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Microbial activity during composting of anthracene-contaminated soil
Chemosphere 52 (2003) 1505–1513 www.elsevier.com/locate/chemosphere
Microbial activity during composting of anthracene-contaminated soil Y. Ma a, J.Y. Zhang b, M.H. Wong a
a,*
Department of Biology, Institute for Natural Resources and Environmental Management, Hong Kong Baptist University, Kowloon Tong, Hong Kong b Department of Environmental Science, Wuhan University, Wuhan 430072, China
Abstract Microbial activity of an anthracene-spiked soil mixed with kitchen waste during laboratory composting at 56–59 C was studied using an in-vessel technology. The effect of old compost containing acclimated microorganisms on the composting efficiency was also investigated. Microbial succession, microbial enzyme activity, microbial diversity and anthracene removal rate were analyzed during 42 days of composting. The results demonstrated that inoculating with old compost increased the amounts of thermophilic microorganisms, but did not significantly increase anthracene removal. A microbial succession from mesophilic bacteria to thermophilic bacteria and thermophilic actinomycetes was observed during composting. Polyphenol oxidase activity decreased while catalase activity varied irregularly. Microbial diversity increased drastically when temperature elevated from 35 to 56 C, but decreased when temperature maintained at 56–59 C. 2003 Elsevier Ltd. All rights reserved. Keywords: Anthracene; Composting; Microbial activity
1. Introduction There are several toxic components in petroleum waste including alkyl compounds, olefin hydrocarbons, and aromatic hydrocarbons. polycyclic aromatic hydrocarbons (PAHs) are resistant to degradation in the natural environment due to their conjugated system of multiple benzene rings. Some PAHs are even known as ÔPriority PollutantsÕ, such as anthracene and benzopyrene, exert a carcinogenic effect and may cause aberrance and mutations in biological tissues. Many techniques have been involved in petroleum (or PAHs-) waste treatment, but composting has become of increasingly interest due to its economical, efficiency and environmental friendly properties (Prince and Sambasivam,
*
Corresponding author. Tel.: +852-3411-7746; fax: +8523411-7743. E-mail address: [email protected] (M.H. Wong).
1993). In-vessel composting is one of the most practical composting technologies, which is mainly performed in a closed vessel, and the abiotic factors can be controlled automatically. The best composting performance can therefore be achieved, and the odour can also be removed effectively (Hay and Kuchenrither, 1990; Davis and Russell, 1993). It has been reported that the coal tar containing a mass of PAHs was transformed to some odour-less substances like humus after being composted at high temperature, PAHs with 2,3,4-rings diminished by 95% (Taddeo, 1989). Hogan (1988) found that the contents of PCB and PAHs in sewage waste decreased drastically through in-vessel composting treatment. The American Air Force attempted to remediate excavated hydrocarbon contaminated soil at McClellan Air Force Base of California, and achieved 90% removal of TPH (total petroleum hydrocarbon) after 90 daysÕ composting (Stefanoff and Garcia, 1995). As a biodegradative process, composting efficiency depends on microbial biomass and activity. The microbes
0045-6535/03/$ - see front matter 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0045-6535(03)00489-2
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are normally derived from composting mass, old compost (acclimated microbial source) and also commercial bacterial strains (Nakasaki et al., 1985a; Brodie, 1994; Carr et al., 1995). The aim of the present work was to study microbial activity during composting of simulated anthracene-contaminated soil. We focused on (1) the composting of simulated anthracene-contaminated soil using in-vessel technology, (2) the effect of inoculating with old compost on anthracene removal, and (3) the microbial succession, microbial enzyme activity, and microbial diversity during composting.
2. Materials and methods 2.1. Composting materials and composter Soil was collected from a flower garden at Wuhan University, China, and screened through a 20-mesh sieve before use (soil properties are shown in Table 1). Anthracene (analytical grade) was dissolved in benzene and sprinkled evenly on garden soil to simulate an anthracene-contaminated soil (5000 mg anthracene kg1 dw). Kitchen waste was a homogeneous mixture of cooked rice and vegetables. Wood chips were collected from a lumberyard at Wuhan University, and also sieved through 2 mm before use. Each of 2.5 kg simulated anthracene soil was mixed with 6.0 kg kitchen waste, wood chips were added at twice volumes of anthracene and kitchen wastes. Composting mass A was inoculated with 0.85 kg old compost (the content of degradable volatile material in the inoculated old compost was negligible compared with the whole composting materials), and composting mass B did not contain any old
Table 1 Initial characteristics of composting mass and basic properties of garden soil
Wet weight Moisture content (%) Volatile material, VM (%) Total organic carbon, TOC (%) Total nitrogen, TN (%) C/N ratio pH Bacteria (cfu g1 dw) Actinomycetes (cfu g1 dw) Fungi (cfu g1 dw)
Composting mass A
Composting mass B
Garden soil
9.36 60.0
8.51 60.0
– 2.98
49.7
48.1
–
27.2
26.5
3.39
0.92
0.94
0.10
29.4 8.10 – –
28.0 7.75 – –
32.4 7.20 2.7E5 5.2E5
–
–
1.9E4
