Effects of compost stability and contaminant concentration on the bioremediation of PAHs-contaminated soil through composting.

The objective of this study was to investigate the effect of two factors: the stability degree (0.37-4.55 mg O(2) g(-1) Organic Matter h(-1)) of different composts derived from the organic fraction of municipal solid wastes and the concentration of a complex mixture of PAHs including fluorene, phenanthrene, anthracene, fluoranthene, pyrene and benzo(a)anthracene in the bioremediation of soil. The two factors were systematically studied applying central composite design methodology. The obtained results demonstrated that compost stability degree was particularly important during the first stage of the process. Stable composts enhanced the levels of degradation in soil-compost mixture and a degradation rate of 92% was achieved in this period, but only 40% was degraded with the least stable compost. The PAHs concentration was also important during the process, since the degradation rates increased with the increase in the PAHs concentration. Moreover, all the individual PAHs demonstrated a notable decrease in their concentrations after the incubation period, but pyrene was degraded to lower levels in some treatments compared to others PAHs.


Introduction 1
The large range of hazardous chemicals with different structures and different 2 toxicity levels that are continuously released from several anthropogenic sources are 3 continuously causing environmental pollution [1]. Polycyclic aromatic hydrocarbons 4 (PAHs) are one of the most encountered pollutants in the ecosystems; as a consequence, 5 soil contamination with these contaminants is a matter of major concern as they can be 6 introduced to the soil by various sources, where these compounds are categorized as 7 toxic for both humans and environment [2]. Indeed, with more concern regarding the 8 ecosystem and more strict regulation like the EU landfill directive (1999/31/EC), which 9 tries to reduce the amount of wastes that can be sent to the landfill, transforming the 10 contaminated soil to such landfills has become limited. Furthermore, the restoration of 11 many contaminated sites is preferable as the available agricultural areas are gradually 12 degraded with time [3]. Accordingly, there is a critical need to develop and implement 13 an effective remediation technology to reduce the threats caused by such contaminants 14 and create a sustainable reuse of soil. 15 Bioremediation can be regarded as an attractive technology that results in the 16 partial or complete biotransformation of many organic contaminants to microbial 17 biomass and stable innocuous end-product. Moreover, this technology is believed to be 18 cost-effective and environmentally accepted [4,5]. The contaminated soil are normally 19 deficient in nutrients that are necessary to support the indigenous microorganisms to 20 develop themselves, or sometimes the microorganisms are only available at low levels 21 that makes the bioremediation process progress at very slow rates [6]. To overcome 22 these conditions, normally the bioremediation of hydrocarbon contaminated soils often 23 rely upon the addition of nutrients or microorganisms (biostimulation and 24 bioaugmentation) [7,8]. Composting as a remediation tool has been considered a 25 P r e -p r i n t suitable technology in the bioremediation of contaminated soils, and it has been used to 1 mitigate these limiting factors, as it also improves the soil properties [9]. On the other 2 hand, applying composting technology provides a sustainable reuse of the organic 3 biodegradable fraction of wastes, which is both microbial and nutrient rich. However, 4 contaminants bioavailability is an important factor affecting microbial degradation rates 5 in soil and sediments as the microorganism are able to attack the target contaminant 6 only when it is dissolved within the materials [10]. Thereby, the selected organic 7 amendment for the bioremediation process should serve to improve and overcome any 8 deficiencies or limitations that influence the process efficiency. One challenge with this 9 type of research is that composting feedstock composition can vary widely from one 10 facility to another; this can affect the chemical and microbial conditions in the 11 amendments [11]. Bioremediation of contaminated soils using composting process 12 depends on a number of physical, chemical, and biological factors that determine the 13 microbial accessibility to the target contaminants [10,12,13], where the amendment 14 properties are of great role in determining the process behaviour. Although various 15 amendments have been applied during the composting of contaminated soil, still much 16 specialized research is needed. Compost stability is one of these factors as this 17 parameter is related to microbial activity within the organic material and it also can be 18 related to the compost composition like humic matter [6]. Until now, no studies were 19 reported that explore this important parameter and its influence on the degradation of 20 PAHs in the soil. For an efficient treatment process, the microbial activity, which is 21 considered the main factor in the bioremediation process, should be maintained at 22 adequate levels [14]. The contaminants concentration is one of the factors that influence 23 the microbial activity as these contaminants may exhibit some toxic or inhibition 24 influence when they exist at high levels. However, low concentration could be below 25 P r e -p r i n t composting process, composts derived from the organic fraction of municipal wastes 23 (OFMSW) were applied during the bioremediation experiments. In this study, five 24 types of OFMSW composts were used during the experimental treatments. The main 25 difference between these composts was the degree of stability "the rate of organic 1 matter decomposition as a result of the microbiological activity" and it is mainly 2 related to the availability of readily degradable substrates. The different levels of 3 stability were determined using the Dynamic Respirometric Index (DRI). Only 4 compost B was obtained from a home composter in the University Autònoma of 5 Barcelona, where the others were obtained from composting plants located in the 6 Barcelona area (Spain). These composts were selected to be characterized by a 7 different degree of stability ranging from full-stable to unstable compost [16,17], 8 which would provide the ability to examine their effects on the degradation of the used 9 PAHs and to relate and predict the effect of their major components as a consequence 10 on the bioremediation process. The main characteristics of the used composts are also 11 presented in Table 1. 12 13

