0 C, 37 0 C, 41 0 C, 44 0 C) and agitation (90 rpm, 114 rpm, 150 rpm, 185 rpm, 210 rpm).Then, the suspension (the cell density measurement) was sentrifused at 2400 g for 20 min to seperate filtrate and supernatant (to endoglucanase enzyme activity and reducing sugar assay).

Cell density, endoglucanase enzyme activity and reducing sugar assay
Cell density of the microbial symbionts of the Macrotermes gilvus Hagen termite's gut-Saccharomyces cerevisiae was done by using spectrophotometer UV-Vis (Parkin Elmer, Model lambda-45) at OD 600 nm (Sakolvaree & Deevong 2016).Endoglucanase enzyme activity was done with 0.5 mL cell-free supernatant added with 0.5 mL CMC (1%, w/v) which was dissolved in buffer pH 7.0, and then was incubated at 37 0 C for 30 min.Reducing sugar assay was done based on standard method by using benedict quantitative reagent, and the absorbance measurement was done by using spectrophotometer UV-Vis (Parkin Elmer, Model lambda-45) at OD 477 nm (Sambo et al., 2015).The sample of reducing sugar was obtained by calculating χ value in a formula that was obtained from the measuring of standard solvent (glucose 0 mmolL -1 , 2.5 mmolL -1 , 5 mmolL -1 , 7.5 mmolL -1 and 10 mmolL -1 ).

RESULTS AND DISCUSSION
Water hyacinth biomass as the result of the delignification that was used in this research obtained cellulose 42.24 ± 0.24%.Similar research that was conducted by Reales-Alfaro et al., (2013) was producing less cellulose with amount of 31.67%.The different percentage of that lignocellulose component was reported by Reales-Alfaro et al., (2013) that because the condition of nutrition habitate affect the metabolism process of the plant that generates different chemical characteristic.On the other hand, these differences might caused by the geographic location and growing condition of water hyacinth (Das et al., 2016).
After that, the cellulose was converted into glucose by cellulase enzyme through simultaneous saccharification and fermentation (SSF).In this research, cellulase enzyme was obtained from the microbial symbionts of the Macrotermes gilvus Hagen termite's gut which was optimized on some factors such as pH, temperature and agitation by central composite design (CCD).The advantage of CCD is representatif in showing optimum condition with less run amount (Montgomery 2013).
Actual and prediction values (from the result of data CCD analyzing) for cell density value, endoglucanase enzyme activity and reducing sugar from the result of SSF sample testing, were presented in Table 1.
Based on the Table 1, it showed that the mean of actual value from those three responses had considered small difference towards the prediction value, thus can be indicated that those three responses has valid value.On the other hand, the testing result of Student's t-test showed that the influence of pH and agitation towards cell density value was significantly different while the temperature had influence that was not too different from the value of P ˃ 0.05.Those results mean that the influence of pH ˃ agitation ˃ temperature towards cell density value.Yet the result of Student's t-test for endoglucanase enzyme activity and reducing sugar shows that only pH wich has significant different, while the temperature and agiation indicated insignificance.It means that pH ˃ temperature ˃ agitation, towards endoglucanase enzyme activity and reducing sugar.
The result of the optimization confirmed that pH has most effect towards cell density value, endoglucanase enzyme activity and reducing sugar.It is because pH influences many chemical reaction by affecting chemical product transport and enzyme that are passing through cell membrane (Liang et al., 2010).Similar research about the influence of pH towards cell density and endoglucanase enzyme activity was reported by Deka et al. (2013) and Sakolvaree & Deevong (2016).
The influence of pH towards cell density value could be seen from the maximum respond that reach 3.50 ± 0.13 selmL -1 (prediction value).It showed that microbial symbionts of Macrotermes gilvus termite's gut was optimum on considered neutral pH.That microbes included in neutrophil microbes with the rate of pH around 5.5-8.0.Sakolvaree & Deevong (2016) explained that microbial symbionts of termite gut can live far below its optimum pH and some of bacteria symbionts reported forming endospore which help the bacteria in it to survive and improve their ability to live in high temperature and extreme condition.That is related with the range of termite gut pH where the microbial symbionts breed.Brune & Ohkuma (2011) reported that the pH of Cubitermes termite's gut are crop (pH 4.0), midgut (pH 4.0-7.0),mixed segment (pH 7.0-12.0),pouch (P1) (pH 12.0), P3 (pH 8.0-12.0),P4 (pH 6.0-8.0) and P5 (pH 4.0-6.0).
Cell density value showed the amount of microbes cells that can produce endoglucanase enzyme.That enzyme has a function to hidrolise CMC that has crystaline structure and hard to dissolve in the water and become the source of carbon needed as the energy and ability to growth (Sakolvaree & Deevong 2016).
Besides the influence of pH, agitation is also confirmed more affecting in obvious towards cell density value than the temperature, otherwise Table 1.Actual and prediction value for cell density (selmL -1 ), endoglucanase enzyme activity (UmL - 1 ) and reducing sugar (mmolL -1 )
Saccharomyces cerevisiae in this research was used as fermentative yeast to degraded glucose into ethanol.Fermentative yeast, such as Saccharomyces sp., has growing characterictic on the base of the medium and forming sediment in the growing assay by Sabouraud Broth (SB) medium (Jumiyati et al., 2012).The limitation of Saccharomyces as fermentative yeast is only be able to degrade reducing sugar such as, glucose, fructose, and sucrose into ethanol, acetate, lactate acid and many others (Scully & Orlygsson 2015).While Chansoliya et al. (2016) explained that Saccharomyces cerevisiae was reported is more potential to be used as fermentation microbes than Zymomonas mobilis.

CONCLUSION
The result of the research shows that pH is really influencing towards the amount of cell density which is asociated with endoglucanase enzyme activity and reducing sugar.Agitation is more affecting after pH towards the amount of cell density than temperature, and so the otherwise for endoglucanase enzyme activity and reducing sugar.It is corresponding with the optimizing result of optimum condition that showed the maximum amount of reducing sugar which was 60.13 mmolL -1 in pH 6.95 at temperature (37 0 C) and agitation (150 rpm).

ACKNOWLEDGEMENT
Thank you for the Research, Technology, and Higher Education Ministry which has been funding and facilitating the implementation of this research through students creativity programe 2015.

Figure 1 .
Figure 1.Interaction of (a) pH and temperature, (b) pH and agitation, (c) temperature and agitation towards the value of reducing sugar (mmolL -1 ) on a maximum point