compost. The old compost was produced from the petroleum waste composting 6 months ago. It was stabilized and contained a mixture of acclimated microorganisms for petroleum degradation (Ma et al., 2000). The C/N ratio was adjusted with Russia compound fertilizer (N–P–K ¼ 16–16–16) to about 30:1. After thoroughly mixed by hand, all composting materials for each composting mass were placed into separate composter. The composter was made of plexiglass (volume: 12.5 l; height/diameter: 2.2) (Fig. 1). The control parameters for composting were moisture: 55–60%, pH: 7.0–8.5, TOC: >20%, and xenobiotic compounds to degradable compounds ratio: 1/30 (Table 1). Composting temperature was maintained at 56.5–59.5 C based on Rutgers aeration mechanism (MacGregor et al., 1981; Zhang et al., 1999; Ma et al., 2000), and the temperature of inner composter was kept at about 40 C by exterior heating. The experiment lasted for 42 days. 2.2. Chemical analysis of composting samples The oven-dried sample was heated at 600–700 C for 2–3 h to determine the volatile material (VM) content. A distilled water suspension (1:10 ratio, air-dried composting material) was used for measuring pH. Organic matter (TOC) was determined by Modified Walkey– Black method using H2 SO4 –K2 Cr2 O7 . Total N was digested with a Kjeldahl procedure and determined by steam distillation (Page et al., 1982). The CO2 produced during composting was absorbed by 2N NaOH solution as shown in Fig. 2, and CO2 yield was measured everyday through titrating against 1N HCl solution (Atchley and Clark, 1979; Lehtokari et al., 1983; Yusuf, 1990; Michel et al., 1995). The air-dried sample was mortared and screened by a 60-mesh sieve, ultrasonic-extracted by methanol for high-performance liquid chromatography (HPLC) determination. HPLC was performed on a Waters 280 system with YWG-C18 column (200 · 4 mm, 10 lm), 991 PDA uv detector (3000 psi, 20 C, 254 nm), U6k sample injector and 510 pump. Mobile phase is 85% methanol:15% water (0.8 ml/min). 2.3. Microbial analysis of composting samples 2.3.1. Microbial enumeration The microbial enumeration was measured with spread plate counting method. The incubation media used for isolation of bacteria, actinomycetes and fungi were peptone beef-extract agar, starch–casein agar and streptomycin–rose bengal agar, respectively (Martin, 1950; Nakasaki et al., 1985a; Carter, 1993). The incubation temperatures for mesophilic and thermophilic microorganisms were 30 and 58 C accordingly. The incubation durations were 2–3 days for mesophilic bacteria, 1–2 days for thermophilic bacteria, 5–7 days for mesophilic actinomycetes, 3–5 days for thermophilic
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Fig. 1. Schematic diagram of a composter. 1. Compost material, 2. Composter chamber, 3. Thermometer hole, 4. Air outlet, 5. Sampling holes, 6. Percolate layer, 7. Air inlet, 8. Water collecting zone, 9. Water outlet, 10. Flow meter, 11. Air compressor, 12. Buffer, 13. Wooden box for heat preservation.
Fig. 2. Flow chart of the laboratory scale system used to determine CO2 yield.
actinomycetes, 2–3 days for mesophilic and thermophilic fungi.
equation was introduced to estimate metabolic activity of microbes (Bach et al., 1985):
2.3.2. Estimation of metabolic activity for different microorganisms At thermophilic stage of composting, thermophilic bacteria and thermophilic actinomycetes were assumed to have specific CO2 evolution rate according to different volatile materials conversions. The following nonlinear
na Ra þ nb Rb ¼ rCO2 na nb
the amount of thermophilic actinomycetes (cfu g1 dw) the amount of thermophilic bacteria (cfu g1 dw)
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Ra Rb rCO2
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CO2 yield of each thermophilic actinomycete (mol CO2 cfu1 ) CO2 yield of each thermophilic bacterium (mol CO2 cfu1 ) CO2 evolution rate (mol CO2 g1 dw)
Each term in the left side of equation defined the CO2 evolution rate by either thermophilic bacteria or thermophilic actinomycetes. Ra and Rb can be calculated when microbial number and CO2 evolution rate for thermophilic bacteria and thermophilic actinomycetes were available at a given conversion of volatile materials.
2.3.3. Catalase activity Each of 2 g air-dried composting samples was weighed into a 150 ml conical flask, added with 40 ml distilled water and 5 ml 0.3% H2 O2 . Then the flask was stopped and shaked at 120 rpm for 30 min, 5 ml 3N H2 SO4 was introduced immediately after shaking to end the reaction. 25 ml of the filtrate was titrated with 0.1N KMnO4 and catalase activity was expressed as ml of 0.1N KMnO4 consumed g1 dry matter (Institute of Soil Science, CAS, 1985; Alef and Nannipieri, 1995).
2.3.4. Polyphenol oxidase activity Each of 1.0 g air-dried samples was weighed into 50 ml volumetric flask, added with 10 ml 1% pyrogallic acid solution, mixed well and incubated at 30 C for 1 h. 2.5 ml of 0.5N HCl was added after incubation, and the mixed solution was extracted by ether. Combined the extract and diluted to 50 ml. The control was conducted using 10 ml distilled water instead of 10 ml 1% pyrogallic acid solution. Polyphenol oxidase activity was determined by colorimetry method at 430 nm, and expressed as mg of purpurogallin g1 dry matter h1 (Institute of Soil Science, CAS, 1985; Alef and Nannipieri, 1995).
2.3.5. Microbial diversity Microbial diversity was assessed by Shannon– Weaver index as described below (Strom, 1985a; Krebs, 1994). Soil extract agar was used as incubation media for enumerating total microorganisms (James, 1958). The temperature for incubation was the same as the temperature of composting pile when sampled. The compost were sampled at 0 day (the initial stage of composting, 38 C), 2 days (temperature just rose to 56 C), 10 and 35 days when temperature was maintained between 56 and 59 C. H0 ¼
S X i¼1
Pi log2 Pi
H0 Pi
S
Shannon–Weaver index the proportion of individuals in the ith species (colony) to total individuals in overall species (colonies) the number of different species (colony types)
2.4. Sampling period design Sampling was performed at intervals when the conversion of volatile materials reached 5%, 10%, 15%, 20%, and 25%, respectively, as well as at the beginning and the end of experiment.