Composting reactors and monitoring instruments 14
The used reactors were Dewar® vessels (4.5-L), which were modified and 15 conditioned to operate in a batch-mode way for the composting experiments. These 16 reactors are thermally isolated, so the process can be kept under the natural composting 17 temperatures, and the influence of ambient temperature can be minimized. Aeration was 18 provided through a pipeline connected to the bottom of the reactor where a plastic mesh 19 is placed to insure a correct distribution of the air through the composting mixture, and 20 the exhausted air exits the reactor through an outlet in the reactor cover. Oxygen 21 concentration was measured by means of an oxygen sensor (Crowcon's Xgard, United 22 Kingdom), where the inlet of the sensor is connected to the reactor outlet and 23 consequently the oxygen percentage in air was determined. Aeration rate and frequency 24 were adjusted to prevent any limitation or excess in the oxygen percentage in the 25 reactors, consequently, sporadically aeration mode was used and the oxygen 1 concentration was well maintained to insure aerobic conditions (more than 10%). 2 Temperature was monitored by Pt-100 sensors (Sensotran, Spain) connected to a data 3 acquisition system (DAS-8000, Desin, Spain) that was connected to a personal 4 computer. The software used (Proasis®Das-Win 2.1, Desin, Spain) also permits to 5 monitor both the temperature and oxygen content in the reactors. These two parameters 6 (temperature and oxygen concentration) are useful to control and follow the process 7 during the different phases. 8 9

Experiments set-up 10
The PAHs were mixed together according to their percentages to be introduced 11 as the target contaminants during the composting process. These contaminants were 12 spiked into soil to have the initial concentration expressed as total of PAHs according to 13 the values determined by the experimental design technique that were decided to be 14 from 0.1g/kg to 2g/kg (dry matter). After this, the contaminated soil was mixed with the 15 organic amendment at ratio of 1:1 (w:w, dry weight). The mixture was then mixed with 16 bulking agent at a ratio 1:1 (V:V) in an attempt to provide proper porosity to maintain 17 aerobic conditions. All the components of the composting treatments were manually 18 mixed according to the aforementioned ratios, resulting in about 3.5 kg that were used 19 in each reactor. The used bulking agent consisted of wood chips and pruning wastes that 20 were not biodegraded under laboratory composting conditions. Water content of the 21 composting mixtures was adjusted to be within the recommended values (50-60%) by 22 adding tap water before incubation. The composting matrix was left under natural 23 composting temperatures. Aeration flow rate and frequency were monitored and 24 adjusted during the process to avoid any limitation or excess in the oxygen 25 concentration that may affect the process. All the composting mixtures were manually 1 prepared according to the proposed values of the experimental design technique (Table  2 2) and were incubated for 30 days. 3 4 2.6. Sampling 5 During the incubation period, the performance of the process was monitored and 6 samples were collected after 10, 20 and 30 days of composting in order to measure the 7 degradation rate in these periods. For sampling, the reactors were opened and the 8 reactor contents were manually well mixed. Then duplicate grab samples (20-30 g) were 9 taken. During each sampling, moisture content was adjusted, if necessary. Thus, the 10 composting mixture was moistened with tap water and remixed well to maintain water 11 content within the optimum values (50-60%). 12 The content of the PAHs in the composting mixture was determined after 9 extraction using a Soxhlet extraction process. Duplicated 10 g samples were extracted 10 using acetone/dichloromethane (1:1 v/v) as solvent during two hours. Afterwards, the 11 solvent was left to evaporate and then the remaining residue (extract) was dissolved in 12 10 ml of dichloromethane. A 1-µl extract of this solution was injected in a gas 13 chromatograph (GC8690N, Agilent, Spain) equipped with flame ionization detector 14 (FID) and a splitless injector. A Zebron ZB-5HT Inferno column (Agilent, Spain) was 15 used. Initial temperature was maintained at 50˚C for 1 min, and then it was increased at 16 a rate of 7˚C/min until 320˚C, then another rate of 20˚C/min until 400˚C was applied 17 and maintained at this final temperature for 5 min. The concentration of the PAHs was 18 determined after the calibration of the method with standard PAHs samples. 19 To investigate the volatilization of the PAHs during the composting process, 20 samples from the exhausted air were collected using Tedlar bag of known volume [21], 21 and then samples of 1 ml of that air were analyzed using the same GC methodology. 22 However, this test was simply used to check if part of the PAHs decrease is caused by 23 volatilization, but the actual amount of the volatilized PAHs could not be determined as 24 only small amounts of some low molecular weight PAHs were detected. 25 1

