Optimizing the impact of temperature on bio-hydrogen production from food waste and its derivatives under no pH control using statistical modelling

The effect of temperature on bio-hydrogen production by co-digestion of sewerage sludge with food waste and its two derivatives, i.e. noodle waste and rice waste, was investigated by statistical modelling. Experimental results showed that increasing temperature from mesophilic (37 C) to thermophilic (55 C) was an effective mean for increasing bio-hydrogen production from food waste and noodle waste, but it caused a negative impact on bio-hydrogen production from rice waste. The maximum cumulative biohydrogen production of 650 mL was obtained from noodle waste under thermophilic temperature condition. Most of the production was observed during the first 48 h of incubation, which continued until 72 h of incubation. The decline in pH during this interval was 4.3 and 4.4 from a starting value of 7 under mesophilic and thermophilic conditions, respectively. Most of the glucose consumption was also observed during 72 h of incubation and the maximum consumption was observed during the first 24 h, which was the same duration where the maximum pH drop occurred. The maximum hydrogen yields of 82.47 mL VS, 131.38 mL COD, and 44.90 mL glucose were obtained from thermophilic food waste, thermophilic noodle waste and mesophilic rice waste, respectively. The production of volatile fatty acids increased with an increase in time and temperature in food waste and noodle waste reactors whereas they decreased with temperature in rice waste reactors. The statistical modelling returned good results with high values of coefficient of determination (R) for each waste type and 3-D response surface plots developed by using models developed. These plots developed a better understanding regarding the impact of temperature and incubation time on bio-hydrogen production trend, glucose consumption during incubation and volatile fatty acids production.


