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Valorization of ash and spent mushroom substrate via solid-state solubilization by Acidithiobacillus ferrooxidans
Waste Management 87 (2019) 612–620
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Valorization of ash and spent mushroom substrate via solid-state solubilization by Acidithiobacillus ferrooxidans Agnieszka Saeid a,⇑, Ami Patel b a b
Department of Advanced Material Technologies, Faculty of Chemistry, Wroclaw University of Science and Technology, Gdanska 7/9, 50-344 Wroclaw, Poland Mansinhbhai Institute of Dairy & Food Technology (MIDFT), Mehsana-384002, Gujarat State, India
a r t i c l e
i n f o
Article history: Received 18 October 2018 Revised 26 February 2019 Accepted 27 February 2019
Keywords: Solubilization of phosphates Acidithiobacillus ferrooxidans Ash SMS Solid-state solubilization Germination test
a b s t r a c t This work describes the possibility of utilization of ash originated from the incineration of sewage sludge from the wastewater treatment plant with the 3rd stage of biological treatment, and spent mushrooms substrate (SMS) as a raw material for the production of the substrate for agriculture and horticulture with the property of slow-releasing of phosphorus via solubilization process. Ash was mixed with SMS in different ratios (1, 5 and 10%), where SMS was used as a substrate/medium – the source of nutrients necessary for the growth of bacteria Acidithiobacillus ferrooxidans (A. ferrooxidans), while the ash was used as a source of phosphorus. Solubilization of phosphorus from ash via solubilization process was conducted for 50 days. During this time pH, conductivity, as well as the concentration of available forms of phosphorus were monitored. Obtained results were compared with the control group deprive inoculation by A. ferrooxidans. The concentration of available to plants phosphorus (express as P2O5) was an average 1.5 times higher in the SMS inoculated with A. ferrooxidans in all considered groups. Observation confirms the possibility of utilization of treated SMS as a substrate in agriculture and horticulture as the utilitarian properties (weight and length of plant/root) of plants obtained in germination test were higher when compared with the control group where SMS without inoculation was used. Ó 2019 Elsevier Ltd. All rights reserved.
1. Introduction Current paper deals with the issue related to the valorization of wastes into valuable products what stay in line with the circular economy. What is more proposed solution could be the method of mitigation of the relation of phosphorus fertilizers production with phosphates industry (Schoumans et al., 2015, Roy, 2017). The phosphate rock is one of the critical raw materials according to an updated list published in 2014, hence alternative phosphorus sources should be proposed with suitable processing methods that allow for its beneficial application (Report on critical raw materials for the EU, 2014). A few European countries made the phosphorus recovery obligatory, such as Switzerland or Germany from sewage sludge and slaughterhouse, since these two flows of phosphorus seem to be the most concentrated ones (European Sustainable Phosphorus Platform, 2015). One of the environmental friendly treatment method of ash described in the literature, is their microbial solubilization that could be used to treat low quality phosphates rock as well as the ⇑ Corresponding author. E-mail addresses: [email protected] (A. Saeid), [email protected] (A. Patel). https://doi.org/10.1016/j.wasman.2019.02.048 0956-053X/Ó 2019 Elsevier Ltd. All rights reserved.
secondary P-bearing raw materials such as ashes, what results with liberation of phosphorus from structure that is not available to plants (Lukashe et al., 2019; Mupambwa et al., 2016; Wyciszkiewicz et al., 2017a, 2017b, 2017c). As a result of this mentioned substrate could be valorized into the phosphorus fertilizers, with the properties of biofertilizers, because of the presence of living beneficial soil organisms within it where in an appropriate manner, and under appropriate conditions, raw materials with microorganisms, along with the nutrient components would be combined (Wyciszkiewicz et al., 2017d, 2015a; 2015b). Another issue concern in this paper is the utilization of spent mushrooms substrate (SMS) as a source of nutrients that maintain the growth of beneficial microorganisms under optimal conditions. Since spent mushrooms substrate (SMS) and its efficient utilization is also an important issue, nowadays widely discussed in the literature (Zhu et al., 2012; Majchrowska-Safaryan and Tkaczuk, 2013; Nakatsuka et al., 2016), attempts to its application as a base, the source of nutrients and matter of keeping the proper conditions of growth of microorganism responsible for phosphorus solubilization from ash, were undertaken (Wyciszkiewicz et al., 2017d). SMS is often used as a one of the component in the cocomposting SMS with sewage sludge (Meng et al., 2018), pig manure (Meng et al., 2018b), dairy manure (Luo et al., 2018), sewage
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sludge, wheat straw (Meng et al., 2017) indicating a synergistic effect of co-digestion with SMS -improvement of maturity of the compost and reduction of emission of nitrogenous gasses and higher methane yield. For the effective use of SMS as animal feed or as a soil conditioner there is need to improve its preservative quality as it typically contains about 60% moisture, it putrefies easily because it contains nutrient-rich organic compounds (Kwak et al., 2008, Tasaki et al., 2014). All features considered as an obstacle in the case of its utilization in solid-state solubilization are considered as beneficial. The result of a combination of SMS treatment along with solidstate solubilization could mitigate the phosphorus problem and reduce the amount of generated and stored wastes originated from the food industry, such as SMS, as well as ashes originated from incineration of waste-sludge form wastewater treatment plant with 3rd stage of biological treatment. The aim of the present paper was to evaluate the effect of solidstate solubilization performed by A. ferrooxidans when as a base SMS mixed with the secondary P-bearing raw material were used on the concentration of available phosphorus (express as a P2O5). Further, obtained substrates based on the SMS (treated and not treated by A. ferrooxidans) were used as the substrate in the germination test to evaluate its influence on the utilitarian properties of the test plant. 2. Materials and methods 2.1. Materials In the experiment, spent mushroom substrate (SMS) (Fig. 1) and ash originated from incineration of waste sludge from wastewater plant Łyna in Olsztyn in Poland was used as a growth medium for A. ferrooxidans. Ash as a phosphate substrate was ground by blender and mixed with SMS. The content of SMS without the addition of A. ferrooxidans and SMS treated is presented in Table 1. 2.2. Bacteria and culture medium Phosphate sources were treated with A. ferrooxidans as a phosphate–solubilizing microorganism. Bacterial strain was obtained from Professor Zygmunt Sadowski from Wroclaw University of Science and Technology. A. ferrooxidans is an autochthonous strain of bacteria (F7-01) isolated from the tailings impoundment ‘‘Iron Bridge”, Poland (Wyciszkiewicz et al., 2017b). The concentration
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of bacteria in suspension that was used in the inoculation was 1.5 g L1 and 5 L of the suspension was used in the experiment as an inoculum (Fig. 1). 2.3. Solubilization experiment The test was conducted in six Eco-Composters with the capacity of 270 L (tumbler composter with the spherical shape) (Fig. 1). Ash (0.1 kg, 0.5 kg and 1 kg) and spent mushroom substrate (SMS) (10 kg) were mixed and soaked with 5 L of A. ferrooxidans suspension. As a control group three composters filled with ash (0.1 kg, 0.5 kg and 1 kg) and SMS (10 kg) mixed and soaked with 5 L of distilled water, were used. Every one or two weeks, samples after mixing of the stock were taken to measure electrical conductivity (EC) and were dried. Available and soluble phosphorus forms in SMS were determined. The solubilization experiment was conducted for 50 days. 2.4. Germination test On 12 dishes with 15 g of universal garden soil, 50 seeds were planted. As a plant, the sunflower was selected. All plates were stratified at 0 °C for three days. The seed trials were prepared according to the International Safe Transit Association (ISTA) Standards. Three groups in four replicates were prepared: control group (standard soil), a group with additional 20 g of untreated SMS and group with 20 g of SMS that was modified by solid-state solubilization of phosphates from ash by A. ferrooxidans. After the 7th day of field trials, the plants were collected, measured and counted. The whole mass of plants was dried to constant weight. 2.5. Extraction of P2O5 In order to investigate the efficiency and consequently bioavailability of phosphorus, P2O5 ammonium citrate and water-soluble fractions were determined. The experiment was carried out in two stages, by 1 h at 65 °C for ammonium citrate and 30 min at 25 °C for water, according to Regulation (EC) No 2003/2003 of the European Parliament and of the Council relating to fertilizers (method 3.1.4 Extraction of phosphorus which is soluble in neutral ammonium citrate, and 3.1.6 Extraction of water-soluble phosphorus). The contribution of soluble and available phosphorus was evaluated by the colorimetric vanadophosphomolybdate method described elsewhere (Wyciszkiewicz et al., 2017d).
Fig. 1. Scheme of the experiments.
