The role of Serendipita indica and Lactobacilli mixtures on mitigating mycotoxins and heavy metals’ risks of contaminated sewage sludge and its composts

Accumulation of the Municipal Sewage Sludge (MSS) is considered as one of the most harmful renewable ecological and human health problems. MSS is a renewable resource that could be used as a soil organic amendment. This study aims to reduce the Heavy Metals (HMs) from the sludge content and sludge compost. Furthermore, this study is considered the first to assess the mycotoxins content in sludge and sludge compost via a new biological treatment using the fungus Serendipita indica or a mixture of lactic acid bacteria, thus providing safer nutrients for the soil amendment for a longer time and preserving human health. The HMs and mycotoxins were determined. The results exhibited that the biotic remediation of bio-solid waste and sewage sludge compost succeeded; a new bio-treated compost with a very low content of heavy metals and almost mycotoxins-free contents was availed. Also, the results indicated that the Lactobacilli mixture realized the best results in reducing heavy metals contents and mycotoxins. Afterward, S. indica. biotic remediation of bio-solid waste and sewage sludge compost minimized the health risk hazards affecting the human food chain, allowing for the different uses of sludge to be safer for the environment.

preparation of samples for mycotoxins detection. Representative homogenized subsamples were taken from six points of the four tested samples.
Determining the mycotoxins content. Preliminary mycotoxins are qualitatively detected using Thin-Layer Chromatography (TLC) according to Gilbert and Anklam 35 . Then, mycotoxins are quantitatively detected using a detection method in soil according to Uhlig et al. 36 with certain modifications. MSS samples, 20 g each, were extracted with 100 ml methanol-water (9:1). Then, the samples were blinded then centrifugated at 3,000 RPM, and each supernatant was purified using Whatman filter paper (40 Millipore's). Mycotoxins occurrence in the samples were determined using TLC plates and two eluents TEF (toluene/ethylacetate /90% formic acid 5:4:1.v/v/v) and TAM (toluene/acetone/methanol 5:3:2, v/v/v) according to 37 was applied. The developer Anis (0.5% p-anisaldehyde in methanol/acetic acid/conc) and sulfuric acid (17:2:1 v/v/v) was heated for eight minutes at a temperature of 130 °C. The dry-developed TLC plates were viewed under longwave (366 nm) and shortwave (254 nm) UV light and compared with the published data on color and Rfg values (Retardation Factors relative to Griseofulvin for some known mycotoxins 37 . The present mycotoxins types are registered. After determining the type of mycotoxins, A phenyl hexyl column was used to separate each mycotoxin in the final MSS extract according to to 38 with certain modifications. Each mycotoxin was analyzed using High-Pressure Liquid Chromatography (HPLC) with fluorescence detection. Mycotoxin standards are brought from Sigma Aldrich, Cairo bureau.

Confirmatory test.
To eliminate other undesired co-extracted matters from MSS, the hair bait analysis technique was applied according to Jogawat et al. and Nosratabadi et al. 29,39 with further development. The pH values of MSS were measured according to 40 . The removal of horse-hair fatty traces was done by soaking them for 24 h in diethyl ether and rinsing them for 4-5 times with distilled water followed by air drying. Keratinophilic fungi were isolated by the hair-baiting technique where horsehair was used as a keratin bait source. 12 sterile Petri dishes were half filled with MSS and three pieces of marked horsehairs tips with colored sterilized stickers which were 2-3 cm each. The stickers were spread over the surface of MSS. The MSS samples were moistened with sterile distilled water and then were baited by burying sterile marked horsehair pieces in the MSS. These dishes were incubated at a temperature of 30 °C for 15 days then the horsehair pieces were gently removed to inoculate three potato dextrose broth conical flasks, which were 100 ml each, supplemented with streptomycin (30 mg/l.) and then incubated at a temperature of 30 °C for 7 days. At the end of the incubation period, the same phenyl hexyl column was used and each mycotoxin was detected using the HPLC apparatus with fluorescence detection according to 38 .
