Improving sewage sludge compost process and quality by carbon sources addition

In present study, the effects of carbon sources on compost process and quality were evaluated in the lab-scale sewage sludge (SS) composting. The composting experiments were performed for 32 days in 5 L reactors. The results showed that carbon sources could change the nitrogen conversion and improve the compost quality. Especially, the readily degradable carbon source could promote organic matter degradation, improve nitrogen conversion process and accelerate compost maturation. The addition of glucose and sucrose could increase dissolved organic carbon, CO2 emission, dehydrogenase activity, nitrification and germination index during the SS composting. That's because glucose and sucrose could be quickly used by microbes as energy and carbon source substance to increase activity of microbes and ammonia assimilation. What's more, the NH3 emission was reduced by 26.9% and 32.1% in glucose and sucrose treatments, respectively. Therefore, the addition of readily degradable carbon source could reduce NH3 emission and improve compost maturity in the SS composting.


Materials and methods
Composting system. The dewatered SS were collected from a local sewage disposal plant in Harbin, China. Pumice was used as bulking agent, which was a light volcanic rock with no organic matter (OM). First 10 kg dewatered SS were mixed with 5 kg pumice, and then the mixture was divided into five parts. One of them was set as the control treatment without extra carbon source, and other four treatments were added 4% (carbon source/SS) sucrose, glucose, cellulose and starch. The composting material of each treatment was mixed evenly before the composting begins and put into respective reactors. The characteristics of raw materials were shown in Table 1. The reactors consisted of plastic cylinders with inner diameter 300 mm and height 600 mm respectively, and the other description were reported in previous literature 5 . In order to reduce the heat loss during composting, the reactors were put into a water tank whose temperature was set below 1∼3 °C of contol treatment 20 . The air was ventilated from the bottom of reactors by a air pump with 0.4 L/min aeration rate.
Sampling protocol. The compost experiment was conducted for 32 days, and the samples were collected on Day 1, 3, 5, 7, 9, 13, 18, 23 and 32, respectively. The composting samples were collected from upper, middle and lower part of every reactor using the methods of quatering. The three 10 g samples were combined into one composite sample, after mixed thoroughly, the composite sample was divided into two parts on average. The first part was used to determine the moisture content through drying by an oven, and then the dried samples were used to determine organic matter (OM) content. The second part was stored at 4 °C for the measurement of dissolved organic carbon (DOC), DHA, NH 4 + , NO 3 − and germination index, and all the samples were carried out in triplicate for variance analysis.
Analysis methods. The OM contents were determined by measuring the loss of dry-solid mass after igniting at 550 °C for 5 h in a muffle furnace. The exhausted gas was obtain through a aluminium sampling bag everyday used the method of Maulini et al. 23 . The NH 3 and CO 2 were trapped by boric acid and natrium hydroxydatum solutions, respectively, then determined by titration 24 . The fresh sample was mixed with distilled water at 1:10 mass ratio (samples:distilled water) and oscillated in a shaker for 1 h. Then the mixture was centrifuged at 12,000 rpm for 5 min and the supernatant was filtered by 0.45 μm filter membrane to obtain the water extracts of sample 25 . The DOC content in the water extracts was measured by a Biotector TOC-B7000 TOC analyser (Hach, America). The DHA of sample was determined by the methods of Tiquia 26 . NH 4 + and NO 3 − were extracted in a 2 M KCl (sample at 1% mass ratio) and determined by colorimetry methods refer to the reports of Belyaeva and Haynes' 27 . The germination indexs (GI) were measured by pakchoi seeds and water extracts, twenty pakchoi seeds were distributed on the filter paper in a sterile dish (9 cm diameter), then 5 mL of the compost water extract was added to the filter paper and incubated at 20 ℃ for 3 days in dark. The computational method according to the author's previous method 5 . The data revealed in present paper were obtained from the average of three parallel samples and then were calculated by Microsoft Excel 2017 and the analyses of statistical were completed by SPSS 16.0.