Introduction
Anaerobic digestion , as a waste management approach, has been in practice for more than a century (McCarty, 1981).It is widely used to treat a variety of solid wastes and wastewater on a small scale as well as on an industrial scale.It has multiple advantages like 30-50 % reduction in waste volume as well as production of valuable byproducts such as methane and hydrogen (Lin et al., 2011).A large amount of organic fraction of municipal solid waste or food waste is produced every year around the world.During 2010, production of food waste in China reached 352 Mt and the major contributor was canteens and restaurants (Tai et al., 2011;Wang et al., 2013).The food waste contains more than 80 % volatile solids (VS) that are biodegradable solids and can be converted into hydrogen or methane easily (Shin et al., 2004;Zhang et al., 2007;Zhu et al., 2008).Several studies represent an increase in bio-hydrogen production from food waste due to the addition of buffers and minerals.Although such C. Arslan et al.: Optimizing the impact of temperature on bio-hydrogen production addition can maintain a specific pH and nutrimental required for optimum bio-hydrogen production, but it also increases the cost of production (Nielsen et al., 2001;Han, 2004).The cost of production can be reduced by adding sewage sludge as a source of Clostridium mix culture (Fang et al., 2006).Nutritional deficiency in food waste was also balanced by adding sewage sludge that made food waste more suitable for bio-hydrogen production (Shin et al., 2004).It means that integrated waste management can be done at a wastewater treatment plant by co-digestion of sewage sludge and food waste.Although sewerage sludge is a good source of Clostridium mix culture, it also contains hydrogen consumers like methanogens.Heat treatment is mostly opted to deactivate hydrogen consumers.The traditional method of placing sewage sludge in boiling water is now no longer in practice and is replaced by microwave heating that provide more uniform heating as compared to the boiling water method (Luo et al., 2010;Wang et al., 2011;Duangmanee et al., 2007).The temperature and time for heat treatment varied from 75 to 121 • C for 15 min to 2 h, but 100 • C for 15 min was mostly reported (Li and Fang, 2007;Fang et al., 2006).
Carbohydrate-rich wastes like food waste are suitable for Clostridium species as stoichiometrically it can produce two moles of hydrogen from one mole of hexose (Payot, 1998).Theoretically, 553 mL hydrogen can be produced by 1 g of polysaccharides if it is totally converted into acetate.The highest practical yield of 346 mL g −1 carbohydrate was achieved by Fang et al. (2006) by using rice as a source of carbohydrate (78 %), pre-treated sewage sludge as a source of Clostridium and adding a variety of nutrients.Rice waste and noodle waste has 40 % share in the total food waste produced in China (Shiwei, 2005).The noodle waste is also rich in carbohydrates, but still there is no research reported on bio-hydrogen production from noodle waste.
Temperature and pH have a great impact on the smooth running of AD (Saraphirom and Reungsang, 2010).Most of the studies reported bio-hydrogen production under mesophilic as well as thermophilic conditions and few were reported under psychrophilic conditions.Lu et al. (2011) developed microbial electrolysis cells (MECs) that could be operated at 9 • C by using Geobacter psychrophilus as dominating population and achieved a hydrogen yield of 0.62 m 3 H 2 m −3 d −1 .Heidrich et al. (2013) further modified MECs to a pilot-scale MEC and achieved bio-hydrogen production of 0.015 LH 2 L −1 d −1 at 25 • C. On the other end, under mesophilic and thermophilic conditions, there is no need for such sophisticated technology and a better bio-hydrogen yield can be achieved by simple reactors or by lab scale batch experiments.The temperature shift from mesophilic to thermophilic conditions can change the rate of hydrogen production during anaerobic digestion (Li and Liu, 2012;Saripan and Reungsang, 2014).Whereas hyper-thermophilic provide better pathogenic destruction but it also decreased the bio-hydrogen production (Sahlström, 2003;Yokoyama et al., 2007).Keeping the same temperature but changing the initial pH from 7 to 8, the bio-hydrogen yield was changed from 64.48 to 55 mL VS −1 under no pH control conditions (Lin et al., 2013b;Nathao et al., 2013).The same yield was increased to 70 mL VS −1 when pH was manually controlled for food waste under thermophilic conditions, which represents the impact of pH management (Shin et al., 2004).The hydrogen production by anaerobic digestion will be further improved if pH lies in the range of 5 to 6 (Radjaram and Saravanane, 2011).The pH of food waste lies in the range of 4 to 5, which further decreases by the production of volatile fatty acids (VFA) to such a level that can inhibit the bacterial growth.The pH can be controlled by automatic pH controllers, addition of nutrients and buffers, manual monitoring and control (Yasin et al., 2011;Zhu et al., 2008;Kim et al., 2004).But all these methods increased the cost of operation.Along with cost, maintaining pH at specific point is not suitable especially when mix culture is used as the response of different microbial stream could be different to same pH level.So, by co-digestion, the pH of the anaerobic digestion process can be improved and it can be further adjusted to a desired initial value by adding HCl or NaOH.After adjusting the desired initial pH under co-digested conditions, the biohydrogen production can be achieved under no pH control conditions, which can reduce the cost of operation (Fang et al., 2006).
The yield of bio-hydrogen is calculated by dividing the cumulative hydrogen produced by VS, chemical oxygen demand (COD) or glucose (Chen et al., 2006;Dong et al., 2009;Fang et al., 2006).The yields are misleading if calculated in terms of added or start up values of VS, COD and glucose as it seems quite impossible that the whole of added material is converted into hydrogen.In this regard the removal quantities of such parameters are the best option to calculate the yield.
The optimization played an important role in bio-hydrogen production and its application with respect to incubation time in combination with temperature is an important factor to get the maximum output with minimum intake of energy.In order to achieve this purpose, statistical modelling is an important tool to study the impact within the experimental range and can be further used for the development of response surface plots (Jo et al., 2008).
This study was designed to investigate the impact of temperature on bio-hydrogen production from co-digestion of sewerage sludge with food waste and its carbohydrate-rich derivatives i.e. rice waste and noodle waste with the help of statistical modelling.The response surface methodology was employed to study the impact of time and temperature on bio-hydrogen production, glucose consumption and VFA production.The pH during incubation was not controlled and the drop of pH during anaerobic digestion was also studied to find an optimum pH range of bio-hydrogen production from food waste derivatives.

Batch experiment for bio-hydrogen production
The waste was collected from student dining at the Nanjing Agricultural University.The food left on the plates after lunch/dinner consisted of rice, noodles, meat, bones, potato and other vegetables.At first, bones and other foreign materials were removed and left over waste was treated as food waste.The food waste was then ground in a meat grinder with an equal amount of water and a resultant slurry was used for bio-hydrogen production (Reungsang et al., 2013).Rice and noodles were removed from collected waste and converted into slurry in the same way opted for food waste.The sludge was obtained from a settling channel and it was washed with tap water and sieved to remove foreign materials (Nathao et al., 2013).The sludge was placed in a preheated oven at 100 • C for 15 min, so that hydrogenotrophic methanogens could be deactivated (Li and Fang, 2007).Some important properties of feed stock and sewerage sludge are enlisted in Table 1.
Two series of experiments were conducted in duplicate in 550 mL digesters with working volume of 400 mL (Hu et al., 2014).In order to achieve 10 % initial TS concentration, water was added along with feedstock and sewerage sludge in the digesters.The feedstock and sewerage sludge were added in equal proportion.As the pH of food waste was not so high even after co-digestion with sewage sludge, the initial pH within reactor was less than 7 which was carefully raised to 7 with the help of 3M NaOH solution (Zhu et al., 2008).Series I was to observe the bio-hydrogen production under mesophilic temperature (37 • C) and series II was to investigate the impact of thermophilic temperature (55 • C) on biohydrogen production potential of feed stock in comparison with that produced under mesophilic conditions.