Screening of total heavy metal elements in the raw MSS samples. Heavy metals were determined in the representative samples using the analytical device ICP-ES Agilant 5100VDV of Inductively Coupled Plasma Atomic Emission Spectroscopy (ICPOES), an analytical technique used for the analysis of trace metals with high accu- www.nature.com/scientificreports/ racy and precision and low detection limit starting at ppb levels to the high concentration of ppm. The metals' analysis process and quality control were carried out according to 41,42  Biotic treatment of raw municipal sewage sludge. The effects of the biotic treatment in reducing mycotoxins contents and heavy metals (HMs) concentration levels in raw MSS. Testing the activity of Serendipita indica in low and high pH solutions. The optimal pH for growing S. indica to obtain the higher fungal growth mat mass was tested to assess the extent of the enhanced probability of reducing mycotoxins and removing HMs.
The experiment was carried out by growing S. indica on Complete Media (CM) 50 ml under many acidic and basic pH values at 4, 5, 7.8, and 8.2, successively, for 15 days at a temperature of 25 °C. At the end of the incubation period, the fungal growth mat mass was dewatered and weighed. Then, the weight of S. indica growth mass was registered as showed in Table 1. After determining the optimal pH for S. indica growth. The Complete Media (CM), 150 ml each, was adjusted at the determined pH then was inoculated with S. indica and incubated at a temperature of 25 °C for 15 days.
The effects of Serendipita indica in reducing mycotoxins contents and heavy metals (HMs) concentration levels in raw MSS. Six cylinders, 2000 ml each, were used in this experiment. Each cup contained 300 g MSS, 1,350 ml deionized sterilized water, and 150 ml medium of S. indica growing on potato dextrose medium broth under the determined pH value. These parameters were utilized to reach a final liquid volume of 1,500 ml. After that, each flask was covered with a plastic cover 3 cm in depth, then incubated for 15 days at a temperature of 25 °C to simulate the real situation in the treatment process a covered tank is used; each tank is 100 kg each. Control samples, three cylindrical cups 1,000 ml each, were used in all the experiments, which consisted of 100 g MSS + 350 ml distilled deionized water or/and 150 ml potato dextrose broth adjusted to a pH value of 4. At the end of the incubation period, the fungal growth mat mass growth was gently removed. The excess water was disposed of, and then the treated sewage sludge silt was obtained. The mycotoxins were detected using the HPLC apparatus with fluorescence detection according to 38 and the heavy contents were gauged according to 41,42 .
The effects of LAB mixture in reducing mycotoxins contents and heavy metals (HMs) concentration levels in raw MSS. Only acidic pH is used in this experiment because the LAB is known to be more active and effective in an acidic pH value of 5 according to literature 43  composting processes of MSS. This experiment's main target was to minimize the mycotoxins and heavy metals contents in Municipal Sewage Sludge (MSS) to reduce the pollution of soils, groundwater, and plants and prevent them from reaching the human food chain. The composting processes of MSS were carried out under aerobic conditions using three composting methods as follows: The roles of conventional MSS compost. Pretreated MSS compost, and biotic MSS compost in reducing mycotoxins contents and heavy metals concentration.
Preparation of rice straw and clayey soil samples. Samples of rice straw and clayey soil were taken for mycotoxins estimation and heavy metals determination according to Ilieva et al. and Zhang et al. 38,41 . Then, the samples were surface sterilized using sodium hypochlorite (75%) and washed with sterilized deionized water three times and oven dried for one hour at a temperature of 45 °C after getting rid of excess water.
the conventional composting processes. The conventional composting process in the field was mimicries under in vitro conditions but within a small scale 45 . The Dried Municipal Sewage Sludge (DMSS) was taken then distributed into a pile. Each pile (40 cm in height × 75 cm in length and 2.25 to 2. 50 kg in weight) contained DMSS covered with a clay soil layer then a rice straw layer was added then another DMSS layer. The process was repeated until getting a large pile or heap as recommended according to 46 was formed. The compost was incubated under aeration conditions at a temperature of 65 °C for 50 days according to Lu et al. 46,47 . At the end of the incubation period, a representative sample was taken for mycotoxins estimation and heavy metals determination according to Ilieva et al. and Zhang et al. 38,41 .