Results and discussion
Organic matter and dehydrogenase activity. The degradation rate of OM directly reflects the metabolism velocity of the microorganisms during the composting process 28 . The initial OM contents in carbon source treatment were higher than that in control treatment which was 61.3% (Fig. 1a). The OM contents in carbon source treatments were in the range of 65.3-67.3%, which showed the difference among them was not significant. The OM degraded rapidly of all treatments during the thermophilic phase of composting, and the OM contents dropped to 45.8% (control), 44.8% (glucose), 47.1% (sucrose), 49.2% (starch) and 51.3% (cellulose) respectively at the end of the thermophilic phase (Day 13). During mesophilic phase and thermophilic phase, the loss of OM contents in the control treatment and the cellulose treatment were 15.5% and 15.4%, respectively. However, the losses in glucose and the sucrose treatment were higher than the other treatments, were 21.9% and 20.2%, respectively. Moreover, the loss of starch treatment was slightly higher than that in control treatment was 16.1%. That's because the glucose and sucrose belong to easily degradable carbon sources which were easy to be used by microorganisms, could increase the activity of composting microorganisms and promote the degradation of OM. Contrarily, owing to the complex macromolecular structure of cellulose that was not easy to be used by microorganisms, the addition of cellulose has the least impact on the degradation rate of OM 11 . At the end of composting, the lowest OM degradation rates also appeared in the glucose and the sucrose treatment, which www.nature.com/scientificreports/ were 37.6% and 38.4%, respectively. However, the differences between the other three treatments were not significant in the range of 40.1-41.2%. Obviously, the addition of glucose and sucrose would promote the metabolic activities of microorganisms and accelerate the degradation of OM in composting. Dehydrogenase is a collective name for a series of metabolic reaction enzymes that catalyze the degradation of OM to produce ATP inside the microbial cells 29 . Therefore, DHA has been recognized as an important parameter that can react to the speed of biochemical reactions during composting 30 . The evolution of DHA is shown in Fig. 1b, the DHA of each treatment was extremely sensitive to temperature, what's more, they showed a positive correlation 31 . The DHA increased in the mesophilic phase and thermophilic phase in all treatments, and reached their peak values in the thermophilic phase, then the DHA gradually decreased in the cooling phase until the end of composting. The results showed that the addition of carbon source significantly increased the DHA, and the different raise between types of carbon sources were significant. The DHA of glucose treatment reached peak on Day 7 and the peak value of 2.35 mg TPF/g h, while the other four treatments reached the peak on Day 9. The peak value of sucrose treatment was 2.44 mg TPF/g h that was the highest, followed by the starch, cellulose and control treatment which were 1.92 mg TPF/g h, 2.13 mg TPF/g h, and 1.45 mg TPF/g h, respectively. Nikaeen et al. 30 also reported that DHA had been increasing during the initial phase of composting, and then gradually decreased with decreasing temperature in the cooling phase. Previous reports had confirmed that DHA showed positive correlation with the culturable microorganisms in composting 5 , therefore, the addition of carbon sources could provide energy materials for the compost microorganisms, promote the microorganisms growth and improve DHA in compost. Similar phenomenon had been found by Zhang et al. 32 , who reported that the nutrients in the bulking agent could promote the metabolism of compost microorganisms and increase their biological activity and biomass. In present study, as readily degradable carbon source glucose and sucrose could be quickly used by microorganisms, so the DHA were stronger than those in other treatments, this results was consistent with previous reports 33,34 . Dissolved organic carbon and CO 2 emission. DOC mainly includes small molecule, simple structure and water soluble carbon source materials, so it is easily used by