Chemical analysis
The volume of bio-hydrogen produced was measured by displacement of 3 % NaOH solution.This concentration of NaOH can remove other gases and water vapours (Fang et al., 2006;Lin et al., 2013a;Saraphirom and Reungsang, 2010).Other parameters like TS, VS, COD, VFA and alkalinity were measured according to standard methods (APHA, 2005).For glucose detection, phenol sulphuric acid method was used (Lay and Fan, 2003).

Modelling of hydrogen production was done by Modified
Gompertz equation (MGE), which was used for cumulative bio-hydrogen measurement (Ramos et al., 2012) where H , t, P , R m , λ and e represent cumulative bio-hydrogen production (mL), incubation time (h), biohydrogen production potential, maximum bio-hydrogen production rate (mL h −1 ), lag phase duration (h) and 2.71828 respectively.The values of H , t, P , R m were solved by using curve fitting tool in (ver.2010a).
In this study, the effects of different types of food waste, temperature and incubation time on bio-hydrogen and VFA production as well as on glucose consumption, were analysed by full quadratic model as shown below (Kim et al., 2008;Jo et al., 2008) where X i and X j are the controlled parameters, which influence Y and a 0 , a ii , a ij , are the offset term, linear and quadratic coefficients respectively.As the waste types are different, so the above model is used, including waste type (n = 3) and excluding waste type (n = 2).