3. Results and discussion 3.1. Changes in microbial populations and CO2 evolution during composting The amounts of bacteria, actinomycetes and fungi at both normal and high temperature were counted. The results showed that mesophilic actinomycetes (MA) and mesophilic fungi (MF) were below the order of 104 cfu g1 dw, and thermophilic fungi (TF) was below the order of 102 cfu g1 dw. Therefore, their contributions were supposed to be very small comparing with mesophilic bacteria (MB), thermophilic bacteria (TB) and thermophilic actinomycetes (TA) (Figs. 3 and 4). Although MB did not increase notably as TB and TA during the composting, their absolute amounts were great. It was interesting to find quite lots of MB existed at thermophilic stage (56–60 C), which might be resulted from their thermotolerance property. It has been observed that the spore ratio of mesophilic bacteria in the samples obtained at 60 C was about 40%, which was helpful to improve their thermotolerance ability (Nakasaki et al., 1985b). The results of thermotolerance test demonstrated that mesophilic bacteria could survive 60 C by forming colonies on composting mass, but no oxygen consumed by them at 60 C. Mesophilic bacteria were considered to have very little contribution in degradation of organic matter during composting (Nakasaki et al., 1985b). Therefore, two main contributors for composting were thermophilic bacteria and thermophilic actinomycetes. When compared Figs. 3a and b, inoculating with old compost was found to significantly increase the initial amounts of thermophilic bacteria and thermophilic actinomycetes (the amounts of TB and TA were 6.55E7 and 1.78E6 for composting mass A, 1.78E7 and 6.02E5 for composting mass B, respectively). The changes of CO2 evolution rate (rCO2 ) during composting were shown in Fig. 5. The rCO2 was defined as moles of CO2 yield hr1 g1 dw. It was found that rCO2 in mass A that inoculated with old compost, was higher than that in mass B. In addition, the curves of rCO2 had
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(a)
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(b)
Fig. 3. Variation of temperature and microbial amounts with sampling intervals in (a) composting mass A and (b) composting mass B.
Table 2 Values of estimated specific CO2 evolution rates of thermophilic actinomycetes Ra and thermophilic bacteria Rb , under different volatile material conversions (Xvm ) Xvm 5%
10%
15%
20%
Ra (mol 3.49E)13 16.42E)13 7.29E)13 1.98E)13 CO2 cfu1 ) Rb (mol 5.82E)13 1.02E)13 0.72E)13 0.78E)13 CO2 cfu1 )
Fig. 4. Variation of CO2 evolution rate and volatile material conversion (Xvm ) with sampling intervals.
Fig. 5. Ratio of estimated CO2 evolution rate attributed to thermophilic bacteria to overall CO2 evolution rate.
two obvious peaks, which may be related to the succession of different microorganisms. 3.2. Estimation of metabolic activity for different microorganisms It was reported that the CO2 evolution rate was mostly associated with degradation of volatile materials in composting mass (Bach et al., 1985). At different conversions of volatile materials, thermophilic bacteria
and thermophilic actinomycetes had different specific CO2 evolution rates as indicated by Rb and Ra , respectively. The Rb and Ra for the present study were calculated and shown in Table 2. The Rb was higher than Ra at initial stage of composting, which showed the metabolic activity of thermophilic bacteria was higher than thermophilic actinomycetes. While after conversion of volatile materials reached 10%, Ra was significantly higher than Rb , implying the metabolic activity of thermophilic actinomycetes exceeded thermophilic bacteria. The nb Rb =rCO2 represented the ratio of CO2 evolution rate attributed to thermophilic bacteria. The CO2 evolution was mainly attributed to thermophilic bacteria at initial stage for composting mass A. By the time conversion of volatile materials had reached 10%, the ratio of nb Rb =rCO2 was less than 0.50, and CO2 evolution attributed more to thermophilic actinomycetes (Fig. 5). There was a similar trend for nb Rb =rCO2 ratio in mass B. Although nb Rb =rCO2 decreased gradually in mass B, it kept higher than 0.50, which suggested that the amount of thermophilic actinomycetes did not increase in the limited period, and inoculating with old compost would be beneficial to increase thermophilic actinomycetes amount. It is well known that actinomycetes grow much slower than most bacteria and fungi, and they are weak competitors for composting at high nutrient level. However, they get more active in the latter period of high temperature when the nutrient level is lower, because they are generally more tolerant to high temperature (Nakasaki et al., 1985c). A mass of white mycelium was
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noticed on the surface of composting mass at the end of experiment, which confirmed the development of actinomycetes during composting. Soil organic materials will be first degraded by bacteria, and actinomycetes played an important role in the degradation of xenobiotic organic compounds (i.e., cellulose, lignin, and chitin) (Ruan, 1990; Martin, 1995). The present results also indicated that the succession of microorganisms during composting was a process from mesophilic bacteria to thermophilic bacteria and thermophilic actinomycetes. Mesophilic bacteria were active at initial stage of composting. Metabolic activity of composting was mainly attributed to thermophilic bacteria at the initial stage of high temperature, and then to thermophilic actinomycetes at the latter stage of high temperature (de Bertoldi et al., 1983). The amount of fungi was lower than 102 cfu g1 dw throughout the experiment, which was probably due to the high moisture content of the composting mass (>60%). In addition, the optimal temperature for TF was 45–50 C (Nakasaki et al., 1985c), and 56.5–59.5 C observed in the present experiment was unfavorable to fungi growth. Finstein et al. (1983) reported that fungi decreased rapidly when temperature approached 60 C, and they even totally disappeared at 65 C.