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Table 1 (a) mineral content of SMS after the solid-state solubilization process; (b) other characteristic parameters of SMS. Element/parameters
Dose of Ash 1%
5%
10%
SMS
SMS treated
SMS
SMS treated
SMS
SMS treated
a) Macroelements, % N,% P2O5 (P) K2O (K) Ca Mg S Na
2.41 1.79 (0.784) 1.55 (1.45) 8.41 0.376 2.936 0.096
2.43 2.40 (1.04) 1.98 (1.86) 9.56 0.441 4.440 0.131
2.25 3.51 (1.53) 2.03 (1.90) 9.52 0.560 3.159 0.163
2.13 4.23 (1.84) 1.73 (1.62) 9.46 0.604 3.024 0.164
2.25 4.17 (1.82) 1.61 (1.51) 10.8 0.574 3.016 0.158
2.24 6.45 (2.81) 2.23 (2.09) 9.15 0.813 3.220 0.216
Microelements, mg kg1 B Ba Be Co Fe, % Mn Mo Sb Se Si
21.5 75.7 0.152 3.57 0.518 209 4.02 12.7 <4.5 (LOD) 486
24.3 83.1 0.122 2.19 1.340 237 7.65 13.1 <4.5 (LOD) 555
21.9 99.7 0.216 1.85 0.804 248 6.57 19.1 30.246 443
24.7 106 0.182 2.28 0.771 258. 4.56 <0.25 (LOD) 20.908 389
22.5 102 0.183 2.02 0.775 250 7.36 2.220 8.263 289
29.8 135 0.216 4.03 1.650 293 5.30 19.114 21.894 472
Toxic metal, mg kg1 Cr Cu Al As Zn Cd Pb
8.88 44.8 2237 6.55 210 <0.010 (LOD) 16.8
9.36 58.1 1764 <0.5 (LOD) 257 0.157 5.79
17.2 99.7 3619 <0.5 (LOD) 369 0.616 2.06
22.3 116 4017 17.3 448 0.501 13.18
21.4 107 3637 <0.5 (LOD) 419 0.508 3.01
38.0 202 5521 11.1 807 0.682 9.99
Others, mg kg1 Ti Tl V Ni Ag
171 19.5 5.15 4.94 0.065
161 10.4 3.58 7.49 0.114
268 15.4 8.973 8.80 0.795
337 3.87 6.979 14.1 1.218
303 <1.0 (LOD) 6.79 11.2 1.20
461 6.97 7.92 15.1 2.12
b) Conductivity0, mS cm1 Conductivity‘, mS cm1 SF, % C/N mg Cd kg1 P2O5
7.4 3.52 28.6 10.1 < 0.1 (LOD)
7.69 3.46 38.9 9.48 6.56
7.0 3.78 17.7 10.4 17.6
8.17 3.33 25.5 10.1 11.9
7.91 3.2 16.2 10.5 12.2
7.78 2.74 20.2 9.49 10.6
LOD – limit of detection.
2.6. C and N content analysis Elemental analysis (CN) of the raw SMS and treated SMS was determined using VarioMacroCubeElementar (C, H, N) analyzer. As a standard solution D-phenylalanine (C = 65.44%; N = 8.48%) was used. 2.7. ICP-OES analysis The samples were mineralized with spectrally pure 69% HNO3 (Suprapur, Merck) in a closed system by using microwave oven Milestone Start D. After mineralization, solutions were diluted to 25 g. The elements concentrations were determined by ICP–OES Varian-Vista MPX (Australia) with ultrasonic nebulizer CETAC U5000AT+. The results are the arithmetic mean of three measurements. 2.8. Statistical methods The results of the experiments were analyzed statistically using the program Statistica ver. 10. The normal distribution of the variable was tested using the Shapiro-Wilk test. The Brown Forsythe test was used to verify the homogeneity of variance (for a normal distribution).
For groups with homogeneous variance, test F of the significance of differences was performed. For distribution other than normal, Kruskal-Wallis test was used. Statistically significant differences were for p < 0.05. 3. Results and discussion The performed experiments were aimed to find whether it is possible to apply A. ferrooxidans in the solubilization of P- bearing secondary raw materials conducted in a solid-state manner. The experiment was divided into two parts. At the first stage, six variations of solubilization processes were conducted that allowed to obtain six substrates which differ with the presence of bacteria and the dosed of the P-bearing source used (Table 1a); in the second stage were tested as a substrate that potentially could be used in agriculture and horticulture. Furthermore, obtained formulations could be defined as fertilizer since the content of phosphorus in form available to plants (expressed as the P2O5) in obtained formulation were 1.2%, 1.3% and 1.7% respectively, for products obtained as a result of solubilization of SMS conducted with addition of 1%, 5% and 10% of ash. While the total content of phosphorus (express as P2O5) was 2.40%, 4.23% and 6.45% for increasing dose of ash applied in the experiments. According to the Regulation of the Minister of Agriculture and Rural Development (Journal of
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Laws 236, 29 October 2004) minimum quality requirements for the content of phosphorus (expressed as the P2O5) in phosphorus fertilizers is 1% (m/m). In this context, the obtained substrate in the form of treated SMS by solubilization process can be classified as a phosphorus fertilizer. In the previous experiments published elsewhere, the level of P2O5 reached 1.3% for dose 5% of addition of poultry bones when as a Phosphorus Solubilizing Bacteria (PSB), Bacillus megaterium was used (Wyciszkiewicz et al., 2016). One of the most concerned issues when considering the safety of the natural environment when applying fertilizers is the content of Cd (Jastrze˛bska et al., 2018). Recently, the European Union (EU) had already begun to apply more stringent restrictions on the content of Cd in fertilizers. Limits in mg Cd per kg P2O5 in fertilizers for EU will decrease gradually 60 40 20 (Rojo, 2017). So far those limit significantly varied between European countries, for example in Finland it is 21.5 mg Cd per kg P2O5, in Sweden 44, in Denmark 48, in Belgium 90, in Austria 120, while in Australia 131 and in Japan 148 mg Cd/kg P2O5 (Roberts, 2014). Cd present in fertilizer is originated from phosphate rock, which nowadays is the main resource used in the phosphorus industry. In the case of secondary raw materials originated for example from food industry exceeding the applicable limits is very rare, what was marked in the Table 1b where the ration of Cd per 1 kg of P2O5 in obtained substrate/fertilizer formulation is lower than 20 mg kg1. Another issue undertaken during the experiments was a C/N ratio evaluation. The obtained ratio for all considered level of addition of ash in the case of SMS with A. ferrooxidans was characterized by lower C/N ration when compared with the control group. Inoculation with A. ferrooxidans resulted in the lowering of carbon content, probably due to its utilization during the growth. The level of nitrogen was almost the same in the control and experimental group. Similar findings were reported elsewhere (Wyciszkiewicz et al., 2017d).
The set of six experiments of treatment of spent mushrooms substrate mixed with the ash applied at three different doses 1, 5 and 10%, was conducted for 50 days. The control group contained the SMS mixed with ash but without A. ferrooxidans. The inoculation of experimental composters by A. ferrooxidans affected the significant increase of P2O5 available to plants as a result of the production of sulphuric acid that was responsible for releasing of phosphorus from not available to plants form. To describe the changes in P2O5 concentrations during solubilization, the proposed model was used (Eq. (1)):
C P2 O5 ¼ f ðt Þ ¼
C Pav2 Oailable t 5 max
ð1Þ
K þt
where the C Pav2 Oailable , mg L1 is the maximum of phosphorus concen5 max tration express as P2O5, K is the constant that expresses time when
. When K, day is lower, the curve C P2 O5 , is equal to ½ of C aPv2 Oailable 5 max changes more rapidly i.e. solubilization is faster. Table 2 collects all evaluated model parameters both with the errors that express the fit of the proposed model to the experimental data. It was found that inoculation of a solid-state reactor filled with SMS and ash by A. ferrooxidans results in the increase of
C aPv2 Oailable by 58.4% in the case of addition of 1%, increase by 58.6% 5 max when 5% was added, and increase by 63.5% when 10% was used. All evaluated parameters were statistically significant with one exception of K constant evaluated for 5% addition of raw materials to the reactor inoculated with A. ferrooxidans. Constant K, was also effected by the inoculation by A. ferrooxidans. Evaluated K values for SMS treated by A. ferrooxidans were lowered by 44%, 19% and 31% when compared with the SMS not treated by A. ferrooxidansfor addition of 1%, 5% and 10% of ash, respectively (Fig. 2). It can be interpreted that the process of releasing available phosphorus (express as the P2O5) is faster when A. ferrooxidans was applied. 3.2. Solubilization factor
3.1. Solubilization The choice of A. ferrooxidans was intentional, because it is well established that this is a harmless mesophilic organism, and since the assumptions of performed studies was to develop the formula of substrate that could serve as a slow released fertilizer, that after application on the soil would be still subjected to action of bacteria, and as a result of this the release of phosphates would be observed. If thermophilic microorganism would be used instead, the issues on survival in the soil would have to be raised especially in the temperate climate zone.
The solubilization effect was expressed as the Solubilization Factor (SF, %) defined as the ratio (expressed as a percentage) of soluble P2O5 present in the solution to phosphorus (expressed as P2O5) introduced to solubilization medium in solid/not available form. Fig. 3 represents the influence of the dose of ash used in the solid-state solubilization process on the SF. The obtained data indicate the correlation between the dose of applied resource and solubilization efficiency what stay in the line with the previous findings (Wyciszkiewicz et al., 2017a; 2017b; 2017c). In this case the correlation was not statistically significant, r = 0.777
Table 2 C Pav ailable O5 max t Parameters of model C P2 O5 ¼ f ðt Þ ¼ 2Kþt describing the kinetics of changes of concentration P2O5 during the solid-state solubilization process (N = 5). italic- statistically significant parameters. Addition of raw materials SMS
1% 5%
p value
R2
v2
7.83 ± 0.83
0.001
0.992
0.238
29.7 ± 6.6 8.01 ± 1.04
0.011 0.002
0.980
0.483
13.3 ± 5.2 10.4 ± 1.0
0.063 0.001
0.983
0.628
K, day
16.3 ± 4.4
0.021
, g kg1 C Pav2 Oailable 5 max K, day
12.4±±1.2
0.000
0.995
0.0373
16.6 ± 4.3 12.7 ± 1.4
0.018 0.001
0.980
0.365
10.9 ± 4.1 17.0 ± 1.9
0.057 0.001
0.991
0.222
11.3 ± 4.0
0.049
Model parameter
Value ± SD
;g kg1 C Pav2 Oailable 5 max K, day , g kg1 C Pav2 Oailable 5 max K, day
10%
SMS treated
1% 5%
C Pav2 Oailable , g kg1 5 max
, g kg1 C Pav2 Oailable 5 max K, day
10%
C Pav2 Oailable , g kg1 5 max K, day
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Fig. 2. Kinetic of releasing of phosphorus (express as P2O5 extracted in neutral ammonium citrate) during the solid-state solubilization.