Effect of temperature and time on bio-hydrogen production
A comparison of actual and MGE modelled bio-hydrogen production under mesophilic and thermophilic conditions is shown in Fig. 1, which shows an early start of bio-hydrogen production in food waste and noodle waste as compared to rice waste.This early production was quantified with the help of MGE as λ shown in Table 2, which clearly shows that the highest lag phase belongs to rice waste under both temperatures.The bio-hydrogen production in food waste continued until 72 h of incubation and this time period for food waste was higher than that observed for noodle waste, but still the cumulative bio-hydrogen production of food waste was the lowest as compared to the other two waste types.Looking at the R m values, it is clear that food waste has the lowest mesophilic R m value of 6.688 mL h −1 among all three wastes that ultimately caused the cumulative bio-hydrogen production to decrease.Only the R m value is not responsible for higher yield as the highest mesophilic R m of 21.05 mL h −1 belongs to noodle waste, but the cumulative bio-hydrogen production of noodle waste was found to be smaller than rice waste having R m value of 16.52 mL h −1 .The rice waste has a higher production because it produced bio-hydrogen for 96 h as compared to noodle waste which produced until 60 h.In fact, mesophilic bio-hydrogen production in noodle waste decreased considerably after 24 h of incubation as compared to thermophilic bio-hydrogen production where the decrease was observed after 36 h of incubation.That is close to each other.It can be seen in Fig. 2 that the difference in active duration was due to pH as it was dropped from 7 to 4.6 in the noodle waste reactor during the first 12 h under mesophilic temperature and it was further dropped to 4.4 whereas a pH drop in thermophilic reactor was 5.3 to 4.9 in the same duration.On the other end, pH drop in rice waste reactor under thermophilic conditions was higher as compared to mesophilic temperature, which ultimately reduced the cumulative bio-hydrogen production that was found in agreement with the finding of Fang et al. (2006).The reduction in bio-hydrogen production with low pH is due to homoacetogenic bacteria, which are more active at low pH (Ramos et al., 2012;Schiel-Bengelsdorf and Dürre, 2012).The trend for pH drop with temperature in food waste reactors was opposite to other reactors and R m increased to a value of 26.42 mL h −1 when temperature was shifted from mesophilic to thermophilic.This increase is due to Thermoanaerobacterium thermosaccharolyticum that grow at higher temperature in food waste and produce more hydrogen (Shin et al., 2004).As a whole, R m values found in the present study were higher than those reported in previous studies (Fang et al., 2006;Ramos et al., 2012).The volume of bio-hydrogen production with time was used to fit in a quadratic model by using solver function MS Excel and the resultant equation obtained was where Y is the predicted bio-hydrogen production; x 1 , x 2 and x 3 are the coded values of incubation time, waste type and temperature respectively.There is a poor relationship between actual and predicted value as the coefficient of determination (R 2 ) was calculated to be 0.5576, which can explain only 55.76 % variability of the response.The diversity among waste type is the main reason for such a low value and this value could be at a higher level if the same waste was used in different proportions as reported in other studies (Kim et al., 2008;Jo et al., 2008).To overcome this problem, quadratic model was again developed for each waste type and the following equations were obtained: Food waste: Noodle waste: Y = 472.5 + 97.5x 1 + 42.5x 2 Rice waste : where x 1 and x 2 are the coded values of time and temperature.All F values represented that the regression model obtained are statistically significant.The F value of food waste  is much higher as compared to other values; this is because of the coefficient of determination that explained 98.58 % variability.The quadratic model obtained from rice waste was not perfectly fit, but it can better explain results as compared to the model developed for all waste types as a whole.
The three-dimensional (3-D) response surfaces and twodimensional (2-D) contours were developed within the experimental range for each waste type by taking bio-hydrogen production as a response by using the above-mentioned equations.The 3-D and 2-D curves of the calculated response showed the interaction of incubation time and temperature in Fig. 3a-c.For food waste, it is clear that the gas production increases with time and temperature from 115 mL at the starting end to 354 mL at the extreme modelled conditions.Between 0 and 24 h of incubation, bio-hydrogen production increased with an increase in temperature for food waste, i.e. 115 mL of bio-hydrogen was produced at 37 • C that increased to 76.09 and 152.17 % at 46 and 55 • C, respectively.During the next 24 h of incubation, bio-hydrogen production reduced with the increase in temperature, i.e. 114.5 mL biohydrogen was produced at 37 • C and 30.78 and 91.22 % reduction was observed at 46 • C and 55 • C, respectively.Even after reduction in bio-hydrogen production between 24 and 48 h of incubation, the cumulative bio-hydrogen production increased with an increase in temperature from food waste.The impact of temperature and time can be better viewed in 2-D contour (Fig. 3a), which shows that the increase in temperature increases bio-hydrogen production more at 24 h as compared to 72 h of incubation.It also revealed the fact that the first 24 h are important for bio-hydrogen production from food waste under thermophilic temperatures and the next 24 www.biogeosciences.net/12/6503/2015/Biogeosciences, 12, 6503-6514, 2015 are important for production under mesophilic temperature, which is in agreement with findings of Shin et al. (2004).
Although noodle waste also produced more bio-hydrogen at elevated temperature, the time effect was opposite to that observed for food waste.The bio-hydrogen production in noodle waste during 0-24 h was 350 mL at 37 • C that was 5.4 and 10.81 % decreased at 46 and 55 • C, respectively.But in the next 24-72 h, there was an increase of 178.57 and 357.14 % at 46 and 55 • C, respectively .As far as rice waste was concerned, temperature has a negative impact on bio-hydrogen production.Between 24 to 48 h, the bio-hydrogen production in rice waste was 131 mL at 37 • C, 114.5 mL at 46 • C and 98 mL at 55 • C. In next 24h, the bio-hydrogen production was reduced to 65.65 %, 75.11 % and 87.76 % at 37 • C, 46 • C and 55 • C, respectively.The reduction in bio-hydrogen production for rice waste was in agreement with previous findings (Fang et al., 2006).The 2-D contours in Fig. 3b and c differentiate the impact of temperature with time on bio-hydrogen production for noodle waste and rice waste as the contour patterns are quite opposite to each other.