Fig. 6. Profile of polyphenol oxidase activity during composting.
Fig. 7. Profile of catalase activity during composting.
3.3. Catalase and polyphenol oxidase activities during composting Microbial enzymes are important factors for microbial metabolic activity. Catalase enhances oxidization of compounds by H2 O2 , and it exists in all microorganisms. Catalase in composting mass is related to microbial activity and respiration. It also reflects the intensity of soil–microbial processes. Polyphenol oxidase is associated with the conversion of aromatic organic compounds, the key components of humus. It was reported that oxidase especially polyphenol oxidase played a role in the process of conversion of aromatic organic compounds to humus in soil, and polyphenol oxidase was negatively correlated to the level of humification (Zhou et al., 1981). Therefore, determination of these two enzyme activities should be helpful to get a better understanding of metabolic process during composting. Polyphenol oxidase activity tended to diminish during composting (Fig. 6). Analysis at the first 6 sampling intervals showed that the decrease were similar in composting mass A and B, but at the last sampling interval, polyphenol oxidase in mass A dropped sharply to 0.09 mg g1 dry matter. It implied that humification of composting mass was improved, and old compost seemed to speed up the humification process. Fig. 7 showed the change of catalase was irregular during the composting, but it was positively correlated with the amount of mesophilic bacteria as shown in Fig. 8, which means catalase activity increased with an increase of meso-
Fig. 8. Relationship between catalase activity and mesophilic bacteria amount during composting.
philes number during composting. Since catalase in composting mass was reacted and determined at normal temperature, catalase activity was supposed to only reflect the intensity of metabolic activity for mesophiles.
3.4. Microbial diversity in composting ecosystem Species diversity generally refers to the number of different species present (species richness) and the relative ‘‘evenness’’ of the number of individuals (or amount of biomass) in each species. The Shannon–Weaver index combines both of these factors and has the advantage of being relatively independent of sample size. The effect of temperature on microbial diversity in composting ecosystem was studied by many researchers (Strom, 1985a;
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Table 3 Microbial diversity at different composting stages Composting pile temperature (C)
Age of compost (d)
Incubation temperature (C)
Plate colonies amount (individual)
Colony types
Shannon– Weaver index (H 0 )
Composting mass A
38 56 56–59 56–59
0 2 10 35
37 56 58 58
120 36 17 24
4 6 5 2
0.52 2.13 1.85 0.54
Composting mass B
38 56 56–59 56–59
0 2 10 35
37 56 58 58
200 46 27 115
5 3 5 2
0.18 1.49 2.18 0.76
Kuter, 1986), but the changes of microbial diversity during the whole composting have never been reported. The changes of Shannon–Weaver index during composting were shown in Table 3. It can be found that at 56 C, Shannon–Weaver index was much higher than that at 38 C. The present study demonstrated that although microbial diversity was low at high temperature, the active amount was great. This result was in line with other reports (Strom, 1978; Strom, 1985b). Shannon– Weaver index diminished at high temperature, but microbial diversity was believed to not always directly correlate with biological productivity (Odum, 1971). This was the case in the present study that diversity index was unrelated with the results of plate counts. Nevertheless, microbial diversity has positive correlation with the stability of microbial community. An active microbial community consisted of different microorganisms that possess different metabolic activities, was highly desired to achieve an effective degradation of various organic compounds during composting. The possible reason for why Shannon–Weaver index diminished at high temperature was as follows. First, the degradable organic materials were relatively high at the initial period of high temperature, which stimulated a high microbial diversity; second, the content of degradable organic matter decreased more quickly while the content of xenobiotic compounds (i.e., anthracene) increased relatively at the latter period of high temperature, and most microorganisms diminished or even disappeared in this period because they cannot use anthracene as direct or indirect carbon source. 3.5. Removal of anthracene The removal rate of anthracene was 55.3% for composting mass A, and 50.5% for composting mass B respectively (Fig. 9). There was no significant difference between treatments inoculating with and without old compost. At the second sampling interval, the concentrations of anthracene in both mass A and B were higher
Fig. 9. Removal rate of anthracene during composting.
than that in mass of starting materials, and they were 107.45% and 104.76% of original concentration respectively. This might be due to two reasons. Firstly, many organic compounds in composting mass were degraded or mineralized by aerobic microorganisms at initial stage, and the total weight of composting mass dropped very quickly, while the degradation of anthracene did not keep pace with the total weight loss. Secondly, the amount of microorganisms that acclimated to high anthracene concentrations was very small at the beginning of composting. It has been observed that microorganisms should adapt to the pollutant and produce induced enzyme before they degrade a particular pollutant, and then can they grow and reproduce themselves by using this pollutant as carbon and energy sources (Atlas, 1993).
4. Conclusions The sequence of microbial succession during composting was from mesophilic bacteria, to thermophilic bacteria and thermophilic actinomycetes. Mesophilic bacteria were most active among the initial stage of composting. When at the earlier stage of high temperature (56–59 C), thermophilic bacteria were the main contributors to composting. The amount of thermophilic actinomycetes increased at the latter period of high temperature, and played an important role in organic
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compounds degradation. Polyphenol oxidase activity was negatively related to the humification of composting mass, and could be used as an indicator represents composting characteristics. While catalase activity only correlated with mesophiles at normal temperature, and could not indicate the whole microbial activities during composting. Microbial diversity increased drastically when temperature rose from 35 to 56 C, and decreased when the temperature maintained at 56–59 C for a prolonged period. Anthracene was removed up to 55.3% by in-vessel composting within 42 days, however inoculating with old compost did not significantly improve anthracene removal.