Fig. 4. The influence of the dose of ash on the final content of P2O5 available to plants (extracted in neutral ammonium citrate), mg kg1, during the solid-state solubilization process.
Table 3 Differences between the parameters: dry weight, length of plant as well as the length of root, determined in germination tests for three experimental groups, control (water), SMS not treated and SMS treated by A. ferrooxidans. Parameter
Fig. 3. The influence of dose of ash used in the solid-state solubilization process, on the solubilization factor (SF, %).
(p = 0.433 for SMS treated) and r = 0.899 (p = 0.289 for SMS). Higher initial dose of phosphorus raw materials causes higher final concentration of available to plants phosphorus (express as P2O5) but at the same time, lower efficiency express as an SF (Table 1b). Fig. 4 represents the influence of the applied dose of P-bearing sources and the presence of bacteria on the changes between the initial and the final concentration of phosphorus available to plants form (express as P2O5). It was shown that the solubilization even without the addition of bacterial cells is performed, probably by the native microbiota of SMS, nevertheless is much less efficient when compared with results obtained for A. ferrooxidans. 3.3. Germination test Obtained substrates after 50 days of incubation of SMS with A. ferrooxidans (SMS treated) or without (SMS non treated), were used as a substrate in the germination test on sunflower seeds. The sum of germinated seeds increase in all the experimental groups (SMS treated and not treated) both with the increase of applied dosage of P-bearing source at the solubilization stage (Table 3), moreover the sum of germinated seeds was higher for the group where the SMS treated by A. ferrooxidans was applied, with exception when
SMS
SMS treated
Median ± SD
Median ± SD
The average amount of germinated seeds (sum of all) Standard soil 6.33 ± 1.53 (19*) 1% 7.33 ± 1.03 (44) 5% 8.50 ± 0.84 (51) 10% 8.67 ± 0.82 (52)
– 8.17 ± 1.17 (49) 8.50 ± 0.84 (51) 8.83 ± 0.41 (53)
Dry weight, g Standard soil 1% 5% 10%
0.232 ± 0.000887 0.0206 ± 0.0016a 0.0214 ± 0.0010b 0.0238 ± 0.0033c
– 0.0243 ± 0.0028a 0.0271 ± 0.0052b 0.0306 ± 0.0059c
Length of plant, cm Standard soil 1% 5% 10%
7.46±±2.24d,e 11.43 ± 1.60d 11.95 ± 2.06 12.5 ± 1.49
– 12.20 ± 1.70e 12.35 ± 1.41 12.1 ± 1.92
Length of root, cm Standard soil 1% 5% 10%
5.31 ± 1.76f 6.39 ± 1.81 6.53 ± 1.53 6.55 ± 1.64
– 6.88 ± 1.44f 6.98 ± 1.29 6.74 ± 1.67
a–f
statistically significant differences p 0.05. N = 3.
*
5% of addition was applied - in this case, the amount of germinated seeds was equal with SMS not treated. The biomasses of plants were measured: length of green part and root as well as the dry weight. The medians of measured parameters were presented in Table 3, both with statistical differences between experimental groups (Fig. 5). Statistically significant differences were found between the dry weight of plants cultivated with the addition of SMS treated and not treated, for all applied doses. Further, the differences between the dry weight of the experimental groups increase as the dose of addition increase. The following increase of dry weight was found: 18%, 27% and 29% for 1%, 5% and 10% of the level of the P-bearing source in the treated SMS. Statistically significant differences were also found between the length of the
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617
Fig. 5. Box and whiskers plot of (a) length of plant (cm) and (b) length of root (cm) - for addition of 1% of ash; (c) length of plant (cm) and (d) length of root (cm) - for addition of 5% of ash; (e) length of plant (cm) and (f) length of root (cm) for addition of 10% of ash.
green part of the plants that have grown on the not treated and treated SMS, and when only standard soil was used. Between the groups where the plants have grown on the standardized soil, length of green part of plant were lower by 63% (p < 0.05) and by 53% (p < 0.05) when compared with plants that have grown on the of SMS treated by A. ferrooxidans 1% and on the SMS not treated, respectively. The lengths of the green part, as well as root,
were higher for the experimental group where the SMS treated was used with one exception of the length of the green part when 10% of SMS was applied, in that case, the group where SMS not treated was used plants were higher by 3%. Addition of treated SMS enriched with 10% of P-bearing source effected in inhibition of the growth of plant. In rest cases the length of the plants and roots were higher for the group where SMS treated was used, but
0.997
1.00
1.00
Zn Si
0.998 1.00
1.00
0.999 1.00
1.00 1.00
1.00
1.00
0.999
1.00
1.00
0.997 0.999
1.00
0.998
1.00
1.00
1.00
0.999 0.999 1.00
0.999
0.997
1.00
1.00
1.00 0.999 0.999 1.00 1.00 0.999 1.00
1.00
1.00 1.00 0.999 0.998
1.00 1.00
0.998
0.998 0.997 0.999
0.999
0.998
Se
1.00 1.00
Sb S
0.997
Pb P Ni Na Mo Mn Mg K Fe
0.998
Cu Cr Co Cd
0.999
Ca Be
0.999 0.997
Ba B
1.00
1.00 1.00
Ag Al As B Ba Be Ca Cd Cr Cu Fe K Mg Mn Na Ni P Pb S Sb Se Si SMS treated
0.999
As Al Ag
0.999 Ca Mo Si
The content of the elements in the plant biomass is strongly controlled by the mechanism of plant metabolism (Lima et al., 2017). The interactions between the elements present in the soil solution can be both antagonistic and synergistic and are involved in the metabolism of more than two elements. The type of interaction effects, whether antagonistic or synergistic are more concentration depended, and its definition is easier when exceeds some critical level of toxicity is observed. Since the applied spent mushrooms substrate did not deliver the nutrients in exceed level, observed interaction presents in the matrix (Table 4) even statistically significant, barely can find its confirmation in the literature (Kabata-Pendias and Pendias, 2000). To compare the influence of the form of substrate used in the germination tests, the ratio of the content of elements to its content in the substrate, which can be defined as a transfer factor was evaluated and presented in Fig. 6. The evaluated factor, in most cases, was comparable between the two considered groups where SMS and treated SMS were used. The significant differences were found for As, K, and Ni when the transfer factor was higher for control (not treated SMS), and for Cd and Se when the factor was higher for treated SMS. Smaller differences were found for Na, Sb, and Si and it was higher for control (not treated SMS) and for Pb when it was higher for SMS treated. It can be concluded that A, K, Ni Na, Sb, and Si are easier up taken from not treated SMS, while Cd, Pb and Se from treated SMS. The statistically significant correlations (Table 4) were found between the content of elements in the substrate (treated and not treated) and in the plants. In the case of not treated SMS by A. ferrooxidans, the statistically significant, negative and strong correlation was found between Ca and Ag, Ba, Be, Cd, Fe, S, Sb and Se, while in the case of treated SMS Ca was statistically correlated with Ag, Al, B, Ba, Ca, Mg and P but these correlations were positive. Probably the presence of A. ferrooxidans in the treated SMS resulted in the changes of the form of other than the Ca nutrients that became more available, since
SMS
3.4. Elemental content of plants
PLANT
those differences were not statistically significant. When taking into account the length of root, the best-applied level of addition of treated SMS would be 5% since in that case the length of root was the highest of all considered cases. Furthermore, for that dose of application the highest length of the green plant was also noticed. Even the highest dry weight was recorded for the addition of 10% of treated SMS. Improved parameters of cultivated plants were observed for the experimental group, where the SMS treated by A. ferrooxidans was used, which suggests that combination of solubilization process with spent mushrooms substrate is a potential method for the valorization of SMS as well as P-bearing secondary raw materials. Solubilization could be an alternative to composting which is widely used as a method of SMS valorization. The composting of SMS is conducted in the windrows with the size is for example of 1.5 m 2.5 m 1.5 m. It would be interesting to perform solubilization when composting occurring simultaneously, it could be possible if thermophilic kind of bacteria would be used since the temperature inside the windrows often exceeded 40 °C (the stage when thermophilic microorganisms are responsible for composting process). So far, in conducted experiments, mesophilic bacteria were used (optimal growth temperature 35 °C) such as B. megaterium (Wyciszkiewcz at al., 2016) and A. ferrooxidans (present paper). In the case of conjugation of SMS utilization with solubilization conducted by mesophilic bacteria, it would be crucial to keep the process temperature at a level when mesophilic organisms conduct the microbial processes. Because the experiment was conducted in the 270 L composter and only for 50 days, the composting was not initiated.
0.999
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Table 4 Correlation matrix of correlation coefficients (only statistically significant) between the content of elements in the plant and in the substrate used in the experiment, separated for treated and not treated SMS.
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619
Fig. 7. Influence of dose of ash on the changes of conductivity as an effect of solubilization performed by A. ferrooxidans (SMS treated) expressed in %.