microorganisms to participate in the biochemical reaction of composting 35 . The change of DOC in the composting could indirectly reflect the metabolism of compost microorganisms, and DOC had a certain relationship with the degradation of OM and the maturity of the compost product, so DOC could also be used to evaluate the stability of composting product 36 . The evolutions of DOC in five treatments are showed in Fig. 2a, the differences of initial DOC concentrations between five  www.nature.com/scientificreports/ treatments were significant, which was closely related to the type of extra carbon source. The concentrations of DOC of the glucose and sucrose treatments were significantly higher than those of other treatments, because the glucose and sucrose were easily degradable carbon source, and their water solubility directly led to the increase in DOC concentration. However, the initial DOC concentration of starch treatment was lower than that of the glucose and sucrose treatments, and cellulose and control treatments had the lowest initial DOC concentrations, because the larger molecular structures in starch and cellulose could not be easily soluble in composting 20 . In the early stages (Day 1-7) of composting, the DOC concentration of all treatments decreased rapidly, especially in the glucose and sucrose treatments, which decreased from 38.6 mg/g and 37.5 mg/g to 22.6 mg/g and 23.4 mg/g, respectively. And the decreases were 16.0 mg/g and 14.1 mg/g, which were much higher than those of other treatments. In addition, the DOC concentrations of glucose and sucrose treatments experienced a slight increase on the Day 7-9 in thermophilic phase, this might be because the readily degradable carbon source promoted the degradation of the original OM of the SS composting and the release rate of DOC from OM degradation was higher than the degradation rate of it. The degradation of DOC in the cellulose treatment was similar to that of the control treatment, which was fast in the mesophilic phase and thermophilic phase of the compost. The DOC concentrations of all treatments remained stable during the cooling phase, and the final DOC concentration was in the range of 18.2-23.9 mg/g. CO 2 was generated during the degradation of OM by microorganisms in the composting, so the release rate of CO 2 could reflect the activity of microorganisms and the degradation rate of OM 37 . The evolutions of the CO 2 emission in the five treatments are shown in Fig. 2b. The change trends of CO 2 emission in all treatments were similar, but the amounts of CO 2 emission were significantly different. All the amounts of CO 2 emission increased rapidly in mesophilic and thermophilic phase, and reached their respective peaks on the Day 8 of composting. Thereinto, the biggest CO 2 emission amount was 536.3 mg/kg/48 h in glucose treatment, followed by 489.6 mg/ kg/48 h in sucrose treatment, 401.2 mg/kg/48 h in starch treatment, 330.6 mg/kg/48 h in cellulose treatment and 269.5 mg/kg/48 h in control treatment. After the peaks, the amount of CO 2 emission in each treatment gradually decreased until the end of composting, and only a small amount of CO 2 was volatilized during Day 20-32 of composting. Obviously, the addition of extra carbon source promotes the CO 2 emission during the composting process. What's more, as easily degradable carbon source, glucose and sucrose were easier to be used by microorganisms, whose promotions for CO 2 emission were stronger than those in other treatments. The accumulative amounts of CO 2 emission were 2557.2 mg/kg and 2576.6 mg/kg in glucose and sucrose treatments, which were 86.9% and 88.3% higher than that in the control treatment at 1368.2 mg/kg, respectively. The accumulative amounts of CO 2 emission in starch and cellulose treatments were 2391.6 mg/kg and 1947.2 mg/kg, respectively, which were lower than those in the glucose and sucrose treatments and higher 74.8% and 42.3% than the control treatment. Because the complex macromolecular structure of cellulose was not easily used by microorganisms, the promotion of CO 2 emission was negligible. Moreover, starch was easier to be used by microorganisms than cellulose, so the CO 2 emission in starch treatment was higher than that in cellulose treatment.