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Microbial activity during composting of anthracene-contaminated soil Y. Ma a, J.Y. Zhang b, M.H. Wong a
a,*
Department of Biology, Institute for Natural Resources and Environmental Management, Hong Kong Baptist University, Kowloon Tong, Hong Kong b Department of Environmental Science, Wuhan University, Wuhan 430072, China
Abstract Microbial activity of an anthracene-spiked soil mixed with kitchen waste during laboratory composting at 56–59 C was studied using an in-vessel technology. The effect of old compost containing acclimated microorganisms on the composting efficiency was also investigated. Microbial succession, microbial enzyme activity, microbial diversity and anthracene removal rate were analyzed during 42 days of composting. The results demonstrated that inoculating with old compost increased the amounts of thermophilic microorganisms, but did not significantly increase anthracene removal. A microbial succession from mesophilic bacteria to thermophilic bacteria and thermophilic actinomycetes was observed during composting. Polyphenol oxidase activity decreased while catalase activity varied irregularly. Microbial diversity increased drastically when temperature elevated from 35 to 56 C, but decreased when temperature maintained at 56–59 C. 2003 Elsevier Ltd. All rights reserved. Keywords: Anthracene; Composting; Microbial activity
1. Introduction There are several toxic components in petroleum waste including alkyl compounds, olefin hydrocarbons, and aromatic hydrocarbons. polycyclic aromatic hydrocarbons (PAHs) are resistant to degradation in the natural environment due to their conjugated system of multiple benzene rings. Some PAHs are even known as ÔPriority PollutantsÕ, such as anthracene and benzopyrene, exert a carcinogenic effect and may cause aberrance and mutations in biological tissues. Many techniques have been involved in petroleum (or PAHs-) waste treatment, but composting has become of increasingly interest due to its economical, efficiency and environmental friendly properties (Prince and Sambasivam,
*
Corresponding author. Tel.: +852-3411-7746; fax: +8523411-7743. E-mail address: [email protected] (M.H. Wong).
1993). In-vessel composting is one of the most practical composting technologies, which is mainly performed in a closed vessel, and the abiotic factors can be controlled automatically. The best composting performance can therefore be achieved, and the odour can also be removed effectively (Hay and Kuchenrither, 1990; Davis and Russell, 1993). It has been reported that the coal tar containing a mass of PAHs was transformed to some odour-less substances like humus after being composted at high temperature, PAHs with 2,3,4-rings diminished by 95% (Taddeo, 1989). Hogan (1988) found that the contents of PCB and PAHs in sewage waste decreased drastically through in-vessel composting treatment. The American Air Force attempted to remediate excavated hydrocarbon contaminated soil at McClellan Air Force Base of California, and achieved 90% removal of TPH (total petroleum hydrocarbon) after 90 daysÕ composting (Stefanoff and Garcia, 1995). As a biodegradative process, composting efficiency depends on microbial biomass and activity. The microbes
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are normally derived from composting mass, old compost (acclimated microbial source) and also commercial bacterial strains (Nakasaki et al., 1985a; Brodie, 1994; Carr et al., 1995). The aim of the present work was to study microbial activity during composting of simulated anthracene-contaminated soil. We focused on (1) the composting of simulated anthracene-contaminated soil using in-vessel technology, (2) the effect of inoculating with old compost on anthracene removal, and (3) the microbial succession, microbial enzyme activity, and microbial diversity during composting.
2. Materials and methods 2.1. Composting materials and composter Soil was collected from a flower garden at Wuhan University, China, and screened through a 20-mesh sieve before use (soil properties are shown in Table 1). Anthracene (analytical grade) was dissolved in benzene and sprinkled evenly on garden soil to simulate an anthracene-contaminated soil (5000 mg anthracene kg1 dw). Kitchen waste was a homogeneous mixture of cooked rice and vegetables. Wood chips were collected from a lumberyard at Wuhan University, and also sieved through 2 mm before use. Each of 2.5 kg simulated anthracene soil was mixed with 6.0 kg kitchen waste, wood chips were added at twice volumes of anthracene and kitchen wastes. Composting mass A was inoculated with 0.85 kg old compost (the content of degradable volatile material in the inoculated old compost was negligible compared with the whole composting materials), and composting mass B did not contain any old
Table 1 Initial characteristics of composting mass and basic properties of garden soil
Wet weight Moisture content (%) Volatile material, VM (%) Total organic carbon, TOC (%) Total nitrogen, TN (%) C/N ratio pH Bacteria (cfu g1 dw) Actinomycetes (cfu g1 dw) Fungi (cfu g1 dw)
Composting mass A
Composting mass B
Garden soil
9.36 60.0
8.51 60.0
– 2.98
49.7
48.1
–
27.2
26.5
3.39
0.92
0.94
0.10
29.4 8.10 – –
28.0 7.75 – –
32.4 7.20 2.7E5 5.2E5
–
–
1.9E4