Fig. 6. The influence of kind of SMS used in germination tests on the transfer of nutrients, as well as toxic elements from the substrate, to the plant biomass.
the content of Ca do not differ significantly between the experimental groups of substrate used in the germination tests (p > 0.05). The treatment of SMS by A. ferrooxidans resulted in the higher dose of available to plants phosphorus (express as the P2O5) as it was mentioned before. As a result of this, few statistically significant correlations were found between the content of phosphorus in the treated substrate and content of Fe, K, Mn and S in the plant’s biomass. In the SMS not treated such correlations were not found. Additionally, in the case of treated SMS, negative and strong statistically significant correlations were found between the Pb present in the substrate and K and S detected in the plant biomass, what demonstrates the toxic effect of Pb.
3.5. Electrical conductivity Fresh, not treated SMS characteristically has a high salt content, measured indirectly by electrical conductivity. The average salinity before the experiment for all experimental groups was 7.66 but at the end of the solubilization process decreased to 3.34, and this change was statistically significant (p < 0.005). When considering two experimental groups it was found that the decrease of salinity in the bacterial treated SMS was higher than that found in the SMS not treated. Fig. 7 shows that in the case of utilization of A. ferrooxidans the decrease of salinity is higher when the dose of applied ash is higher. While in the case of not treated SMS by A. ferrooxidans the opposite relationship was observed. The direction factors of regression curves determined for both discussed cases is equal – 1.49 (p = 0.059) for SMS not treated (negative correlation) and 1.09 (p = 0.0082) for SMS treated (positive correlation). This difference confirms the importance of the application of bacteria in the reducing of the salinity. A. ferrooxidans successfully utilize and decrease the content of inorganic as well as organic substances delivered by SMS. Salinity stress is a common environmental problem and an important factor limiting crop production. Salinity affects all plants in three ways: osmotic stress, toxic ion stress, and nutritional imbalances. Dissolved salts in the nutrient solution exert an osmotic effect that reduces the availability of free (unbound) water through physical processes that impair water extraction from the soil by roots (Syvertsen and Garcia-Sanchez, 2014). Spent mush-
rooms substrate recognized as wastes originated from agro-food industry is produced every year at the significant amount (Phan and Sabaratnam, 2012), and its no utilization can cause environmental problems for example due to nitrate lixiviation to aquifers (González-Marcos et al., 2015). Another crucial issue is its high salt content (Zhang et al., 2012) and moisture which are a significant limitation of its wide application in agriculture. At the same time SMS is rich in nutrients significant for plant nutrition such as N, P, K, and organic matter (Wyciszkiewicz et al. 2016). Application of spent mushrooms substrate as a base and at the same time as a source of nutrients crucial for the growth of bacteria, in the solubilization process is the promising approach for the valorization of SMS as well as P-bearing secondary raw materials in the form of substrate that has great properties as a soil conditioner or soil amendments used in the horticulture and agriculture. The previous finding proved that solubilization conducted by Bacillus megaterium in solid state manner where SMS was used, resulted in the double increase of the amount of available to plants P2O5 what was confirmed within germination tests where the plant cultivated on the SMS treated by B. megaterium was higher and heavier; moreover, the intensity of green color was improved as well (Wyciszkiewicz et al. 2016). 4. Conclusions During conducted experiments, it was confirmed that it is possible to utilize SMS as a base for solubilization conducted in a solidstate manner as a source of nutrients for growth of A. ferrooxidans that was responsible for solubilization of phosphorus delivered in the form of ash. The concentration of available phosphorus evaluated from solubilization process was higher in all considered doses of applied ash when compared with the control group. Obtained substrates from the control and experimental group were used in the germination test. Improved parameters of cultivated plants were observed for the experimental group, where the SMS treated by A. ferrooxidans was used, suggests that combination of solubilization process with spent mushrooms substrate is a potential method for the valorization of SMS as well as P-bearing secondary raw materials. Acknowledgments This project is financed within the framework of Grant PBS 2/ A1/11/2013 entitled: ‘‘Phosphorus renewable raw materials – a
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resource base for new generation of fertilizers.” awarded by the National Centre for Research and Development, Poland and grand entitled: ‘‘Biofortification of plant biomass with selenium by utilization of biofertilizers obtained via microbiological solubilisation – BioSeFert” awarded by the National Centre for Research and Development in Poland. Appendix A. Supplementary material Supplementary data to this article can be found online at https://doi.org/10.1016/j.wasman.2019.02.048. References European Sustainable Phosphorus Platform, Scope and News. Switzerland makes phosphorus recycling obligatory; 2015. González-Marcos, A., Alba-Elías, F., Martínez-de-Pisón, F.J., Alfonso-Cendón, J., Castejón-Limas, M., 2015. Composting of spent mushroom substrate and winery sludge. Compost Sci. Util. 23, 58–65. https://doi.org/10.1080/ 1065657X.2014.975868. Jastrze˛bska, M., Saeid, A., Kostrzewska, M.K., Bas´ladyn´ska, S., 2018. New phosphorus biofertilizers from renewable raw materials in the aspect of cadmium and lead contents in soil and plants. Open Chem. 6, 35–49. https://doi.org/10.1515/ chem-2018-0004. Kabata-Pendias, A., Pendias, H., 2000. Trace elements in soils and plants. CRC Press, London. Kwak, W.S., Jung, S.H., Kim, Y.I., 2008. Broiler litter supplementation improves storage and feed-nutritional value of sawdust-based spent mushroom substrate. Bioresour. Technol. 99, 2947–2955. https://doi.org/10.1016/j. biortech.2007.06.021. Lima, M.F., de Eloy, N.B., Siqueira, J.A.B., Inzé, D., Hemerly, A.S., Ferreira, P.C.G., 2017. Molecular mechanisms of biomass increase in plants. Biotechnol. Res. Innov. 1, 14–25. Lukashe, N.S., Mupambwa, H.A., Green, E., Mnkeni, P.N.S., 2019. Inoculation of fly ash amended vermicompost with phosphate solubilizing bacteria (Pseudomonas fluorescens) and its influence on vermi-degradation, nutrient release and biological activity. Waste Manage. 83, 14–22. https://doi.org/10.1016/j. wasman.2018.10.038. Luo, X., Yuan, X., Wang, S., Sun, F., Hou, Z., Hu, Q., Zhai, L., Cui, Z., Zou, Y., 2018. Methane production and characteristics of the microbial community in the codigestion of spent mushroom substrate with dairy manure. Bioresour. Technol. 250, 611–620. https://doi.org/10.1016/j.biortech.2017.11.088. Majchrowska-Safaryan, A., Tkaczuk, C., 2013. Possibility to use the spent mushroom substrate in soil fertilization as one of its disposal methods. J. Res. Appl. Agric. Eng 58, 57–62. Meng, L., Li, W., Zhang, S., Wu, Ch., Lv, L., 2017. Feasibility of co-composting of sewage sludge, spent mushroom substrate and wheat straw. Bioresour. Technol. 226, 39–45. https://doi.org/10.1016/j.biortech.2016.11.054. Meng, L., Zhang, S., Gong, H., Zhang, X., Wu, C., Lia, W., 2018a. Improving sewage sludge composting by addition of spent mushroom substrate and sucrose. Bioresour. Technol. 253, 197–203. https://doi.org/10.1016/j. biortech.2018.01.015. Meng, X., Liua, B., Xi, C., Luo, X., Yuan, X., Wang, X., Zhu, W., Wang, H., Cui, Z., 2018b. Effect of pig manure on the chemical composition and microbial diversity during co-composting with spent mushroom substrate and rice husks. Bioresour. Technol. 251, 22–30. https://doi.org/10.1016/j.biortech.2017.09.077.