Effect of temperature on bio-hydrogen yield
The bio-hydrogen yield was calculated by dividing the P value on Table 2 with VS fed , VS removed , COD removed and glucose removed .The bio-hydrogen yield calculated on the basis of VS fed lay in the range achieved by Lin et al. (2013b) and temperature impact on yield was the same as observed for P .The yield on the basis of VS consumed represented uptake efficiency of VS during anaerobic digestion.The increase in P with temperature for food waste was 23.41 % whereas the yield increased by 2.86 % only that indicated the efficient removal of VS at higher temperature.The biohydrogen yield calculated for FW on the basis of VS removed lay in the range used by Kim et al. (2004).Using the same scale of VS removed , bio-hydrogen yield for rice waste decreased 47.37 % with an increase in temperature, whereas the decrease in P was 78.81 % that represented a decrease in removal of VS at elevated temperature which was in agreement with the findings of Fang et al. (2006).As compared to food waste and noodle waste, the increase in P and yield calculated by VS removed was close, but it revealed the fact that VS removal efficiency slightly decreased at an elevated temperature.
When the yield measuring scale was shifted from VS removed to COD removed , the results represented quite a different picture of temperature impact.The increase in temperature from 37 to 55 • C increased 42.41 % bio-hydrogen yield calculated on the basis of COD removed for food waste.The increase in bio-hydrogen production due to same increase in temperature from 37 to 55 • C was 23.37 %.Such a difference in yield and production increment represented a decrease in COD removal efficiency at an elevated temperature for food waste.For rice waste, the decrease in yield was 61 %, which was close to a 78 % decrease in P .Increasing temperature also increased the yield for noodle waste to 20 %, which was smaller than the increase in P , representing a higher rate of COD removal at elevated temperature.All the bio-hydrogen yields calculated on the basis of COD removed lay in the range calculated by Tawfik and El-Qelish (2014).
Glucose removal efficiency for food waste decreased with an increase in temperature as the increase in P was 23.41 against 42.19 % when bio-hydrogen yield was calculated on the basis of glucose removed .Whereas the change in yield for noodle waste and rice waste was close to the change observed for P .The decrease in glucose concentration was close to that observed in previous studies (Abdeshahian et al., 2014;Kapdan and Kargi, 2006).The yield calculated on a glucose basis was further studied on a daily basis and it was observed that the highest yield of 33 mL glucose −1 removed for 0-24 h duration belonged to noodle waste under mesophilic condition.During the next 24 h period, the highest yield of 400 mL glucose −1 removed was achieved by noodle waste under thermophilic temperature, which was close to the finding of Fang et al. (2006) but still smaller than the theoretical yield of 553 mL g −1 carbohydrate.The yield for rice waste also increased under both temperatures but it was much higher at mesophilic as 184.37 mL glucose −1 removed against 24.99 mL glucose −1 removed at thermophilic temperature.During 24 to 72 h of incubation, the yield in all reactors reduced except noodle waste under mesophilic conditions.As a whole, 24-48 h duration of incubation was found to be more important for bio-hydrogen production from glucose.The production of glucose modelled by quadratic equation using previ-ously defined notation as As the coefficient of determination is not so high so quadratic modelling was done for each waste type as Food waste : Y = 7.820 − 3.561x 1 + 0.412x 2 The 3-D response plots and contours for glucose removal were developed by the above models (Fig. 5).It was observed that in the first 24 h of incubation, the rate of utilization of glucose increased, with an increase in temperature up to 55 • C for food waste, decreased for noodle waste, and remained almost unaffected for rice waste.The sequence for glucose utilization rate was in the rank of NW > FW > RW.
Between 24 and 48 h, glucose utilization rate decreased for food waste and increased for noodle and rice waste under mesophilic and thermophilic conditions.Between 48 and 72 h, rate of utilization remained the same as previous one but rank was slightly changed as FW > RW > NW.With an increase in temperature, between 24 and 72 h, the rate of glucose utilization decreased for food waste but increased for noodle and rice waste.As a whole, the glucose consumption at the end of incubation was higher at 37 • C as compared to 55 • C for food waste.At the end of incubation, noodle waste and rice waste represented quite opposite picture of glucose consumption with temperature as observed for food waste.
The contours represented a better understanding for glucose consumption and the contour varied in different manners for each waste type as shown in Fig. 5.