compost. The old compost was produced from the petroleum waste composting 6 months ago. It was stabilized and contained a mixture of acclimated microorganisms for petroleum degradation (Ma et al., 2000). The C/N ratio was adjusted with Russia compound fertilizer (N–P–K ¼ 16–16–16) to about 30:1. After thoroughly mixed by hand, all composting materials for each composting mass were placed into separate composter. The composter was made of plexiglass (volume: 12.5 l; height/diameter: 2.2) (Fig. 1). The control parameters for composting were moisture: 55–60%, pH: 7.0–8.5, TOC: >20%, and xenobiotic compounds to degradable compounds ratio: 1/30 (Table 1). Composting temperature was maintained at 56.5–59.5 C based on Rutgers aeration mechanism (MacGregor et al., 1981; Zhang et al., 1999; Ma et al., 2000), and the temperature of inner composter was kept at about 40 C by exterior heating. The experiment lasted for 42 days. 2.2. Chemical analysis of composting samples The oven-dried sample was heated at 600–700 C for 2–3 h to determine the volatile material (VM) content. A distilled water suspension (1:10 ratio, air-dried composting material) was used for measuring pH. Organic matter (TOC) was determined by Modified Walkey– Black method using H2 SO4 –K2 Cr2 O7 . Total N was digested with a Kjeldahl procedure and determined by steam distillation (Page et al., 1982). The CO2 produced during composting was absorbed by 2N NaOH solution as shown in Fig. 2, and CO2 yield was measured everyday through titrating against 1N HCl solution (Atchley and Clark, 1979; Lehtokari et al., 1983; Yusuf, 1990; Michel et al., 1995). The air-dried sample was mortared and screened by a 60-mesh sieve, ultrasonic-extracted by methanol for high-performance liquid chromatography (HPLC) determination. HPLC was performed on a Waters 280 system with YWG-C18 column (200 · 4 mm, 10 lm), 991 PDA uv detector (3000 psi, 20 C, 254 nm), U6k sample injector and 510 pump. Mobile phase is 85% methanol:15% water (0.8 ml/min). 2.3. Microbial analysis of composting samples 2.3.1. Microbial enumeration The microbial enumeration was measured with spread plate counting method. The incubation media used for isolation of bacteria, actinomycetes and fungi were peptone beef-extract agar, starch–casein agar and streptomycin–rose bengal agar, respectively (Martin, 1950; Nakasaki et al., 1985a; Carter, 1993). The incubation temperatures for mesophilic and thermophilic microorganisms were 30 and 58 C accordingly. The incubation durations were 2–3 days for mesophilic bacteria, 1–2 days for thermophilic bacteria, 5–7 days for mesophilic actinomycetes, 3–5 days for thermophilic
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Fig. 1. Schematic diagram of a composter. 1. Compost material, 2. Composter chamber, 3. Thermometer hole, 4. Air outlet, 5. Sampling holes, 6. Percolate layer, 7. Air inlet, 8. Water collecting zone, 9. Water outlet, 10. Flow meter, 11. Air compressor, 12. Buffer, 13. Wooden box for heat preservation.
Fig. 2. Flow chart of the laboratory scale system used to determine CO2 yield.
actinomycetes, 2–3 days for mesophilic and thermophilic fungi.
equation was introduced to estimate metabolic activity of microbes (Bach et al., 1985):
2.3.2. Estimation of metabolic activity for different microorganisms At thermophilic stage of composting, thermophilic bacteria and thermophilic actinomycetes were assumed to have specific CO2 evolution rate according to different volatile materials conversions. The following nonlinear
na Ra þ nb Rb ¼ rCO2 na nb
the amount of thermophilic actinomycetes (cfu g1 dw) the amount of thermophilic bacteria (cfu g1 dw)
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Ra Rb rCO2
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CO2 yield of each thermophilic actinomycete (mol CO2 cfu1 ) CO2 yield of each thermophilic bacterium (mol CO2 cfu1 ) CO2 evolution rate (mol CO2 g1 dw)
Each term in the left side of equation defined the CO2 evolution rate by either thermophilic bacteria or thermophilic actinomycetes. Ra and Rb can be calculated when microbial number and CO2 evolution rate for thermophilic bacteria and thermophilic actinomycetes were available at a given conversion of volatile materials.
2.3.3. Catalase activity Each of 2 g air-dried composting samples was weighed into a 150 ml conical flask, added with 40 ml distilled water and 5 ml 0.3% H2 O2 . Then the flask was stopped and shaked at 120 rpm for 30 min, 5 ml 3N H2 SO4 was introduced immediately after shaking to end the reaction. 25 ml of the filtrate was titrated with 0.1N KMnO4 and catalase activity was expressed as ml of 0.1N KMnO4 consumed g1 dry matter (Institute of Soil Science, CAS, 1985; Alef and Nannipieri, 1995).
2.3.4. Polyphenol oxidase activity Each of 1.0 g air-dried samples was weighed into 50 ml volumetric flask, added with 10 ml 1% pyrogallic acid solution, mixed well and incubated at 30 C for 1 h. 2.5 ml of 0.5N HCl was added after incubation, and the mixed solution was extracted by ether. Combined the extract and diluted to 50 ml. The control was conducted using 10 ml distilled water instead of 10 ml 1% pyrogallic acid solution. Polyphenol oxidase activity was determined by colorimetry method at 430 nm, and expressed as mg of purpurogallin g1 dry matter h1 (Institute of Soil Science, CAS, 1985; Alef and Nannipieri, 1995).
2.3.5. Microbial diversity Microbial diversity was assessed by Shannon– Weaver index as described below (Strom, 1985a; Krebs, 1994). Soil extract agar was used as incubation media for enumerating total microorganisms (James, 1958). The temperature for incubation was the same as the temperature of composting pile when sampled. The compost were sampled at 0 day (the initial stage of composting, 38 C), 2 days (temperature just rose to 56 C), 10 and 35 days when temperature was maintained between 56 and 59 C. H0 ¼
S X i¼1
Pi log2 Pi
H0 Pi
S
Shannon–Weaver index the proportion of individuals in the ith species (colony) to total individuals in overall species (colonies) the number of different species (colony types)
2.4. Sampling period design Sampling was performed at intervals when the conversion of volatile materials reached 5%, 10%, 15%, 20%, and 25%, respectively, as well as at the beginning and the end of experiment.