Other characteristics 2
The other characteristics of both soil and organic co-substrates including: 3 moisture content, organic matter content (OM), pH, electrical conductivity, organic 4 carbon, Kjeldahl nitrogen and humic matter fraction were determined on collected 5 samples according to standard methods [22]. To simplify the recording of the conditions and processing of the experimental 18 data, the factor levels were coded with the notations (-α, -1, 0, +1, α). The value of 19 α=1.414 was determined according to the number of the studied factors (2). The 20 experiment design technique was carried out as it is explained in the literature [23,24]. 21 Table 2  In this study several types of OFMSW compost were evaluated as an organic 9 amendment during the composting of PAHs-contaminated soil. Obviously, the available 10 high content of organic matter in these composts in comparison with the available 11 amount in the soil (Table 1) is thought to be needed to support and develop the 12 microbial activity during the bioremediation process. Meanwhile, these composts are 13 suggested to provide valuable populations of microorganisms that presumably can 14 degrade the contaminant [6]. One of the most important characteristics of the applied 15 amendments that play a major role in the remediation process is the humic matter 16 portion [24,25]. Analysis of the composts showed that humic matters are available 17 among them, but their portions are different according to their stability degree. Indeed, 18 humic matter as part of the compost organic matter was found to be increased with 19 stability degree in the sense that, the more stable the compost was the higher humic 20 matter content was observed. However, compost C deviated from this fact to a small 21 extent, which might be attributed to its high content of organic matter ( Table 1). The 22 other characteristics are almost considered within the acceptable levels for such process. 23 Generally, the growth factors in the used compost are better than those in the soil; 24 P r e -p r i n t consequently, these organic amendments may have a major role in improving the 1 degradation of the contaminants. x x Depending on these equations the regression coefficients (R) of Y 10 , Y 20 and Y 30 were 11 0.83, 0.53 and 0.6 respectively. The obtained regression coefficient after 10 days 12 represents a good regression model, whereas those of 20 and 30 day represent a non 13 perfect regression. In all cases the P values describing the significance levels were not 14 concluding (P>0.05). However, although these statistical values are not within the 15 preferable values to describe the process, the constant variance test indicated that these 16 can be used to predict the degradation rates within ±10%. 17 18

The composting process 19
Temperature variations of the composting materials with time in some 20 experimental runs are illustrated in Figure 1. A lag phase was observed even though its 21 duration varied among the treatments, but it was clear in run 9 where about two days 22 were needed to stimulate the microorganisms. As a result of the microbial activity, 23 temperature began to rise to thermophilic ranges that were achieved during the first 24 week in the treatments when less stable composts were used, but it was always in the 25 mesophilic ranges when more stable composts. It is believed that the achieved 1 thermophilic temperatures in the beginning of composting were attributed to the 2 sufficient amounts of easily degradable materials [19,26]. This assumption agreed with 3 results obtained regarding the organic matter degradation (Figure 2) that showed a 4 notable decrease in this stage for the less stable composts. Moreover, high aeration and 5 frequencies were needed during the first stage of the process especially in the less stable 6 composts (data not shown), which implied that the microbial activity was intense. 7 However, as the materials became more stable and the process entered to the cooling 8 phase, less amount of air was needed. The temperature increases as well as the reduction 9 in the organic matter fraction during the whole process are the most important evidences 10 of the process [17,26]. 11 12