The composition process with pretreated MSS. The modified composition process was carried out using pretreated DMSS with S. indica and/or LAB mixture under the same in vitro conditions with the same steps of the www.nature.com/scientificreports/ conventional composition process as mentioned above. After 50 days, a representative sample was taken for mycotoxins detection and heavy metals determination as mentioned above.
The biotic composition processes. The biotic composition process was carried out using pretreated DMSS with S. indica and/or LAB mixture with the addition of S. indica and/or LAB mixture cultures as follows: pieces of sterile gauze (10 × 20 cm each) were planted separately in 500 ml broth culture of each of the LAB mixture and/ or S. indica and incubated for seven days at a temperature of 25 °C. At the end of the incubation period, the inoculated gauze was gently removed and placed between layers of the compost pile as an additional layer with a ratio of 1:6 layers under the same in vitro conditions with the same steps in both conventional composition and pretreated methods. After 50 days of incubation under aeration conditions, according to Stegenta et al. 5 the gauze was removed and representative samples were taken for mycotoxins detection and heavy metals determination as mentioned above.
Statistical analysis. The first experiment was carried out in three replications in a completely randomized design. Meanwhile, the second experiment was carried out in three versions in a randomized complete-blocks design. The data were subjected to statistical analysis using a Costat computer package (CoHort Software, Berkeley, CA, USA). One-way completely randomized and/or randomized complete blocks ANOVA were used and a comparison between the resulting data was done using Least Significant Difference (LSD) and the correlation between the tested treatment and the removal process was calculated according to Duncan's multiple range test, which was applied to compare the treatment mean values according to 48 . ethical approval. "This article does not contain any studies with human participants performed by any of the authors".

Results
The current results will be divided into two partitions as follows: experiment i: assessment of mycotoxins contents and total heavy metals concentration in raw municipal sludge and testing the effects of biotic treatments on reducing their contents. The effect of the medium's pH value on the growth of S. indica. During the current study, different pH values were tested to study their effect on the growth of S. indica. The results in Table 1 and Fig. 2 illustrate that the difference in pH values significantly affected the S. indica growth and pH value 4 achieved the highest fungal growth mat mass (9.72 g), while pH value 8.2 recorded the lowest value which was 7.81 (g) with L.S.D. 0.05 = 1.13. So, the growth medium of S. indica, which will be used in all the experiments of this current study, will be adjusted to pH value 4.
Estimation of mycotoxins contents in raw municipal sludge. The results in Table 1 and Fig. 3 illustrate the following: The occurrence of two mycotoxins imposes the probability of being produced by more than one fungus. So, the action of S. indica and LAB mixture against these fungi was different. Moreover, the increase in afLAB 1 and viomelline concentrations during the incubation in the PDA medium indicated that the natural biotic flora of MSS is still active after all the treatment steps such as coagulation treatments with polyacrylamide. Furthermore, in the case of aflatoxin B 1 , the reduction of afLAB 1 amounts by both S. indica (in acidic pH) and/or LAB mixture was not significantly different. The results in Table 1 indicated that S. indica was more effective in reducing viomelline than LAB mixture which indicated that the capability of the fungus in reducing mycotoxins varied according to the mycotoxin type. The sensitivity of fungus-producing mycotoxins to S. indica antibiosis and LAB's enzyme system was evident.
According to results as illustrated in Table 1 and Fig. 3, the aflatoxin B 1 was 69 part per billion (ppb) and viomelline was 3,400 ppb in raw sludge (the control group), with the selected pH value 4 as pointed in Fig. 3. Using S. indica decreased the Aflatoxin B 1 to 6.240 ppb compared with 6.93 ppb under LAB mixture with no significant variations between both treatments. Also, these results detected that S. indica decreased the viomelline (219 ppb) more than the LAB mixture which was 293.34 ppb as recorded in Table 1. While under the high Table 1. Effect of biotic treatments on mycotoxin contents in sludge. Treatments with the same letters are not significantly different. where the pearson Product Moment Correlation coefficient of the two mycotoxins and the inhibition process are significant were S.E of r 0.103593for aflatoxin and 0.1177833for viomellin and p(r = 0) 0.000*** for both mycotoxins.