Mupambwa, H.A., Ravindran, B., Mnkeni, P.N.S., 2016. Potential of effective microorganisms and Eisenia fetida in enhancing vermi-degradation and nutrient release of fly ash incorporated into cow dung-paper waste mixture. Waste Manage. 48, 165–173. Nakatsuka, H., Oda, M., Hayashi, Y., Tamura, K., 2016. Effects of fresh spent mushroom substrate of Pleurotusostreatus on soil micromorphology in Brazil. Geoderma 269, 54–60. https://doi.org/10.1016/j.geoderma.2016.01.023. Phan, C.W., Sabaratnam, V., 2012. Potential uses of spent mushroom substrate and its associated lignocellulosic enzymes. Appl. Microbiol. Biotechnol. 96 (4), 863– 873. https://doi.org/10.1007/s00253-012-4446-9. Report on critical raw materials for the EU. Report of the Ad hoc Working Group on defining critical raw materials. European Commission; 2014. Roberts, T.L., 2014. Cadmium and phosphorus fertilizers: the issues and the science. 2nd international symposium on innovation and technology in the phosphate industry. Procedia Eng. 83, 52–59. https://doi.org/10.1016/j. proeng.2014.09.012. Rojo, J., 2017. MEPs vote for faster cadmium phase-down in fertilisers. ENDS Europe, 31. Roy, E.D., 2017. Phosphorus recovery and recycling with ecological engineering: a review. Ecol. Eng. 98, 213–227. https://doi.org/10.1016/j.ecoleng.2016.10.076. Schoumans, O.F., Bouraoui, F., Kabbe, C., Oenema, O., Dijk, K.C., 2015. Phosphorus management in Europe in a changing world. Ambio 44, 180–192. https://doi. org/10.1007/s13280-014-0613-9. Syvertsena, J.P., Garcia-Sanchez, F., 2014. Multiple abiotic stresses occurring with salinity stress in citrus. Environ. Exper. Bot. 103, 128–137. Tasaki, Yuji, Kozuka, K., Mochida, K., Sugawara, M., 2014. Effect of Sawdust-Based Spent Mushroom Substrate Treated with Steam on Rat Growth Performance. J. Food Sci. Technol. 20 (2), 493–497. https://doi.org/10.3136/fstr.20.493. Wyciszkiewicz, M., Saeid, A., Chojnacka, K., 2017a. In situ solubilization of phosphorus bearing raw materials by Bacillus megaterium. Eng. Life Sci. 17, 749–758. https://doi.org/10.1002/elsc.201600191. Wyciszkiewicz, M., Saeid, A., Malinowski, P., Chojnacka, K., 2017b. Valorization of phosphorus secondary raw materials by Acidithiobacillus ferrooxidans. Molecules 22, 473. https://doi.org/10.3390/molecules22030473. Wyciszkiewicz, M., Saeid, A., Chojnacka, K., 2017c. Solubilization of renewable phosphorus sources with organic acids produced by Bacillus megaterium. JRM. https://doi.org/10.7569/JRM.2017.634132. Wyciszkiewicz, M., Saeid, A., Samoraj, M., Chojnacka, K., 2017d. Solid-state solubilization of bones by B. megaterium in spent mushroom substrate as a medium for a phosphate enriched substrate. J. Chem. Technol. Biotechnol. 92, 1397–1405. https://doi.org/10.1002/jctb.5135. Wyciszkiewicz, M., Saeid, A., Chojnacka, K., Górecki, H., 2015a. New generation of phosphate fertilizer from bones, produced by bacteria. Open Chem. 13, 951– 958. https://doi.org/10.1515/chem-2015-0113. Wyciszkiewicz, M., Saeid, A., Chojnacka, K., Górecki, H., 2015b. Production of phosphate biofertilizers from bones by phosphate-solubilizing bacteria Bacillus megaterium. Open Chem. 13, 1063–1070. https://doi.org/10.1515/chem-20150123. Wyciszkiewicz, M., Saeid, A., Dobrowolska-Iwanek, J., Chojnacka, K., 2016. Utilization of microorganisms in the solubilization of low-quality phosphorus raw material. Ecol. Eng. 89, 109–113. https://doi.org/10.1016/j. ecoleng.2016.01.065. Zhang, R.-H., Duan, Z.-Q., Li, Z.-G., 2012. Use of spent mushroom substrate as growing media for tomato and cucumber seedlings. Pedosphere 22, 3333–3342. https://doi.org/10.1016/S1002-0160(12)60020-4. Zhu, H.-J., Sun, L.-F., Zhang, Y.-F., Zhang, X.-L., Qiao, J.J., 2012. Conversion of spent mushroom substrate to biofertilizer using a stress-tolerant phosphatesolubilizing Pichia farinose FL7. Bioresour Technol. 111, 410–416. https://doi. org/10.1016/j.biortech.2012.02.042.
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Valorization of ash and spent mushroom substrate via solid-state solubilization by Acidithiobacillus ferrooxidans Agnieszka Saeid a,⇑, Ami Patel b a b
Department of Advanced Material Technologies, Faculty of Chemistry, Wroclaw University of Science and Technology, Gdanska 7/9, 50-344 Wroclaw, Poland Mansinhbhai Institute of Dairy & Food Technology (MIDFT), Mehsana-384002, Gujarat State, India
a r t i c l e
i n f o
Article history: Received 18 October 2018 Revised 26 February 2019 Accepted 27 February 2019
Keywords: Solubilization of phosphates Acidithiobacillus ferrooxidans Ash SMS Solid-state solubilization Germination test
a b s t r a c t This work describes the possibility of utilization of ash originated from the incineration of sewage sludge from the wastewater treatment plant with the 3rd stage of biological treatment, and spent mushrooms substrate (SMS) as a raw material for the production of the substrate for agriculture and horticulture with the property of slow-releasing of phosphorus via solubilization process. Ash was mixed with SMS in different ratios (1, 5 and 10%), where SMS was used as a substrate/medium – the source of nutrients necessary for the growth of bacteria Acidithiobacillus ferrooxidans (A. ferrooxidans), while the ash was used as a source of phosphorus. Solubilization of phosphorus from ash via solubilization process was conducted for 50 days. During this time pH, conductivity, as well as the concentration of available forms of phosphorus were monitored. Obtained results were compared with the control group deprive inoculation by A. ferrooxidans. The concentration of available to plants phosphorus (express as P2O5) was an average 1.5 times higher in the SMS inoculated with A. ferrooxidans in all considered groups. Observation confirms the possibility of utilization of treated SMS as a substrate in agriculture and horticulture as the utilitarian properties (weight and length of plant/root) of plants obtained in germination test were higher when compared with the control group where SMS without inoculation was used. Ó 2019 Elsevier Ltd. All rights reserved.
1. Introduction Current paper deals with the issue related to the valorization of wastes into valuable products what stay in line with the circular economy. What is more proposed solution could be the method of mitigation of the relation of phosphorus fertilizers production with phosphates industry (Schoumans et al., 2015, Roy, 2017). The phosphate rock is one of the critical raw materials according to an updated list published in 2014, hence alternative phosphorus sources should be proposed with suitable processing methods that allow for its beneficial application (Report on critical raw materials for the EU, 2014). A few European countries made the phosphorus recovery obligatory, such as Switzerland or Germany from sewage sludge and slaughterhouse, since these two flows of phosphorus seem to be the most concentrated ones (European Sustainable Phosphorus Platform, 2015). One of the environmental friendly treatment method of ash described in the literature, is their microbial solubilization that could be used to treat low quality phosphates rock as well as the ⇑ Corresponding author. E-mail addresses: [email protected] (A. Saeid), [email protected] (A. Patel). https://doi.org/10.1016/j.wasman.2019.02.048 0956-053X/Ó 2019 Elsevier Ltd. All rights reserved.
secondary P-bearing raw materials such as ashes, what results with liberation of phosphorus from structure that is not available to plants (Lukashe et al., 2019; Mupambwa et al., 2016; Wyciszkiewicz et al., 2017a, 2017b, 2017c). As a result of this mentioned substrate could be valorized into the phosphorus fertilizers, with the properties of biofertilizers, because of the presence of living beneficial soil organisms within it where in an appropriate manner, and under appropriate conditions, raw materials with microorganisms, along with the nutrient components would be combined (Wyciszkiewicz et al., 2017d, 2015a; 2015b). Another issue concern in this paper is the utilization of spent mushrooms substrate (SMS) as a source of nutrients that maintain the growth of beneficial microorganisms under optimal conditions. Since spent mushrooms substrate (SMS) and its efficient utilization is also an important issue, nowadays widely discussed in the literature (Zhu et al., 2012; Majchrowska-Safaryan and Tkaczuk, 2013; Nakatsuka et al., 2016), attempts to its application as a base, the source of nutrients and matter of keeping the proper conditions of growth of microorganism responsible for phosphorus solubilization from ash, were undertaken (Wyciszkiewicz et al., 2017d). SMS is often used as a one of the component in the cocomposting SMS with sewage sludge (Meng et al., 2018), pig manure (Meng et al., 2018b), dairy manure (Luo et al., 2018), sewage
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sludge, wheat straw (Meng et al., 2017) indicating a synergistic effect of co-digestion with SMS -improvement of maturity of the compost and reduction of emission of nitrogenous gasses and higher methane yield. For the effective use of SMS as animal feed or as a soil conditioner there is need to improve its preservative quality as it typically contains about 60% moisture, it putrefies easily because it contains nutrient-rich organic compounds (Kwak et al., 2008, Tasaki et al., 2014). All features considered as an obstacle in the case of its utilization in solid-state solubilization are considered as beneficial. The result of a combination of SMS treatment along with solidstate solubilization could mitigate the phosphorus problem and reduce the amount of generated and stored wastes originated from the food industry, such as SMS, as well as ashes originated from incineration of waste-sludge form wastewater treatment plant with 3rd stage of biological treatment. The aim of the present paper was to evaluate the effect of solidstate solubilization performed by A. ferrooxidans when as a base SMS mixed with the secondary P-bearing raw material were used on the concentration of available phosphorus (express as a P2O5). Further, obtained substrates based on the SMS (treated and not treated by A. ferrooxidans) were used as the substrate in the germination test to evaluate its influence on the utilitarian properties of the test plant. 2. Materials and methods 2.1. Materials In the experiment, spent mushroom substrate (SMS) (Fig. 1) and ash originated from incineration of waste sludge from wastewater plant Łyna in Olsztyn in Poland was used as a growth medium for A. ferrooxidans. Ash as a phosphate substrate was ground by blender and mixed with SMS. The content of SMS without the addition of A. ferrooxidans and SMS treated is presented in Table 1. 2.2. Bacteria and culture medium Phosphate sources were treated with A. ferrooxidans as a phosphate–solubilizing microorganism. Bacterial strain was obtained from Professor Zygmunt Sadowski from Wroclaw University of Science and Technology. A. ferrooxidans is an autochthonous strain of bacteria (F7-01) isolated from the tailings impoundment ‘‘Iron Bridge”, Poland (Wyciszkiewicz et al., 2017b). The concentration
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of bacteria in suspension that was used in the inoculation was 1.5 g L1 and 5 L of the suspension was used in the experiment as an inoculum (Fig. 1). 2.3. Solubilization experiment The test was conducted in six Eco-Composters with the capacity of 270 L (tumbler composter with the spherical shape) (Fig. 1). Ash (0.1 kg, 0.5 kg and 1 kg) and spent mushroom substrate (SMS) (10 kg) were mixed and soaked with 5 L of A. ferrooxidans suspension. As a control group three composters filled with ash (0.1 kg, 0.5 kg and 1 kg) and SMS (10 kg) mixed and soaked with 5 L of distilled water, were used. Every one or two weeks, samples after mixing of the stock were taken to measure electrical conductivity (EC) and were dried. Available and soluble phosphorus forms in SMS were determined. The solubilization experiment was conducted for 50 days. 2.4. Germination test On 12 dishes with 15 g of universal garden soil, 50 seeds were planted. As a plant, the sunflower was selected. All plates were stratified at 0 °C for three days. The seed trials were prepared according to the International Safe Transit Association (ISTA) Standards. Three groups in four replicates were prepared: control group (standard soil), a group with additional 20 g of untreated SMS and group with 20 g of SMS that was modified by solid-state solubilization of phosphates from ash by A. ferrooxidans. After the 7th day of field trials, the plants were collected, measured and counted. The whole mass of plants was dried to constant weight. 2.5. Extraction of P2O5 In order to investigate the efficiency and consequently bioavailability of phosphorus, P2O5 ammonium citrate and water-soluble fractions were determined. The experiment was carried out in two stages, by 1 h at 65 °C for ammonium citrate and 30 min at 25 °C for water, according to Regulation (EC) No 2003/2003 of the European Parliament and of the Council relating to fertilizers (method 3.1.4 Extraction of phosphorus which is soluble in neutral ammonium citrate, and 3.1.6 Extraction of water-soluble phosphorus). The contribution of soluble and available phosphorus was evaluated by the colorimetric vanadophosphomolybdate method described elsewhere (Wyciszkiewicz et al., 2017d).