NH 4 + , NH 3 emission, NO 3 − , Nitrification index. NH 4 + would be produced through the ammonification
of microorganisms with OM degradation in the SS composting process 27 . The evolutions of NH 4 + are shown in Fig. 3a, the NH 4 + concentrations of all treatments increased rapidly during the mesophilic phase and reached their respective peaks on the Day 9 of thermophilic phase. In besides, the change trend of each treatment was similar, because the increase in NH 4 + concentration was mainly caused by the rapid degradation of nitrogencontaining organic compounds 38 . The NH 4 + concentrations of control treatment were significantly higher than those of the carbon source treatments, and it's peak value was 4.21 g/kg, which were 1.22, 1.28, 1.08 and 1.04 times of that in glucose, sucrose, starch, and cellulose treatments, respectively. In the mesophilic phase, with the temperature increased the microorganisms multiplied rapidly, the DHA also increased rapidly (introduced in "Organic matter and dehydrogenase activity") and the ammonification of microorganisms played a major role in nitrogen conversion process. The peak concentrations of NH 4 + of the glucose and sucrose treatments were much lower than that of the control treatment, because the microorganisms could more easily use the degradable carbon sources and promote the ammonia assimilation, that transformed the NH 4 + -N into biological nitrogen. On the contrary, the contents of DOC in the cellulose and control treatments were lower than the other treatments (introduced in "Dissolved organic carbon and CO 2 emission"), so a large amount of nitrogen were converted into NH 4 + by ammonification. After the peak values, the NH 4 + concentrations in all treatments decreased rapidly, because high temperature and the gradually rising pH value promoted the conversion of NH 4 + to NH 3 and released into the atmosphere eventually 24 . Only a small NH 4 + could be detected at the end of composting, and the NH 4 + concentration in control and the cellulose treatments were slightly higher than those in other treatments, which were 0.65 g/kg and 0.69 g/kg, respectively. Ros et al. 39 also reported that the NH 4 + concentration gradually increased during the first 3-6 weeks of composting, and then gradually decreased due to NH 3 emission until the end of composting. NH 3 emission is one of the main reasons for the nitrogen loss, and the too low carbon-nitrogen ratio of the sewage sludge is an important factor that causes a large amount of NH 3 emission during SS composting process 40 . Adding extra carbon source could effectively control nitrogen loss in SS composting 22 , however, the effects of different carbon sources on NH 3 emission were different.
The NH 3 emission rate increased rapidly with the temperature rising in the early stage, then reached their respective peaks in thermophilic phase. The peak value of NH 3 emission of control treatment was 320.7 mg/ kg/48 h, followed by 290.6 mg/kg/48 h in cellulose treatment, 239.5 mg/kg/48 h in starch treatment, 197.5 mg/ kg/48 h in glucose treatment and 185.8 mg/kg/48 h in sucrose treatment (Fig. 3b). The emission rate of NH 3 had a positive correlation with the concentration of NH 4 + in the composting. Therefore, the NH 3 emission amount of each treatment increased sharply when the concentrations of NH 4 + -N were high in the thermophilic phase, www.nature.com/scientificreports/ and the NH 3 emission was also gradually flattening when the concentration of NH 4 + decreased in the cooling phase. The addition of carbon source changed the metabolic pathway of nitrogen, concretely, it could affect the microbial ammonification which led directly to NH 3 emission during the composting process. For example, the peak of the sucrose treatment appeared on Day 12, and the peaks of the other 4 treatments appeared on Day 10.
Moreover, the addition of extra carbon source significantly inhibited the NH 3 emission, especially in the readily degradable carbon source treatments, the NH 3 emission peaks were reduced by 38.4% and 42.3% in glucose and sucrose treatments compared with the control treatment, respectively.
In the whole composting process, the cumulative NH 3 emission of sucrose treatment was 900.3 mg/kg, which was the least of five treatments, followed by 968.8 mg/kg in glucose treatment, 1168.5 mg/kg in starch treatment, 1278.3 mg/kg in cellulose treatment and 1325.9 mg/kg in control treatment. In addition, the cumulative NH 3 emission was decreased by 32.1%, 26.9%, 11.8% and 3.6% compared with the control treatment in sucrose, glucose, starch and cellulose treatments, respectively. That's because glucose and sucrose were easily used by microorganisms, and could participate in the biochemical reaction of composting faster. Moreover, the readily degradable carbon source could increase the DOC concentration (introduced in "Dissolved organic carbon and CO 2 emission") and improve the biodegradability of the carbon source, so more NH 4 + was converted to bio-nitrogen fixed in the compost.
