Phosphate rock reduces the bioavailability of heavy metals byinfluencing the bacterial communities during aerobic composting

CUI Hu,OU YangWANG Li-xiaYAN Bai-xingLI Ying-xin,DING Da-wei

1 Key Laboratory of Wetland Ecology and Environment,Northeast Institute of Geography and Agroecology,Chinese Academy of Sciences,Changchun 130102,P.R.China

2 Jilin Provincial Engineering Center of CWs Design in Cold Region &Beautiful Country Construction,Northeast Institute of Geography and Agroecology,Chinese Academy of Sciences,Changchun 130102,P.R.China

3 University of Chinese Academy of Sciences,Beijing 100049,P.R.China

Abstract Available information on the microbial mechanisms associated with heavy metal (HM) passivation during co-composting amended with phosphate rock (PR) remains limited.Thus,this study investigated the dynamic changes in bacterial communities and HM-fractions (Zn,Cu,Cd,Cr and Pb) during swine manure composting with maize straw,and ascertained the bacterial influence on HM-passivation.The results demonstrated that the addition of PR improved HM-passivation,especially for Zn and Cd,with their bioavailability factors (BFs) reduced by 247.41 and 176.25%,respectively.As for bacterial communities,the proportion of Firmicutes decreased,while the proportions of Proteobacteria,Bacteroidetes,Deinococcus-Thermus and Gemmatimonadetes increased in all treatments.PR significantly changed the primary bacterial phyla in the thermophilic phase.Bacteroidetes were the main bacterial component controlling the passivation of Zn,Cu and Cr,while Deinococcus-Thermus mainly regulated the mobility of Zn and Pb,and Proteobacteria only dominated the transformation among Cd-fractions.These results may provide a reference for the use of HM-passivation techniques during composting.

Keywords:aerobic composting,heavy metals,bacterial community,phosphate rock

1.Introduction

Heavy metals (HMs) are non-biodegradable pollutants that restrict eco-friendly utilization of livestock manure for agricultural production (Harounet al.2009).Universally,HM-phytotoxicity is more closely related to the specific HM-fractions than the total content (Amiret al.2005;Wanget al.2016).Composting is a well-developed method to reduce HM-bioavailability by regulating the transformations among HM-fractions (Chen Y Ret al.2019).Passivators,such as phosphate rock (PR),have been introduced to make HM-passivation more effective (Chenet al.2019;Wanget al.2019).Previous research has shown that a significant decrease in HM-bioavailability can be obtained through co-composting systems amended with PR (20%)(Wanget al.2013),as it may contribute to increasing the highest temperature and lengthening the thermophilic phase of composting (Caoet al.2004).Additionally,composting accelerates the mobilization of phosphorus in PR,which in turn helps to supply nutrients to microorganisms and promote organic matter decomposition;this results in the conversion of available HM-fractions to non-available HMfractions (Luet al.2014).The possible mechanisms for HM-retention by PR include:1) surface ion exchange and complexation;2) precipitation of some amorphous to poorly crystalline,mixed metal phosphates;or 3) substitution of calcium in PR by other metals during recrystallization (Luet al.2014).However,there is limited research on the microbial mechanisms associated with HM-passivation during composting with PR.

Composting,a widely recommended technology for organic waste recycling,is characterized as a biologicallydominated decomposition process in which organic matter is converted to humus-like products (Enejiet al.2003;Jindoet al.2012).This process involves the microbiallycontrolled mineralization and humification of organic matter;the former causes the release of metal cations bound to organic carbon,while the latter accelerates the chelation of negative-valence fatty acids with metal cations to synthesize various compounds (Li Jet al.2019).Generally,bacteria are sensitive to environmental factors,such as pH and nutrient stoichiometry.For example,Amiret al.(2005)reported that the optimal pH range for bacterial development in composting windrows is 6.0–7.5.In addition,Chenet al.(2017) concluded that bacterial composition was significantly correlated with the carbon-to-nitrogen ratio(C:N),and showed that the contribution of temperature to community succession was weaker in composting that was amended with a carbon-rich additive.Further,Tanget al.(2019) found that Firmicutes could control the phytotoxicity of chromium and lead by affecting organic matter,whereas the control of phytotoxicity of zinc and copper was dominated by Bacteroidetes and Actinobacteria.PR,as an alkaline phosphate-rich additive,will lead to a dynamic change in the micro-environment of composting windrows.Therefore,further research efforts into determining the microbial mechanisms of HM passivation within aerobic composting systems amended with PR,through the regulation of pH and nutrient stoichiometry,may greatly enhance the assessment and control of the end-product value and microbial community.