Fig. 1. Scheme of the experiments.
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Table 1 (a) mineral content of SMS after the solid-state solubilization process; (b) other characteristic parameters of SMS. Element/parameters
Dose of Ash 1%
5%
10%
SMS
SMS treated
SMS
SMS treated
SMS
SMS treated
a) Macroelements, % N,% P2O5 (P) K2O (K) Ca Mg S Na
2.41 1.79 (0.784) 1.55 (1.45) 8.41 0.376 2.936 0.096
2.43 2.40 (1.04) 1.98 (1.86) 9.56 0.441 4.440 0.131
2.25 3.51 (1.53) 2.03 (1.90) 9.52 0.560 3.159 0.163
2.13 4.23 (1.84) 1.73 (1.62) 9.46 0.604 3.024 0.164
2.25 4.17 (1.82) 1.61 (1.51) 10.8 0.574 3.016 0.158
2.24 6.45 (2.81) 2.23 (2.09) 9.15 0.813 3.220 0.216
Microelements, mg kg1 B Ba Be Co Fe, % Mn Mo Sb Se Si
21.5 75.7 0.152 3.57 0.518 209 4.02 12.7 <4.5 (LOD) 486
24.3 83.1 0.122 2.19 1.340 237 7.65 13.1 <4.5 (LOD) 555
21.9 99.7 0.216 1.85 0.804 248 6.57 19.1 30.246 443
24.7 106 0.182 2.28 0.771 258. 4.56 <0.25 (LOD) 20.908 389
22.5 102 0.183 2.02 0.775 250 7.36 2.220 8.263 289
29.8 135 0.216 4.03 1.650 293 5.30 19.114 21.894 472
Toxic metal, mg kg1 Cr Cu Al As Zn Cd Pb
8.88 44.8 2237 6.55 210 <0.010 (LOD) 16.8
9.36 58.1 1764 <0.5 (LOD) 257 0.157 5.79
17.2 99.7 3619 <0.5 (LOD) 369 0.616 2.06
22.3 116 4017 17.3 448 0.501 13.18
21.4 107 3637 <0.5 (LOD) 419 0.508 3.01
38.0 202 5521 11.1 807 0.682 9.99
Others, mg kg1 Ti Tl V Ni Ag
171 19.5 5.15 4.94 0.065
161 10.4 3.58 7.49 0.114
268 15.4 8.973 8.80 0.795
337 3.87 6.979 14.1 1.218
303 <1.0 (LOD) 6.79 11.2 1.20
461 6.97 7.92 15.1 2.12
b) Conductivity0, mS cm1 Conductivity‘, mS cm1 SF, % C/N mg Cd kg1 P2O5
7.4 3.52 28.6 10.1 < 0.1 (LOD)
7.69 3.46 38.9 9.48 6.56
7.0 3.78 17.7 10.4 17.6
8.17 3.33 25.5 10.1 11.9
7.91 3.2 16.2 10.5 12.2
7.78 2.74 20.2 9.49 10.6
LOD – limit of detection.
2.6. C and N content analysis Elemental analysis (CN) of the raw SMS and treated SMS was determined using VarioMacroCubeElementar (C, H, N) analyzer. As a standard solution D-phenylalanine (C = 65.44%; N = 8.48%) was used. 2.7. ICP-OES analysis The samples were mineralized with spectrally pure 69% HNO3 (Suprapur, Merck) in a closed system by using microwave oven Milestone Start D. After mineralization, solutions were diluted to 25 g. The elements concentrations were determined by ICP–OES Varian-Vista MPX (Australia) with ultrasonic nebulizer CETAC U5000AT+. The results are the arithmetic mean of three measurements. 2.8. Statistical methods The results of the experiments were analyzed statistically using the program Statistica ver. 10. The normal distribution of the variable was tested using the Shapiro-Wilk test. The Brown Forsythe test was used to verify the homogeneity of variance (for a normal distribution).
For groups with homogeneous variance, test F of the significance of differences was performed. For distribution other than normal, Kruskal-Wallis test was used. Statistically significant differences were for p < 0.05. 3. Results and discussion The performed experiments were aimed to find whether it is possible to apply A. ferrooxidans in the solubilization of P- bearing secondary raw materials conducted in a solid-state manner. The experiment was divided into two parts. At the first stage, six variations of solubilization processes were conducted that allowed to obtain six substrates which differ with the presence of bacteria and the dosed of the P-bearing source used (Table 1a); in the second stage were tested as a substrate that potentially could be used in agriculture and horticulture. Furthermore, obtained formulations could be defined as fertilizer since the content of phosphorus in form available to plants (expressed as the P2O5) in obtained formulation were 1.2%, 1.3% and 1.7% respectively, for products obtained as a result of solubilization of SMS conducted with addition of 1%, 5% and 10% of ash. While the total content of phosphorus (express as P2O5) was 2.40%, 4.23% and 6.45% for increasing dose of ash applied in the experiments. According to the Regulation of the Minister of Agriculture and Rural Development (Journal of
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Laws 236, 29 October 2004) minimum quality requirements for the content of phosphorus (expressed as the P2O5) in phosphorus fertilizers is 1% (m/m). In this context, the obtained substrate in the form of treated SMS by solubilization process can be classified as a phosphorus fertilizer. In the previous experiments published elsewhere, the level of P2O5 reached 1.3% for dose 5% of addition of poultry bones when as a Phosphorus Solubilizing Bacteria (PSB), Bacillus megaterium was used (Wyciszkiewicz et al., 2016). One of the most concerned issues when considering the safety of the natural environment when applying fertilizers is the content of Cd (Jastrze˛bska et al., 2018). Recently, the European Union (EU) had already begun to apply more stringent restrictions on the content of Cd in fertilizers. Limits in mg Cd per kg P2O5 in fertilizers for EU will decrease gradually 60 40 20 (Rojo, 2017). So far those limit significantly varied between European countries, for example in Finland it is 21.5 mg Cd per kg P2O5, in Sweden 44, in Denmark 48, in Belgium 90, in Austria 120, while in Australia 131 and in Japan 148 mg Cd/kg P2O5 (Roberts, 2014). Cd present in fertilizer is originated from phosphate rock, which nowadays is the main resource used in the phosphorus industry. In the case of secondary raw materials originated for example from food industry exceeding the applicable limits is very rare, what was marked in the Table 1b where the ration of Cd per 1 kg of P2O5 in obtained substrate/fertilizer formulation is lower than 20 mg kg1. Another issue undertaken during the experiments was a C/N ratio evaluation. The obtained ratio for all considered level of addition of ash in the case of SMS with A. ferrooxidans was characterized by lower C/N ration when compared with the control group. Inoculation with A. ferrooxidans resulted in the lowering of carbon content, probably due to its utilization during the growth. The level of nitrogen was almost the same in the control and experimental group. Similar findings were reported elsewhere (Wyciszkiewicz et al., 2017d).
The set of six experiments of treatment of spent mushrooms substrate mixed with the ash applied at three different doses 1, 5 and 10%, was conducted for 50 days. The control group contained the SMS mixed with ash but without A. ferrooxidans. The inoculation of experimental composters by A. ferrooxidans affected the significant increase of P2O5 available to plants as a result of the production of sulphuric acid that was responsible for releasing of phosphorus from not available to plants form. To describe the changes in P2O5 concentrations during solubilization, the proposed model was used (Eq. (1)):
C P2 O5 ¼ f ðt Þ ¼
C Pav2 Oailable t 5 max
ð1Þ
K þt
where the C Pav2 Oailable , mg L1 is the maximum of phosphorus concen5 max tration express as P2O5, K is the constant that expresses time when
. When K, day is lower, the curve C P2 O5 , is equal to ½ of C aPv2 Oailable 5 max changes more rapidly i.e. solubilization is faster. Table 2 collects all evaluated model parameters both with the errors that express the fit of the proposed model to the experimental data. It was found that inoculation of a solid-state reactor filled with SMS and ash by A. ferrooxidans results in the increase of
C aPv2 Oailable by 58.4% in the case of addition of 1%, increase by 58.6% 5 max when 5% was added, and increase by 63.5% when 10% was used. All evaluated parameters were statistically significant with one exception of K constant evaluated for 5% addition of raw materials to the reactor inoculated with A. ferrooxidans. Constant K, was also effected by the inoculation by A. ferrooxidans. Evaluated K values for SMS treated by A. ferrooxidans were lowered by 44%, 19% and 31% when compared with the SMS not treated by A. ferrooxidansfor addition of 1%, 5% and 10% of ash, respectively (Fig. 2). It can be interpreted that the process of releasing available phosphorus (express as the P2O5) is faster when A. ferrooxidans was applied. 3.2. Solubilization factor
3.1. Solubilization The choice of A. ferrooxidans was intentional, because it is well established that this is a harmless mesophilic organism, and since the assumptions of performed studies was to develop the formula of substrate that could serve as a slow released fertilizer, that after application on the soil would be still subjected to action of bacteria, and as a result of this the release of phosphates would be observed. If thermophilic microorganism would be used instead, the issues on survival in the soil would have to be raised especially in the temperate climate zone.