Mycotoxins
Raw sludge (control) The effects of biotic treatments on heavy metals concentration in raw sludge. The results are exhibited in Table 2 and Fig. 4 illustrated that the effectiveness of each treatment in removing heavy metal (HMs) elements varied according to the element type. Furthermore, in the case of S. indica, pH = 4 was more effective in reducing the HMs concentration except for cadmium which showed the lowest value at 2.41 compared with the control value 3.43. The removal of arsenic (As) was higher than mercury Hg > Cd > Zn > Al > Cu > Pb > Fe. An efficacy percentage of 96.59% was evident for heavy metal concentrations reduction. Whereas, at basic pH values, the S. indica efficiency for removing or digesting heavy metals was as the following order: As > Hg > Cd > Al > Zn > Pb > Fe > Cu. These heavy metals have an atomic density higher than 4 g/cm 3 , and these include lead (Pb), cadmium (Cd), zinc (Zn), mercury (Hg), arsenic (As), copper, and Iron. The plant absorption of these minerals and their accumulation in food crops are damaging human health.
In the case of the LAB mixture. This treatment realized the highest efficacy ratio in removing Al, Cd, Cu, and Fe in comparison with S. indica. The efficacy of the LAB mixture and S. indica in removing arsenic was the highest. Unfortunately, despite the success of both the LAB mixture and S. indica in reducing concentrations of heavy metals in raw sludge, the concentrations of these heavy metals, except Cd and Pb, are still high compared to the permissible levels whether in air 50 , plants, or soil. On the other hand, for the treated MSS which could be added to soil to fertilize woody trees, the biological treatment of MSS with S. indica was more effective in reducing arsenic (As) than the LAB mixture. Meanwhile, the LAB mixture was only effective in reducing Al to be in the permissible level of HMs in soil (10,000-300,000 ppm). Both biotic treatments reduce Cd and Pb concentrations to be within the allowable limits of the European Union standards (3 and 300, respectively).
EXPERIMENT II: the effects of composting processes of raw municipal sewage sludge on mycotoxins and total heavy metals contents. Mycotoxins were determined quantitatively using HPLC and heavy metals were determined using ICPOES as stated in Table 3. It was shown that the mycotoxins contents (ppb) were viomelline (4,903.78 ppb), aflatoxins (20.90), and moniliformin (2,688) in rice straw, while viomelline and moniliformin were 0.0 in clay soil and aflatoxins was 2.293 ppb. Also, the data in Table 3 recorded the element concentrations. The results in Table 4 exhibited that the conventional process can reduce MSS mycotoxins content by 36.23%, but it was more effective in reducing viomelline than aflatoxin by 79.45%, which indicated that aflatoxin may be more heat stable than viomelline.
The results, as exhibited in Table 5, illustrated that the primary biotic treatment before the composting process with probiotics or/and S. indica reduced the concentrations of the occurred mycotoxins more than the conventional compost. S. indica was more effective in reducing the three occurred mycotoxins more than the LAB mixture. The efficacy ratio of mycotoxins reduction was higher in aflatoxin followed by viomelline then moniliformin. The biotic composting process realized the higher mycotoxins reduction percentages in comparison with the other composition processes. The composting process with S. indica eliminated aflatoxins followed by moniliformin then viomelline, whereas LAB was the best in completely removing viomelline followed by aflatoxins, and the biotic composition process was safer to be added to soil followed by pretreated composting process Our results coincide with those of who reported that Lactobacilli could remove aflatoxin B 1 with a removal rate of 50-80%. But our treatments with the LAB mixture and/or S.indica are approximately similar to those of Huang et al. ratios (65.68 for LAB to 68.28 for S.indica) but the biotic composting process was more effective (99.26) in case of LAB to − 100% in case of S.indica. Furthermore, according to Gourama and Bullerman 28 , The mixture of Lactobacilli species not only inhibited aflatoxin production but also completely inhibited the germination of mold