PAHs degradation 13
The degradation of the PAHs was assessed during the entire incubation period 14 (30 days). Figure 3 presents the remaining PAHs throughout the different runs. By the 15 end of the incubation period, high rates of degradation (76.11%-96.53% as total PAHs) 16 were achieved among all the experiments except the run 6 where only 45.8% was 17 achieved. For instance, during the first 10 days the highest rate of degradation (92%) 18 was observed in run 2, whereas the lowest rate (18%) was in run 6 during this period, 19 and in the other runs it was within 40%-80%. During the remaining period, a low rate of 20 degradation was observed. The contrast among the different treatments could be clearly 21 visualized during the first 10 days of incubation as different degradation rates were 22 obtained. Thus, the compost stability appeared as an effective factor especially when 23 treatments with the same concentration are to be compared (run 5, 8 and 9). In run 9, 24 which had the most active compost, almost 40% of the PAHs were degraded during that 25 period (10 days). However, almost the double degradation rates were obtained when 1 more stable compost was used. It is worthy to indicate that less stable composts got 2 stabilized with more incubation time. Consequently, degradation rates with these 3 composts improved with time, for instance, degradation rate of 76% after 30 days was 4 obtained in run 9. Nevertheless, this rate of degradation was still less than those 5 obtained under the same conditions when more stable compost was supplied. On the 6 other hand, the rates of degradation were found to be varied under the different 7 concentrations, which imply that the degradation process is also influenced by this 8 factor. In general, the PAHs degradation was influenced by the two factors, where the 9 used amendments were able to enhance the degradation process to a great extent. In general, the rate of degradation during the first 10 days was faster compared 23 to the rest incubation period where less degradation occurred. In this sense, the organic 24 contaminants could be sequestrated into the matrix of the soil as this process is 25 generally a function of time [28]. Analysis of exhausted air samples indicated that very 1 small amounts of some low molecular weight PAHs were volatilized during the 2 thermophilic stages (especially for temperatures over 50˚C), thus a portion of these 3 PAHs reduction is due to volatilization but this amount is so small compared to that 4 resulting from the biodegradation as these elevated temperatures remained for about one 5 week in the reactors with less stable compost. However, the majority of the experiments 6 were in the mesophilic ranges ( Figure 1) where no or negligible volatilization occurred. 7 It was also reported that although a portion of the total petroleum hydrocarbons 8 reduction is due to volatilization, abiotic loss has been reported to be generally less than 9 10% at 25˚C in the first 30 days [29]. 10 11