The solubilization effect was expressed as the Solubilization Factor (SF, %) defined as the ratio (expressed as a percentage) of soluble P2O5 present in the solution to phosphorus (expressed as P2O5) introduced to solubilization medium in solid/not available form. Fig. 3 represents the influence of the dose of ash used in the solid-state solubilization process on the SF. The obtained data indicate the correlation between the dose of applied resource and solubilization efficiency what stay in the line with the previous findings (Wyciszkiewicz et al., 2017a; 2017b; 2017c). In this case the correlation was not statistically significant, r = 0.777
Table 2 C Pav ailable O5 max t Parameters of model C P2 O5 ¼ f ðt Þ ¼ 2Kþt describing the kinetics of changes of concentration P2O5 during the solid-state solubilization process (N = 5). italic- statistically significant parameters. Addition of raw materials SMS
1% 5%
p value
R2
v2
7.83 ± 0.83
0.001
0.992
0.238
29.7 ± 6.6 8.01 ± 1.04
0.011 0.002
0.980
0.483
13.3 ± 5.2 10.4 ± 1.0
0.063 0.001
0.983
0.628
K, day
16.3 ± 4.4
0.021
, g kg1 C Pav2 Oailable 5 max K, day
12.4±±1.2
0.000
0.995
0.0373
16.6 ± 4.3 12.7 ± 1.4
0.018 0.001
0.980
0.365
10.9 ± 4.1 17.0 ± 1.9
0.057 0.001
0.991
0.222
11.3 ± 4.0
0.049
Model parameter
Value ± SD
;g kg1 C Pav2 Oailable 5 max K, day , g kg1 C Pav2 Oailable 5 max K, day
10%
SMS treated
1% 5%
C Pav2 Oailable , g kg1 5 max
, g kg1 C Pav2 Oailable 5 max K, day
10%
C Pav2 Oailable , g kg1 5 max K, day
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Fig. 2. Kinetic of releasing of phosphorus (express as P2O5 extracted in neutral ammonium citrate) during the solid-state solubilization.
Fig. 4. The influence of the dose of ash on the final content of P2O5 available to plants (extracted in neutral ammonium citrate), mg kg1, during the solid-state solubilization process.
Table 3 Differences between the parameters: dry weight, length of plant as well as the length of root, determined in germination tests for three experimental groups, control (water), SMS not treated and SMS treated by A. ferrooxidans. Parameter
Fig. 3. The influence of dose of ash used in the solid-state solubilization process, on the solubilization factor (SF, %).
(p = 0.433 for SMS treated) and r = 0.899 (p = 0.289 for SMS). Higher initial dose of phosphorus raw materials causes higher final concentration of available to plants phosphorus (express as P2O5) but at the same time, lower efficiency express as an SF (Table 1b). Fig. 4 represents the influence of the applied dose of P-bearing sources and the presence of bacteria on the changes between the initial and the final concentration of phosphorus available to plants form (express as P2O5). It was shown that the solubilization even without the addition of bacterial cells is performed, probably by the native microbiota of SMS, nevertheless is much less efficient when compared with results obtained for A. ferrooxidans. 3.3. Germination test Obtained substrates after 50 days of incubation of SMS with A. ferrooxidans (SMS treated) or without (SMS non treated), were used as a substrate in the germination test on sunflower seeds. The sum of germinated seeds increase in all the experimental groups (SMS treated and not treated) both with the increase of applied dosage of P-bearing source at the solubilization stage (Table 3), moreover the sum of germinated seeds was higher for the group where the SMS treated by A. ferrooxidans was applied, with exception when
SMS
SMS treated
Median ± SD
Median ± SD
The average amount of germinated seeds (sum of all) Standard soil 6.33 ± 1.53 (19*) 1% 7.33 ± 1.03 (44) 5% 8.50 ± 0.84 (51) 10% 8.67 ± 0.82 (52)
– 8.17 ± 1.17 (49) 8.50 ± 0.84 (51) 8.83 ± 0.41 (53)
Dry weight, g Standard soil 1% 5% 10%
0.232 ± 0.000887 0.0206 ± 0.0016a 0.0214 ± 0.0010b 0.0238 ± 0.0033c
– 0.0243 ± 0.0028a 0.0271 ± 0.0052b 0.0306 ± 0.0059c
Length of plant, cm Standard soil 1% 5% 10%
7.46±±2.24d,e 11.43 ± 1.60d 11.95 ± 2.06 12.5 ± 1.49
– 12.20 ± 1.70e 12.35 ± 1.41 12.1 ± 1.92
Length of root, cm Standard soil 1% 5% 10%
5.31 ± 1.76f 6.39 ± 1.81 6.53 ± 1.53 6.55 ± 1.64
– 6.88 ± 1.44f 6.98 ± 1.29 6.74 ± 1.67
a–f
statistically significant differences p 0.05. N = 3.
*
5% of addition was applied - in this case, the amount of germinated seeds was equal with SMS not treated. The biomasses of plants were measured: length of green part and root as well as the dry weight. The medians of measured parameters were presented in Table 3, both with statistical differences between experimental groups (Fig. 5). Statistically significant differences were found between the dry weight of plants cultivated with the addition of SMS treated and not treated, for all applied doses. Further, the differences between the dry weight of the experimental groups increase as the dose of addition increase. The following increase of dry weight was found: 18%, 27% and 29% for 1%, 5% and 10% of the level of the P-bearing source in the treated SMS. Statistically significant differences were also found between the length of the
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617
Fig. 5. Box and whiskers plot of (a) length of plant (cm) and (b) length of root (cm) - for addition of 1% of ash; (c) length of plant (cm) and (d) length of root (cm) - for addition of 5% of ash; (e) length of plant (cm) and (f) length of root (cm) for addition of 10% of ash.
green part of the plants that have grown on the not treated and treated SMS, and when only standard soil was used. Between the groups where the plants have grown on the standardized soil, length of green part of plant were lower by 63% (p < 0.05) and by 53% (p < 0.05) when compared with plants that have grown on the of SMS treated by A. ferrooxidans 1% and on the SMS not treated, respectively. The lengths of the green part, as well as root,
were higher for the experimental group where the SMS treated was used with one exception of the length of the green part when 10% of SMS was applied, in that case, the group where SMS not treated was used plants were higher by 3%. Addition of treated SMS enriched with 10% of P-bearing source effected in inhibition of the growth of plant. In rest cases the length of the plants and roots were higher for the group where SMS treated was used, but
0.997
1.00
1.00
Zn Si
0.998 1.00
1.00
0.999 1.00
1.00 1.00
1.00
1.00
0.999
1.00
1.00
0.997 0.999
1.00
0.998
1.00
1.00
1.00
0.999 0.999 1.00
0.999
0.997
1.00
1.00
1.00 0.999 0.999 1.00 1.00 0.999 1.00
1.00
1.00 1.00 0.999 0.998
1.00 1.00
0.998
0.998 0.997 0.999
0.999
0.998
Se
1.00 1.00
Sb S
0.997
Pb P Ni Na Mo Mn Mg K Fe
0.998
Cu Cr Co Cd
0.999
Ca Be
0.999 0.997
Ba B
1.00
1.00 1.00
Ag Al As B Ba Be Ca Cd Cr Cu Fe K Mg Mn Na Ni P Pb S Sb Se Si SMS treated
0.999
As Al Ag
0.999 Ca Mo Si
The content of the elements in the plant biomass is strongly controlled by the mechanism of plant metabolism (Lima et al., 2017). The interactions between the elements present in the soil solution can be both antagonistic and synergistic and are involved in the metabolism of more than two elements. The type of interaction effects, whether antagonistic or synergistic are more concentration depended, and its definition is easier when exceeds some critical level of toxicity is observed. Since the applied spent mushrooms substrate did not deliver the nutrients in exceed level, observed interaction presents in the matrix (Table 4) even statistically significant, barely can find its confirmation in the literature (Kabata-Pendias and Pendias, 2000). To compare the influence of the form of substrate used in the germination tests, the ratio of the content of elements to its content in the substrate, which can be defined as a transfer factor was evaluated and presented in Fig. 6. The evaluated factor, in most cases, was comparable between the two considered groups where SMS and treated SMS were used. The significant differences were found for As, K, and Ni when the transfer factor was higher for control (not treated SMS), and for Cd and Se when the factor was higher for treated SMS. Smaller differences were found for Na, Sb, and Si and it was higher for control (not treated SMS) and for Pb when it was higher for SMS treated. It can be concluded that A, K, Ni Na, Sb, and Si are easier up taken from not treated SMS, while Cd, Pb and Se from treated SMS. The statistically significant correlations (Table 4) were found between the content of elements in the substrate (treated and not treated) and in the plants. In the case of not treated SMS by A. ferrooxidans, the statistically significant, negative and strong correlation was found between Ca and Ag, Ba, Be, Cd, Fe, S, Sb and Se, while in the case of treated SMS Ca was statistically correlated with Ag, Al, B, Ba, Ca, Mg and P but these correlations were positive. Probably the presence of A. ferrooxidans in the treated SMS resulted in the changes of the form of other than the Ca nutrients that became more available, since
SMS
3.4. Elemental content of plants
PLANT
those differences were not statistically significant. When taking into account the length of root, the best-applied level of addition of treated SMS would be 5% since in that case the length of root was the highest of all considered cases. Furthermore, for that dose of application the highest length of the green plant was also noticed. Even the highest dry weight was recorded for the addition of 10% of treated SMS. Improved parameters of cultivated plants were observed for the experimental group, where the SMS treated by A. ferrooxidans was used, which suggests that combination of solubilization process with spent mushrooms substrate is a potential method for the valorization of SMS as well as P-bearing secondary raw materials. Solubilization could be an alternative to composting which is widely used as a method of SMS valorization. The composting of SMS is conducted in the windrows with the size is for example of 1.5 m 2.5 m 1.5 m. It would be interesting to perform solubilization when composting occurring simultaneously, it could be possible if thermophilic kind of bacteria would be used since the temperature inside the windrows often exceeded 40 °C (the stage when thermophilic microorganisms are responsible for composting process). So far, in conducted experiments, mesophilic bacteria were used (optimal growth temperature 35 °C) such as B. megaterium (Wyciszkiewcz at al., 2016) and A. ferrooxidans (present paper). In the case of conjugation of SMS utilization with solubilization conducted by mesophilic bacteria, it would be crucial to keep the process temperature at a level when mesophilic organisms conduct the microbial processes. Because the experiment was conducted in the 270 L composter and only for 50 days, the composting was not initiated.