spores. www.nature.com/scientificreports/ The data in Table 6 and Fig. 5 demonstrated the followings results: The composting process of MSS was vital to the minimization of the HMs concentrations more than raw MSS. The HMs removing effectiveness and efficiency occurred in this order: the biotic composting process > pretreated composting process > conventional composting process. Each composting process differed in their HMs-removing efficacy according to the removal elements. In the case of the pretreated composting process, the LAB mixture was more effective than S. indica when interacting with Al and Cd, whereas this effect was similar to S. indica's considering As, Fe, and Cu. On the other hand, the removal efficiency of S. indica was higher in the case of Pb, Zn, and Hg. In the case of the biotic composting process, the LAB mixture was significantly effective in reducing Al, Fe, zinc, and Pb rather more than S. indica's binding efficacy. Meanwhile, both of the LAB mixture and S. indica were similarly effective in reducing As, Cd, Cu, and Hg. All resulting heavy metal elements concentration, except Al, were below their normal range in soil and plant according to their permissible limits recommended by WHO, FAO, Dutch, Austrian, Russian, and European Union standards. These results, as illustrated in Tables 5 and 6 and Fig. 5, indicated also that the biotic compositing can provide nutrients for soil amendment safely. These findings were closely in agreement with those of 51 who reported that the microbial addition during composting is considered highly effective due to the production enhancing of different enzymes resulting in rate acceleration of waste degradation. After 25 days, the biodegradation ability of these microbes was not affected after being added to the soil. Our results match 51 who used Aspergillus niger to removing heavy metals via a bioleaching process. Even though the performance of their method was more effective in reducing Pb and Zn more than our treatments, our method was more effective in reducing Fe, Cu, Cd, As, and Al. Moreover, the bioleaching mechanism augments the metal solubilization which increases their mobilities and, consequently, their bioavailability. Alternatively, biological agents, such as Phanerochates chrysosporium and S.indica which have been used in removing Pb and Cu, respectively 31 , can accumulate the high concentration of heavy metals in the nontoxic forms and be capable of reducing their toxic effects by using them during their physiological metabolism.On the other hand, the LAB mode of action in removing heavy metals was based on H.M binding capacity 22 which decreased their bioavailability.

Discussion
The usage of the LAB mixture or S.indica in removing mycotoxins not only minimized or inhibited the mycotoxins naturally contaminated Mss but also exhibited how their metabolites affect the molds' cell wall. Meanwhile, the competition between S. indica and other filamentous fungi-contaminated Mss on carbon sources caused a sort of carbon starvation which induced autolysis, cell wall degradation biopolymers, and mycotoxin production. Moreover, in this study, we tested different pH values to study their effects on the growth of S. indica. The current results showed the difference in pH values significantly affected the S. indica growth and pH value 4 achieved the highest mass mat growth (9.72 g). These findings agreed with those of 31 . For the estimation of mycotoxins contents in raw municipal sludge, the results showed that S. indica and the LAB mixture against these fungi were not similar and the increase in afLAB 1 and viomelline concentrations indicated that the natural biotic flora of MSS is still active after all the treatments applied in the treatment station. Furthermore, in the case of aflatoxin B1, the reduction of af LAB 1 amounts by both S. indica (in acidic pH) and/or LAB mixture was not significantly different. These findings were matched with those of 52,53 who found that fungal endophyte reduced trichothecenes and zeralenone in maize. Our results are also in harmony with those of 52,53 who mentioned that lactic acid bacteria prevents the Aspergillus flavus growth and the produced mycotoxin because of their ability to bind with aflatoxins in aqueous solutions.