To address these issues,the present study was conducted with the following aims:1) to investigate the dynamic changes in HM-fractions (Zn,Cu,Cd,Cr and Pb) and the bacterial community during swine manure(SM) composting;and 2) to identify the dominant factors controlling the transformations among HM-fractions and ascertain the passivation mechanism of HM which is driven by bacteria.The results may provide guidance for the use of HM-passivation techniques during composting.

2.Materials and methods

2.1.Composting procedure

Aerobic co-composting was undertaken in six medium-scale PVC reactors (100 L).Composting materials,including SM and maize straw (MS) (chopped into 1–2 cm pieces),were homogenized (2:3 by weight) and split into two piles to obtain well-proportioned mixtures with a low C:N in order to deal with more livestock manure to relieve the environmental pressure caused by it.One treatment was mixed with PR at a ratio of 10% (w/w),resulting in experimental treatment(PR);the other treatment free of PR served as control (CK).Each treatment was set up in triplicate.The chemical parameters are shown in Table 1.Deionized water was added to adjust the moisture content to approximately 60%.Composting piles were manually turned once a week to ensure the oxygen supply and the homogeneity of feedstock over a 52-day composting process.Temperatures were monitored at different areas (top,middle and bottom layers)and the average was calculated.Composting samples were obtained after 0,7,11,19,25,35,43 and 52 days,and mixed thoroughly before division into two sub-samples.One sub-sample was stored at–80°C prior to bacterial high-throughput sequencing analysis,and the other was air-dried and crushed through a 100-mesh screen for later physico-chemical parameter analysis.

2.2.Sequential HM-extraction

A modified BCR sequential extraction procedure was conducted to determine the HM-fractions (Chen X Met al.2019),which can be defined as follows:1) exchangeablefraction-bound to carbonates (Exc);2) reducible fractionbound to Fe and Mn oxides (Red);3) oxidizable fractionbound to OM and sulfides (Oxi);and 4) residual fraction(Res).The Exc-fraction and Red-fraction were extracted with acetic acid and hydroxylamine,respectively.The Oxi-fraction was removed using ammonium acetate after oxidation with H2O2.Similar to total content,the Res-fraction was obtained after digestion with HNO3,HF and HClO4.The Exc-fraction and Red-fraction of HM show mobility,while the Oxi-fraction and Res-fraction are assessed as nonbioavailable fractions of HMs.Therefore,the bioavailability factor (BF) was calculated as the ratio of the metal contents in the Exc-fraction and Red-fraction to the total metal content(Nomedaet al.2008).

Table 1 Physicochemical properties of composting raw materials (dry matter basis)1)

2.3.Bacterial high-throughput sequencing

Bacterial community composition was evaluated using PCR amplification and 16S sequencing in triplicate.Total bacterial DNA was extracted with an E.Z.N.A Soil DNA Kit (OMEGA,USA),following the manufacturer’s instructions as described in Maoet al.(2019).PCR amplification was conducted for sequencing the V3–V4 regions.High-throughput (IIIumina Miseq) sequencing was performed in Beijing Allwegene Tech,Ltd.(Beijing,China).The detailed procedure followed the method of Chen X Met al.(2019).Trimmomatic Software(version 0.33) was used to filter low quality reads,and the filtered reads were merged into single,longer sequences with FLASH (version 1.2.11).

2.4.Fourier-transformed infrared (FTIR) spectroscopy

Freeze-dried compost (1 mg) was mixed with water-free potassium bromide (KBr,200 mg),and then adequately powered (<2 μm) in an agate mortar to homogenize the samples.The mixtures were pelleted on an oil press at 10 MPa to form a transparent sheet.Infrared transmission adsorption spectra were obtained on a Thermo Nexus 8700(4 cm–1resolution,32 scans,4 000–400 cm–1) (Hagemannet al.2018).

2.5.Physico-chemical analysis

Three replicates were used in all parameter analyses,and results are reported as mean value±standard deviation.The pH and EC were analyzed using a multi-parameter water quality detector (520M-01A,LTD).Total carbon (TC)and total nitrogen (TN) were measured using a Shimadzu TC-TN analyzer (Shimadzu Corp.,Kyoto,Japan) based on a non-dispersive infrared method.Zn,Cu,Cd,Cr and Pb were determined with inductively coupled plasma mass spectrometry (ICP-MS).

2.6.Statistical analysis

All graphical figures were produced in Origin Pro 2017.Non-metric multidimensional scaling analyses (NMDS) were conducted in R 3.6.1 to show the differences in the bacterial communities.One-way analysis of variance (ANOVA)was performed in SPSS 25.0 to identify the significance levels of differences in microbial compositions between the CK and PR treatments.Redundancy analyses (RDA)were carried out in Canoco 5.0 to determine multivariate relationships among BFs,bacterial communities and chemical parameters.Path analyses (PA) were performed in AMOS 20.0 coupled with the maximum likelihood evaluation program.