0.999
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Table 4 Correlation matrix of correlation coefficients (only statistically significant) between the content of elements in the plant and in the substrate used in the experiment, separated for treated and not treated SMS.
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619
Fig. 7. Influence of dose of ash on the changes of conductivity as an effect of solubilization performed by A. ferrooxidans (SMS treated) expressed in %.
Fig. 6. The influence of kind of SMS used in germination tests on the transfer of nutrients, as well as toxic elements from the substrate, to the plant biomass.
the content of Ca do not differ significantly between the experimental groups of substrate used in the germination tests (p > 0.05). The treatment of SMS by A. ferrooxidans resulted in the higher dose of available to plants phosphorus (express as the P2O5) as it was mentioned before. As a result of this, few statistically significant correlations were found between the content of phosphorus in the treated substrate and content of Fe, K, Mn and S in the plant’s biomass. In the SMS not treated such correlations were not found. Additionally, in the case of treated SMS, negative and strong statistically significant correlations were found between the Pb present in the substrate and K and S detected in the plant biomass, what demonstrates the toxic effect of Pb.
3.5. Electrical conductivity Fresh, not treated SMS characteristically has a high salt content, measured indirectly by electrical conductivity. The average salinity before the experiment for all experimental groups was 7.66 but at the end of the solubilization process decreased to 3.34, and this change was statistically significant (p < 0.005). When considering two experimental groups it was found that the decrease of salinity in the bacterial treated SMS was higher than that found in the SMS not treated. Fig. 7 shows that in the case of utilization of A. ferrooxidans the decrease of salinity is higher when the dose of applied ash is higher. While in the case of not treated SMS by A. ferrooxidans the opposite relationship was observed. The direction factors of regression curves determined for both discussed cases is equal – 1.49 (p = 0.059) for SMS not treated (negative correlation) and 1.09 (p = 0.0082) for SMS treated (positive correlation). This difference confirms the importance of the application of bacteria in the reducing of the salinity. A. ferrooxidans successfully utilize and decrease the content of inorganic as well as organic substances delivered by SMS. Salinity stress is a common environmental problem and an important factor limiting crop production. Salinity affects all plants in three ways: osmotic stress, toxic ion stress, and nutritional imbalances. Dissolved salts in the nutrient solution exert an osmotic effect that reduces the availability of free (unbound) water through physical processes that impair water extraction from the soil by roots (Syvertsen and Garcia-Sanchez, 2014). Spent mush-
rooms substrate recognized as wastes originated from agro-food industry is produced every year at the significant amount (Phan and Sabaratnam, 2012), and its no utilization can cause environmental problems for example due to nitrate lixiviation to aquifers (González-Marcos et al., 2015). Another crucial issue is its high salt content (Zhang et al., 2012) and moisture which are a significant limitation of its wide application in agriculture. At the same time SMS is rich in nutrients significant for plant nutrition such as N, P, K, and organic matter (Wyciszkiewicz et al. 2016). Application of spent mushrooms substrate as a base and at the same time as a source of nutrients crucial for the growth of bacteria, in the solubilization process is the promising approach for the valorization of SMS as well as P-bearing secondary raw materials in the form of substrate that has great properties as a soil conditioner or soil amendments used in the horticulture and agriculture. The previous finding proved that solubilization conducted by Bacillus megaterium in solid state manner where SMS was used, resulted in the double increase of the amount of available to plants P2O5 what was confirmed within germination tests where the plant cultivated on the SMS treated by B. megaterium was higher and heavier; moreover, the intensity of green color was improved as well (Wyciszkiewicz et al. 2016). 4. Conclusions During conducted experiments, it was confirmed that it is possible to utilize SMS as a base for solubilization conducted in a solidstate manner as a source of nutrients for growth of A. ferrooxidans that was responsible for solubilization of phosphorus delivered in the form of ash. The concentration of available phosphorus evaluated from solubilization process was higher in all considered doses of applied ash when compared with the control group. Obtained substrates from the control and experimental group were used in the germination test. Improved parameters of cultivated plants were observed for the experimental group, where the SMS treated by A. ferrooxidans was used, suggests that combination of solubilization process with spent mushrooms substrate is a potential method for the valorization of SMS as well as P-bearing secondary raw materials. Acknowledgments This project is financed within the framework of Grant PBS 2/ A1/11/2013 entitled: ‘‘Phosphorus renewable raw materials – a
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resource base for new generation of fertilizers.” awarded by the National Centre for Research and Development, Poland and grand entitled: ‘‘Biofortification of plant biomass with selenium by utilization of biofertilizers obtained via microbiological solubilisation – BioSeFert” awarded by the National Centre for Research and Development in Poland. Appendix A. Supplementary material Supplementary data to this article can be found online at https://doi.org/10.1016/j.wasman.2019.02.048. References European Sustainable Phosphorus Platform, Scope and News. Switzerland makes phosphorus recycling obligatory; 2015. González-Marcos, A., Alba-Elías, F., Martínez-de-Pisón, F.J., Alfonso-Cendón, J., Castejón-Limas, M., 2015. Composting of spent mushroom substrate and winery sludge. Compost Sci. Util. 23, 58–65. https://doi.org/10.1080/ 1065657X.2014.975868. Jastrze˛bska, M., Saeid, A., Kostrzewska, M.K., Bas´ladyn´ska, S., 2018. New phosphorus biofertilizers from renewable raw materials in the aspect of cadmium and lead contents in soil and plants. Open Chem. 6, 35–49. https://doi.org/10.1515/ chem-2018-0004. Kabata-Pendias, A., Pendias, H., 2000. Trace elements in soils and plants. CRC Press, London. Kwak, W.S., Jung, S.H., Kim, Y.I., 2008. Broiler litter supplementation improves storage and feed-nutritional value of sawdust-based spent mushroom substrate. Bioresour. Technol. 99, 2947–2955. https://doi.org/10.1016/j. biortech.2007.06.021. Lima, M.F., de Eloy, N.B., Siqueira, J.A.B., Inzé, D., Hemerly, A.S., Ferreira, P.C.G., 2017. Molecular mechanisms of biomass increase in plants. Biotechnol. Res. Innov. 1, 14–25. Lukashe, N.S., Mupambwa, H.A., Green, E., Mnkeni, P.N.S., 2019. Inoculation of fly ash amended vermicompost with phosphate solubilizing bacteria (Pseudomonas fluorescens) and its influence on vermi-degradation, nutrient release and biological activity. Waste Manage. 83, 14–22. https://doi.org/10.1016/j. wasman.2018.10.038. Luo, X., Yuan, X., Wang, S., Sun, F., Hou, Z., Hu, Q., Zhai, L., Cui, Z., Zou, Y., 2018. Methane production and characteristics of the microbial community in the codigestion of spent mushroom substrate with dairy manure. Bioresour. Technol. 250, 611–620. https://doi.org/10.1016/j.biortech.2017.11.088. Majchrowska-Safaryan, A., Tkaczuk, C., 2013. Possibility to use the spent mushroom substrate in soil fertilization as one of its disposal methods. J. Res. Appl. Agric. Eng 58, 57–62. Meng, L., Li, W., Zhang, S., Wu, Ch., Lv, L., 2017. Feasibility of co-composting of sewage sludge, spent mushroom substrate and wheat straw. Bioresour. Technol. 226, 39–45. https://doi.org/10.1016/j.biortech.2016.11.054. Meng, L., Zhang, S., Gong, H., Zhang, X., Wu, C., Lia, W., 2018a. Improving sewage sludge composting by addition of spent mushroom substrate and sucrose. Bioresour. Technol. 253, 197–203. https://doi.org/10.1016/j. biortech.2018.01.015. Meng, X., Liua, B., Xi, C., Luo, X., Yuan, X., Wang, X., Zhu, W., Wang, H., Cui, Z., 2018b. Effect of pig manure on the chemical composition and microbial diversity during co-composting with spent mushroom substrate and rice husks. Bioresour. Technol. 251, 22–30. https://doi.org/10.1016/j.biortech.2017.09.077.
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