Our results indicated that S. indica was more effective in reducing viomelline than LAB mixture and these results are meaningful like those of 54 which found that the LAB kefir binding aflatoxinB 1 is more effective than zearalenone and ochratoxin. Our results also coincided with 55,56 who mentioned that the LAB fermentum, LAB gallinarum, and LAB reuetri were only capable of binding aflatoxinB 1 among seven isolates, and the LAB plantarum strains produced enzymes such as α-amylase, esterase, lipase, and α-glucosidase which can differ in their activities according to the fungus-producing mycotoxin. According to our study on the effect of biotic treatments    www.nature.com/scientificreports/ arsenic (As) than the LAB mixture. Meanwhile, the LAB mixture was only effective in reducing Al to be within the permissible level of HMs in soil (10,000-300,000 ppm as maintained by 57 . This finding was closely in agreement with 58 who reported that that natural occurrence safe levels of Cd in Ethiopian soil were 2.3-2.5, whereas our resulted Cd concentration levels after biotic treatments were 1.8-2.4 and Pb were 209.9-220.6. Other HMs were higher than the permissible limits, which may occur due to the short period of incubation. This indicates that this biotic remediation process (after this applied incubation period) was not enough for removing all the HMs and for the safe addition of treated MMs to soil. Our results exhibited that pH values have an important effect on HMs concentrations. This finding was generally in harmony with those of 57 who reported that pH values significantly affect the solubility and mobility of As, Cr, Pb Cu, Ni, and Zn metals. Our data matched those of 59 who mentioned that Hg was adsorbed at low pH values more than high pH values. On the other hand, the determined heavy metals in raw MSS, which can be used as biofuel or to generate electricity, were present in very high concentrations. This indicates that this treatment of Mss was not enough to mitigate the HM risks for human health and air pollution. Our recorded data matched those of 60 who mentioned that air pollution is caused by the high levels of heavy metals in the atmosphere. Also, the values matched with those of the permissible levels of the European Commission. Furthermore, the permissible level of each heavy metal intake and uptake via respiratory, exposure, and dietary routes of humans differed between countries 61 . To study the effect of composting processes of raw municipal sewage sludge on mycotoxins and total heavy metals contents, the mycotoxins heavy metals were determined and the results showed that the mycotoxins contents of viomelline to be 4,903.78 ppb, aflatoxins 20.90, and moniliformin 2,688 in rice straw, while in clay soil and aflatoxins the value was 2.293 ppb. The results exhibited that the conventional process can reduce MSS mycotoxins content by 36.23%. Our findings were identical with those of 62 who mentioned that  Table 5. Comparison between the effects of the three-composting process on mycotoxins reduction. Where, ERMR % = efficacy ratio of mycotoxin reduction, treatments with different letters are significantly different. Data are analyzed as Anova Randomized complete blocks design with highly significant, p*** where the pearson Product Moment Correlation coefficient of the three mycotoxins and the inhibition process are significant were S.E of r 0.0649895 for aflatoxin 0.064468 for viomellin and 0.064468 for moniliformin so P(r = 0) = 0.0001***.  www.nature.com/scientificreports/ the reduction of mycotoxins is generally correlated with the degree of heat applied in the process; however, heat energy may not cause complete elimination of mycotoxins during the incubation period. Most mycotoxins can be reduced at temperatures above 150 °C. Our results can relatively coincide with 5 and 30 in their target to recycle the bio-MSS waste into a useful and beneficial product for soil. All resulting heavy metal elements concentration, except Al, were below their normal range in soil and plant according to their permissible limits recommended  www.nature.com/scientificreports/ by WHO, FAO, Dutch, Austrian, Russian, and European Union standards 63 . So, this conventional composting process was safer for soil addition. Our findings were in agreement with those of 64 who reported that composting process serve to eliminate heavy metals and phytotoxic substance. The treatment of raw primary treated MSS or bio-solids with probiotic (LAB mixture) and/or fungus was effective in reducing both of mycotoxins and heavy metals, but, unfortunately, the concentration of some heavy metals after the biotic treatments are still higher than the allowable limits in air, soil, and plants, which may be the case due to the short period of treatment. Our results showed that the composting process of MSS was vital to reducing biotic and abiotic pollutants. Furthermore, the biotic composition process was the best treatment in removing both mycotoxins and heavy metals from municipal sewage sludge. Although the levels of heavy metals vary greatly in their concentrations among different Mediterranean soils, our obtained HMs concentration in the present study are below the mentioned concentrations in different studies. The resulting MSS compost became safer for soil. Our study does not encourage the usage of raw municipal sewage sludge as woody trees fertilizer but tries to solve its harmful usage effects to the soil. According to European Union legislation on sewage sludge management, the sewage sludge can be added to the soil for a maximum of three years only, but our bioticaltreated compost can be used to save soil amendments with stabilization of organic substrates. According to 65 , the composting of MSS is recommended as a way of transforming a less stabilized waste into a beneficial material. The biotic composting treatment with S. indica or the LAB mixture is recommended; they are very cheap and economically available composting processes, especially in developed countries. Composting is considered to be a vital technology to recycle biodegradable waste while generating a useful product.