The nitrification in the composting process could be evaluated by detecting the NO 3 − concentration. As shown in Fig. 3c, the NO 3 − concentrations of all the treatments were lower than the NH 4 + concentrations throughout the composting process, especially during mesophilic phase and thermophilic phase. The NO 3 − concentration was closely related to the activity of nitrifying microorganisms, but the activity of nitrifying microorganisms was inhibited by high temperature and high NH 4 + concentration, that's why the NO 3 − concentration were so low during the thermophilic phase. During the first 7 days of composting, the NO 3 − concentration of each treatment was at a low level (0.07-0.16 g/kg). The NO 3 − concentrations in carbon source treatment was slightly higher than that in control treatment, then difference among the carbon source treatments was not significant. During the cooling phase, the NO 3 − concentrations of all treatments began to increase as the temperature decreased. The NO 3 − concentrations of glucose and sucrose treatment was significantly higher than that of control, while the differences among starch, cellulose and control treatment were not significant. The NO 3 − concentrations of all treatments reached a stable state at end of composting, and the highest NO 3 − concentration was 0.56 g/kg in sucrose treatment, followed by 0.43 g/kg in glucose treatment, 0.31 g/kg in starch treatment, 0.24 g/kg in cellulose treatment and 0.25 g/kg in control treatment. With the decrease of temperature and the NH 3 emission in the cooling phase, the increase of nitrification in all the treatments caused NO 3 − concentration raised, and the difference between glucose and sucrose treatments was not significant. Their change trends were similar, could be because the glucose and sucrose as easily degradable carbon sources were exhausted during mesophilic phase and thermophilic phase.  , which had been widely used in the evaluation of compost maturity 41 . As shown in Fig. 3d, the nitrification index of each treatment reached its peak in thermophilic phase, that's because the OM rapidly degraded during this period and the NH 4 + concentration remained at a high level. After the thermophilic phase, the nitrification index decreased rapidly as the temperature decreased, because the degradation rate of OM became slower, and the compost gradually completed maturation. Moreover, all the treatments reached maturity on Day 23 of the composting. According to Das et al. 's reports, the nitrification index of less than 0.5 indicated that the compost had reached "completely mature" state at the end of composting, the nitration index between 0.5 and 3.0 indicated that the compost had reached a "mature" state, and the nitrification index greater than 3.0 indicated that the compost had not reached a mature. In present study, all carbon source treatments reached maturity on Day 18 of composting. And the minimum nitrification index was 1.60 in glucose treatment, followed by 1.78 in sucrose treatment, 2.43 in starch treatment and 2.85 in cellulose treatment, while the control treatment was still in immature state with a nitration index of 3.69.
Germination index. Germination index (GI) is a biological activity index commonly used to evaluate compost maturity, and it can intuitively reflect the phytotoxicity change of compost. As shown in Fig. 4, all the GI decreased during first two days of composting, which was due to the production of some NH 4 + and lowmolecular-weight short-chain volatile fatty acids in the early stage of composting. Similar phenomenon had been found by Guo et al. 42 , who reported that the seed GI was at a low level in early stage when they studied the co-composting of corn straw and pig manure. It's reported that when the GI of the compost material exceeded 80%, it indicated that the compost product had no phytotoxicity and reached the maturity state 43 . The GI of all treatments increased rapidly after Day 3 of composting, which might be due to toxic substances degradation and NH 3 emission. Importantly, the GI of the carbon source treatment was higher than that of control, then GI of the carbon source treatments reached over 80% on Day 18 of composting, concretely, the highest GI was 115.2% in sucrose treatment, followed by 108.9% in glucose treatment, 88.3% in starch treatment, 81.6% in cellulose treatment, and only 74.7% in control treatment. The GI of all treatments exceeded 80% on Day 23 of composting, indicating all the treatments reached no phytotoxicity and mature state. Obviously, the addition of extra carbon sources, especially easily degradable carbon sources, could promote the compost maturation, and the compost could dephytotoxicize earlier.

Conclusions
The addition of extra carbon sources could improve the nitrogen conversion and compost quality. Especially, glucose and sucrose, as readily degradable carbon source promoted OM degradation and the maturation of composting, increased DOC contents, CO 2 emission, DHA, nitrification index and GI in the SS compost. Above all, the addition of sucrose and glucose reduced the NH 3 emission by 32.1% and 26.9%, respectively. Nevertheless, it's recommended that suitable alternatives for glucose and sucrose, such as beet pulp and molasses wastes containing readily degradable carbon sources should be researched to decrease production costs in future research.  www.nature.com/scientificreports/