3. Results and discussion 3.1. Changes in microbial populations and CO2 evolution during composting The amounts of bacteria, actinomycetes and fungi at both normal and high temperature were counted. The results showed that mesophilic actinomycetes (MA) and mesophilic fungi (MF) were below the order of 104 cfu g1 dw, and thermophilic fungi (TF) was below the order of 102 cfu g1 dw. Therefore, their contributions were supposed to be very small comparing with mesophilic bacteria (MB), thermophilic bacteria (TB) and thermophilic actinomycetes (TA) (Figs. 3 and 4). Although MB did not increase notably as TB and TA during the composting, their absolute amounts were great. It was interesting to find quite lots of MB existed at thermophilic stage (56–60 C), which might be resulted from their thermotolerance property. It has been observed that the spore ratio of mesophilic bacteria in the samples obtained at 60 C was about 40%, which was helpful to improve their thermotolerance ability (Nakasaki et al., 1985b). The results of thermotolerance test demonstrated that mesophilic bacteria could survive 60 C by forming colonies on composting mass, but no oxygen consumed by them at 60 C. Mesophilic bacteria were considered to have very little contribution in degradation of organic matter during composting (Nakasaki et al., 1985b). Therefore, two main contributors for composting were thermophilic bacteria and thermophilic actinomycetes. When compared Figs. 3a and b, inoculating with old compost was found to significantly increase the initial amounts of thermophilic bacteria and thermophilic actinomycetes (the amounts of TB and TA were 6.55E7 and 1.78E6 for composting mass A, 1.78E7 and 6.02E5 for composting mass B, respectively). The changes of CO2 evolution rate (rCO2 ) during composting were shown in Fig. 5. The rCO2 was defined as moles of CO2 yield hr1 g1 dw. It was found that rCO2 in mass A that inoculated with old compost, was higher than that in mass B. In addition, the curves of rCO2 had
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(a)
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(b)
Fig. 3. Variation of temperature and microbial amounts with sampling intervals in (a) composting mass A and (b) composting mass B.
Table 2 Values of estimated specific CO2 evolution rates of thermophilic actinomycetes Ra and thermophilic bacteria Rb , under different volatile material conversions (Xvm ) Xvm 5%
10%
15%
20%
Ra (mol 3.49E)13 16.42E)13 7.29E)13 1.98E)13 CO2 cfu1 ) Rb (mol 5.82E)13 1.02E)13 0.72E)13 0.78E)13 CO2 cfu1 )
Fig. 4. Variation of CO2 evolution rate and volatile material conversion (Xvm ) with sampling intervals.
Fig. 5. Ratio of estimated CO2 evolution rate attributed to thermophilic bacteria to overall CO2 evolution rate.
two obvious peaks, which may be related to the succession of different microorganisms. 3.2. Estimation of metabolic activity for different microorganisms It was reported that the CO2 evolution rate was mostly associated with degradation of volatile materials in composting mass (Bach et al., 1985). At different conversions of volatile materials, thermophilic bacteria
and thermophilic actinomycetes had different specific CO2 evolution rates as indicated by Rb and Ra , respectively. The Rb and Ra for the present study were calculated and shown in Table 2. The Rb was higher than Ra at initial stage of composting, which showed the metabolic activity of thermophilic bacteria was higher than thermophilic actinomycetes. While after conversion of volatile materials reached 10%, Ra was significantly higher than Rb , implying the metabolic activity of thermophilic actinomycetes exceeded thermophilic bacteria. The nb Rb =rCO2 represented the ratio of CO2 evolution rate attributed to thermophilic bacteria. The CO2 evolution was mainly attributed to thermophilic bacteria at initial stage for composting mass A. By the time conversion of volatile materials had reached 10%, the ratio of nb Rb =rCO2 was less than 0.50, and CO2 evolution attributed more to thermophilic actinomycetes (Fig. 5). There was a similar trend for nb Rb =rCO2 ratio in mass B. Although nb Rb =rCO2 decreased gradually in mass B, it kept higher than 0.50, which suggested that the amount of thermophilic actinomycetes did not increase in the limited period, and inoculating with old compost would be beneficial to increase thermophilic actinomycetes amount. It is well known that actinomycetes grow much slower than most bacteria and fungi, and they are weak competitors for composting at high nutrient level. However, they get more active in the latter period of high temperature when the nutrient level is lower, because they are generally more tolerant to high temperature (Nakasaki et al., 1985c). A mass of white mycelium was
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noticed on the surface of composting mass at the end of experiment, which confirmed the development of actinomycetes during composting. Soil organic materials will be first degraded by bacteria, and actinomycetes played an important role in the degradation of xenobiotic organic compounds (i.e., cellulose, lignin, and chitin) (Ruan, 1990; Martin, 1995). The present results also indicated that the succession of microorganisms during composting was a process from mesophilic bacteria to thermophilic bacteria and thermophilic actinomycetes. Mesophilic bacteria were active at initial stage of composting. Metabolic activity of composting was mainly attributed to thermophilic bacteria at the initial stage of high temperature, and then to thermophilic actinomycetes at the latter stage of high temperature (de Bertoldi et al., 1983). The amount of fungi was lower than 102 cfu g1 dw throughout the experiment, which was probably due to the high moisture content of the composting mass (>60%). In addition, the optimal temperature for TF was 45–50 C (Nakasaki et al., 1985c), and 56.5–59.5 C observed in the present experiment was unfavorable to fungi growth. Finstein et al. (1983) reported that fungi decreased rapidly when temperature approached 60 C, and they even totally disappeared at 65 C.