Effect of compost stability on PAHs degradation 12
The response of the PAHs degradation percentages under different composts 13 stability degrees denoted by DRI (0.37-4.55 mg O 2 g -1 OM h -1 ) are illustrated in Figure  14 4. As shown, the process response is changed when different composts were used as 15 well as during the different composting stages indicating that the compost stability is 16 one of the factors that influence the process performance. This effect was significant 17 during the first stage of the composting process (10 days) as the applied composts had 18 to pass different stages according to their composition and dominant microbial 19 enrichment. With compost E (run 9), which is the most active one, only 40.13% of the 20 total PAHs was degraded comparing to the other composts (A and C) under the same 21 conditions (run 5 and 8) where 70.4% and 73.79% of degradation were achieved 22 respectively. The same fact was also observed in run 1 and 3 which demonstrated more 23 degradation as more stable compost was used. Without doubt, these observations are of 24 great interest when the efficiency of the composting technology is to be evaluated with 25 other technologies. However, the highest rate of degradation (92.2%) was observed in 1 run 2 indicating that the PAHs concentration has its influence in this case. For instance, 2 Oleszczuk [30], observed that the influence of the composting process on the 3 contribution of the potentially bioavailable fraction of the PAH depended on the stage 4 of the experiment. 5 Regarding the composting process and as commented before, the thermophilic 6 temperatures are thought to be not suitable for the microorganisms needed to attack the 7 PAHs contaminants as the lowest degradation rate was observed (run 9). These 8 temperatures were reported to inhibit the degradation process [31-33]. Contrarily, the 9 mesophilic temperatures obtained under the same conditions gave higher rates of 10 degradation indicating that these temperatures and the dominant microorganisms under 11 these conditions are preferable for degradation of such compounds. However, other 12 studies are not coincident with these observations [34]. 13 The organic matter decrease was proportional with the stability degree, where 14 more reduction occurred in compost E. This reduction shows that these types of 15 composts still have a considerable amount of easily degradable matter which was 16 preferable by the microorganisms rather than other sources of nutrients like PAHs. 17 Accordingly, the mass loss after composting is a suitable evidence indicating the bio-18 oxidation of the composting matrix. 19 Among the most important properties correlated with the compost stability is the 20 available amount of the humic matter as part of the organic matter. This matter was 21 found to increase with stable compost (Table 1) where more stable composts had more 22 humic matter. Sorption of the organic contaminants with the soil particles usually 23 decreases the degradation rates as these contaminants become less accessible to the 24 microorganism. However, it was found that the humic matter increases the 25 bioavailability of the organic compounds and it can behave as surfactant during the 1 remediation process, which reduces the bond between the soil and PAHs. The more 2 degradation rates with more stable compost confirm this hypothesis and the PAHs were 3 easily desorbed from the soil particles thereby the degradation was stimulated. Plaza et 4 al. [25], demonstrated that during the composting process, the changed underwent by 5 the humic matter are expected to facilitate the microbial accessibility to PAHs. These 6 experiments concurred well and confirm such suggestions. It was clear in the remaining 7 composting period that the degradation process continued among all the experiments 8 and better results had been obtained with more incubation time as the organic matter got 9 more stabilized and the microorganisms were more acclimatized especially with less 10 stable compost. The humic matter found to be more effective to increase the degradation 11 rates than the high temperatures although it is well-known that the produced high 12 temperatures usually increase the kinetics and desorption of such compounds. 13 concentrations. The lowest degradation rate (18%) after 10 day of incubation was 19 observed in 6 th run that has the lowest concentration (0.1g/kg), where the highest rate 20 (92.21%) was obtained in the 2 nd run that has a concentration of 1.7g/kg, where 21 degradation rate of 66.5% was achieved with the highest used concentration (2g/kg) 22 during the same incubation period. By the end of the process (30 days), the degradation 23 rate in the 6 th run (0.1g/kg) still maintained its order as the lowest achieved rate 24 (45.8%), where it was able to achieve 80.9% with the highest applied concentration 25 (2g/kg). However, when comparing the rate obtained with highest concentration, it was 1 less than the other obtained rates where less concentration were used, therefore, the 2 concentration levels are considered crucial when composting process is to be used. For 3 this reason, when low concentrations are present, these concentrations are thought to be 4 below the levels that are assumed to begin the degradation process as the 5 microorganisms start with easily available materials and as these materials depleted 6 quickly before the degradation take place, it will be difficult to keep the required 7 activity. These results agreed with those obtained in [15], where concentration of low 8 PAH did not degrade even when the system was supplanted with additional carbon 9 sources. Furthermore, JØrgensen et al. [35] argued that the degradation of hydrocarbons 10 is governed by first-order kinetics, where the degradation rate of a compound is 11 proportional to its concentration. However, this argument may be validated to some 12 limits as the microbial activity could be affected (retardation or inhibition) when high 13 concentration is available. In this study when the results of high concentrations are 14 compared to other presenting lower concentrations, it is better to assume that retardation 15 conditions were noted. 16

Conclusions 18
During the composting process, it was clear that the potentially available PAHs are 19 influenced by the compost stability and the composting stages as a consequence. 20 Accordingly, the following conclusions were deduced: 21 1. The observed different behaviors during the first 10 days of composting 22 demostrate that less stable compost is not adequate for this type of remediation, 23 but more stable ones can promote the degradation quickly when the process is 24 well controlled. 25 1 available for degradation by the microbial activity. Indeed, humic matter was 2 more effective to accelerate the degradation rates than the high temperatures. 3 3. By the end of the process, experiments with the less stable compost were able to 4 improve their behavior as the composted materials were more stable, but their 5 results were still less favorable than those obtained with more stable compost. 6 4. PAHs concentration was found to influence the process mainly when low 7 concentrations are available, where the lowest degradation rate was obtained. 8 5. Both of the studied factors (compost stability and PAHs concentration) had a 9 direct effect on the process behavior; therefore, before carrying out the 10 composting process, initial knowledge about the available conditions may help 11 to have an estimation about the process performance and consequently the 12 P r e -p r i n t     P r e -p r i n t