Effect of temperature on VFA production
The VFA revealed an increase with time as reported by Lin et al. (2013b), which is shown in Fig. 6.In the present study, it is observed that VFA in food waste and noodle waste increased with an increase in temperature from 37 to 55 • C but decreased for rice waste that lay in the range calculated by Shin et al. (2004).It can be seen in Fig. 6 that between 24 and 48 h, an increase in VFA was much higher in food waste under thermophilic conditions as compared to mesophilic conditions.During the same interval, bio-hydrogen production almost ceased in the thermophilic food waste reactor whereas it was continuously producing in mesophilic food waste reactor.One of the possible reasons for this reduction is the conversion of glucose to VFA at this stage by homoacetogenic bacteria that reached up to such a level where bio-hydrogen production was not feasible under thermophilic conditions, whereas the VFA production in the mesophilic FW reactor was much smaller than that observed under thermophilic reactor, because of which production continued in the mesophilic reactor (Zhang et al., 2014).The higher concentration of VFA together with low pH can be inhibitory to bacteria that can cause unfavourable physical changes in the cell.By such physical changes, excessive energy is required to pump ions and that energy can be available at a higher temperature.So it increased the yield at elevated temperatures, as observed in the case of food waste and noodle waste (Gottschalk, 1986;Zoetemeyer, 1982;Switzenbaum, 1990).The higher concentration of VFA can also be used as an indicator for higher production of bio-hydrogen as observed by Dong et al. (2009).In the present study, the order to VFA production and cumulative bio-hydrogen production was the same i.e.NW 55 The quadratic model was tried to fit for VFA production data in the same way as opted for glucose and the resultant equation was as follows: Here, again the coefficient of determination is not so high due to the variability of waste type, so the model was repeated for each waste as The 3-D response plots and contours for VFA production on the basis of the above models are shown in Fig. 7.The 3-D contours for food waste and noodle waste seem almost same but the contour lines for both varied in a different manner.Although the production of VFA increased with time and temperature in all reactors but the intensity of change is different for each waste type as observed in Fig. 7.It can be observed from Fig. 7 that the production of VFA has increased for food waste and noodle waste when temperature was increased from 37 to 55 • C.Although with time, the VFA concentration increased, but the rate by which VFAs produced was decreased with time, i.e.VFA production between 24 and 48 h was greater than that produced between 48 and 72 h and this trend continued until 120 h for food waste and noodle waste.As a whole, more VFA was produced between 24 and 120 h under mesophilic temperature as compared to thermophilic temperature in food waste reactor, but as a whole, mesophilic VFA production was found to be less than that of thermophilic as reported by Gadow et al. (2012).It is because of the fact that between 0 and 24 h duration, thermophilic VFA production was much higher than that produced under mesophilic conditions in food waste.By controlling the VFA production during this interval, the yield of bio-hydrogen can be increased for FW as it stopped too early in thermophilic food waste reactor as compared to mesophilic food waste reactor.Thermophilic VFA production was higher than mesophilic VFA production in noodle waste reactor.On the other end, mesophilic VFA production was higher than that produced under thermophilic conditions for RW and VFA increased with time in the same manner as observed for food waste under 40 • C. Between 40 and 55, • C, the VFA trend for rice waste remained the same as of food waste until 96 h after which it started to decrease until 120 h.

Conclusion
Food waste and its two major derivatives, i.e. noodle waste and rice waste, were co-digested with sewerage sludge to produce bio-hydrogen with an initial pH of 7 under mesophilic and thermophilic conditions.The pH was not controlled throughout the incubation.The most effective VS removal was observed in noodle waste reactor that produced the highest experimental cumulative bio-hydrogen of 656.5 mL under thermophilic conditions.The food waste possessed the highest bio-hydrogen yield calculated on the basis of VS removed that represents an efficient conversion of VS into bio-hydrogen.The increase in temperature within the studied range increased the bio-hydrogen production in food waste and noodle waste reactors.The rice waste reactor represented the negative impact of increasing temperature on bio-hydrogen and VFA production.Thermophilic conditions should be preferred for bio-hydrogen production as most of the time food waste is used as feed stock.The quadratic modelling returned good results that were close to experimental ones, when it was done for each waste type of bio-hydrogen, VFA production and glucose removal.The response surface plots and contour plots within the experimental range adequately explained the effect of temperature and time on studying parameters and helped to develop better understanding regarding the variation among the studied parameters especially when the different treatments represented similar trends.VFA production in rice waste reactor changes the trend after 40 • C that was identified only due to quadratic modelling.The lowest limit of pH for bio-hydrogen production was identified as 4.3 and 4.4 for mesophilic and thermophilic temperatures respectively.
Author contributions.C. Arslan and A. Sattar, designed the research and performed all the lab works.C. Ji provided the financial and technical support for designing and conducting research as well as supervised the whole research process.S. Sattar developed and customized 3-D surface plots and assisted in manuscript preparation.K. Yousaf assisted the lab works and analysis.S. Hashim performed the statistical analysis.C. Arslan wrote the manuscript with comments from all authors and A. Sattar finalized the manuscript under the supervision and guidelines of C. Ji.

Figure 1 .
Figure 1.Mesophilic and thermophilic bio-hydrogen production with time.

Figure 2 .
Figure 2. Effect of temperature on pH drop during incubation.

Table 1 .
Properties of test materials.

Table 2 .
Kinetic parameters and bio-hydrogen yield.

Table 3 .
Comparison of bio-hydrogen yield.