Fig. 6. Profile of polyphenol oxidase activity during composting.
Fig. 7. Profile of catalase activity during composting.
3.3. Catalase and polyphenol oxidase activities during composting Microbial enzymes are important factors for microbial metabolic activity. Catalase enhances oxidization of compounds by H2 O2 , and it exists in all microorganisms. Catalase in composting mass is related to microbial activity and respiration. It also reflects the intensity of soil–microbial processes. Polyphenol oxidase is associated with the conversion of aromatic organic compounds, the key components of humus. It was reported that oxidase especially polyphenol oxidase played a role in the process of conversion of aromatic organic compounds to humus in soil, and polyphenol oxidase was negatively correlated to the level of humification (Zhou et al., 1981). Therefore, determination of these two enzyme activities should be helpful to get a better understanding of metabolic process during composting. Polyphenol oxidase activity tended to diminish during composting (Fig. 6). Analysis at the first 6 sampling intervals showed that the decrease were similar in composting mass A and B, but at the last sampling interval, polyphenol oxidase in mass A dropped sharply to 0.09 mg g1 dry matter. It implied that humification of composting mass was improved, and old compost seemed to speed up the humification process. Fig. 7 showed the change of catalase was irregular during the composting, but it was positively correlated with the amount of mesophilic bacteria as shown in Fig. 8, which means catalase activity increased with an increase of meso-
Fig. 8. Relationship between catalase activity and mesophilic bacteria amount during composting.
philes number during composting. Since catalase in composting mass was reacted and determined at normal temperature, catalase activity was supposed to only reflect the intensity of metabolic activity for mesophiles.
3.4. Microbial diversity in composting ecosystem Species diversity generally refers to the number of different species present (species richness) and the relative ‘‘evenness’’ of the number of individuals (or amount of biomass) in each species. The Shannon–Weaver index combines both of these factors and has the advantage of being relatively independent of sample size. The effect of temperature on microbial diversity in composting ecosystem was studied by many researchers (Strom, 1985a;
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Table 3 Microbial diversity at different composting stages Composting pile temperature (C)
Age of compost (d)
Incubation temperature (C)
Plate colonies amount (individual)
Colony types
Shannon– Weaver index (H 0 )
Composting mass A
38 56 56–59 56–59
0 2 10 35
37 56 58 58
120 36 17 24
4 6 5 2
0.52 2.13 1.85 0.54
Composting mass B
38 56 56–59 56–59
0 2 10 35
37 56 58 58
200 46 27 115
5 3 5 2
0.18 1.49 2.18 0.76
Kuter, 1986), but the changes of microbial diversity during the whole composting have never been reported. The changes of Shannon–Weaver index during composting were shown in Table 3. It can be found that at 56 C, Shannon–Weaver index was much higher than that at 38 C. The present study demonstrated that although microbial diversity was low at high temperature, the active amount was great. This result was in line with other reports (Strom, 1978; Strom, 1985b). Shannon– Weaver index diminished at high temperature, but microbial diversity was believed to not always directly correlate with biological productivity (Odum, 1971). This was the case in the present study that diversity index was unrelated with the results of plate counts. Nevertheless, microbial diversity has positive correlation with the stability of microbial community. An active microbial community consisted of different microorganisms that possess different metabolic activities, was highly desired to achieve an effective degradation of various organic compounds during composting. The possible reason for why Shannon–Weaver index diminished at high temperature was as follows. First, the degradable organic materials were relatively high at the initial period of high temperature, which stimulated a high microbial diversity; second, the content of degradable organic matter decreased more quickly while the content of xenobiotic compounds (i.e., anthracene) increased relatively at the latter period of high temperature, and most microorganisms diminished or even disappeared in this period because they cannot use anthracene as direct or indirect carbon source. 3.5. Removal of anthracene The removal rate of anthracene was 55.3% for composting mass A, and 50.5% for composting mass B respectively (Fig. 9). There was no significant difference between treatments inoculating with and without old compost. At the second sampling interval, the concentrations of anthracene in both mass A and B were higher
Fig. 9. Removal rate of anthracene during composting.
than that in mass of starting materials, and they were 107.45% and 104.76% of original concentration respectively. This might be due to two reasons. Firstly, many organic compounds in composting mass were degraded or mineralized by aerobic microorganisms at initial stage, and the total weight of composting mass dropped very quickly, while the degradation of anthracene did not keep pace with the total weight loss. Secondly, the amount of microorganisms that acclimated to high anthracene concentrations was very small at the beginning of composting. It has been observed that microorganisms should adapt to the pollutant and produce induced enzyme before they degrade a particular pollutant, and then can they grow and reproduce themselves by using this pollutant as carbon and energy sources (Atlas, 1993).
4. Conclusions The sequence of microbial succession during composting was from mesophilic bacteria, to thermophilic bacteria and thermophilic actinomycetes. Mesophilic bacteria were most active among the initial stage of composting. When at the earlier stage of high temperature (56–59 C), thermophilic bacteria were the main contributors to composting. The amount of thermophilic actinomycetes increased at the latter period of high temperature, and played an important role in organic
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compounds degradation. Polyphenol oxidase activity was negatively related to the humification of composting mass, and could be used as an indicator represents composting characteristics. While catalase activity only correlated with mesophiles at normal temperature, and could not indicate the whole microbial activities during composting. Microbial diversity increased drastically when temperature rose from 35 to 56 C, and decreased when the temperature maintained at 56–59 C for a prolonged period. Anthracene was removed up to 55.3% by in-vessel composting within 42 days, however inoculating with old compost did not significantly improve anthracene removal.
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