3.Results

3.1.Physico-chemical parameters

According to changes in temperature,the composting process can be divided into three phases:mesophilic phase (<50°C,day 1 to 10),thermophilic phase (>50°C,day 11 to 23) and maturation phase (<50°C,day 24 to 56).The temperature in the PR treatment was higher than that in the CK treatment during the thermophilic phase(Fig.1).The thermophilic phase in all treatments remained above 50°C for 13 days,suggesting that the composting product reached standard sterilization temperatures(GB7959-2012).In the mesophilic phase,the pH decreased initially before increasing and reaching the minimum at day 7,while EC presented a unimodal tendency and peaked at day 7.Thereafter,pH increased until a decrease was observed at day 43,while EC increased continuously.Compared to the CK treatment,EC was lower in the PR treatment,but pH was not.

Throughout the composting process,the TC value decreased continuously,while the TN value decreased initially before increasing.Compared to the CK treatment,the TC and TN values were significant lower in the PR treatment(Fig.2).The C:N ratio was unimodal,peaking at day 19,and showed no significant difference between the CK and PR treatments.

3.2.FTIR spectroscopy

Fig.1 Dynamic changes in temperature,pH and electrical conductivity during the composting period.CK and PR represent nonamended treatment and phosphate rock-amended treatment,respectively.Bars are SD (n=3).

Fig.2 Dynamic changes in total carbon,total nitrogen and carbon-to-nitrogen ratio during the composting process.CK and PR represent non-amended treatment and phosphate rock-amended treatment,respectively.Bars are SD (n=3).

Fig.3 Fourier-transformed infra-red (FTIR) adsorption spectra of compost.CK and PR represent non-amended treatment and phosphate rock-amended treatment,respectively.

FTIR spectra derived from different composting samples showed similar peak patterns (Fig.3).An intense transmittance peak at around 3 370 cm–1,assigned to carbohydrate O–H stretching,including cellulose,hemicellulose,monosaccharide and organic acids,decreased with composting time,and was lower in the PR treatment.This result indicated that PR promoted the decomposition of simple organic matter with low molecular weights.Two minor transmittance peaks present at 2 920 and 2 850 cm–1,attributed to aliphatic C–H stretching,decreased during the composting process and were lower in the PR treatment,while aromatic C=C bending and alkene C=C stretching (1 650 and 1 440 cm–1) were higher in the PR treatment than those in the CK treatment,suggesting that PR accelerated the conversion of saturated carbon into unsaturated carbon.C–O stretching (1 010 cm–1),most likely from polysaccharides,showed no significant difference between the CK and PR treatments.Benzene substitutes,possessing–Ar–X stretching with transmittance peaks at 570 and 470 cm–1,were higher in the PR treatment in the maturation phase,indicating that microbial metabolic activity resulted in an increase in aromatic substances owing to the addition of PR.

3.3.Microbial communities

In the mesophilic phase,the bacterial community structure changed markedly as composting time increased,but no significant difference was observed between the CK and PR treatments (Fig.4).In the thermophilic phase,the bacterial community structure in the PR treatment became significantly different from that in the CK treatment,while there was no significant difference in bacterial community structure during the maturation phase.These results indicate that the addition of PR significantly changed bacterial community structure only in the thermophilic phase.

There was a significant difference in bacterial community composition between the CK and PR treatments (Fig.5).In the mesophilic phase,Firmicutes were the dominant component of the bacterial community,accounting for more than (92.90±0.84)% of total bacteria.In the thermophilic phase,the proportions of Proteobacteria and Bacteroidetes increased slightly in the PR treatment,but not in the CK treatment.In the maturation phase,Firmicutes decreased to less than (15.05±4.05)%,while Proteobacteria,Bacteroidetes,Gemmatimonadetes and Deinococcus-Thermus increased to more than (19.41±2.40),(29.09±1.12),(6.33±0.21) and (13.14±2.62)%,respectively,across all treatments.Together,these results suggest that the CK and PR treatments showed similar changes in their bacterial communities,but that the addition of PR caused differences in the Proteobacteria and Bacteroidetes populations during the thermophilic phase of composting(Fig.6).

3.4.HM-fractions

Fig.4 Analysis of the difference in bacterial community structure during the composting process based on nonmetric multidimensional scaling (NMDS) method.CK and PR represent non-amended treatment and phosphate rockamended treatment,respectively.

During the composting process,total contents of Zn,Cu,Cd,Cr and Pb were all increased,and they were higher in the CK treatment in comparison with the PR treatment.In terms of HM-fractions,there were different responses to PR addition.Exc-Zn and Red-Zn were found to be the main Zn-fractions.The addition of PR slightly stimulated the conversion of Exc-Zn and Red-Zn to Oxi-Zn.The primary fractions of Cu were Exc-Cu,Red-Cu and Oxi-Cu.Co-composting transformed Exc-Cu and Red-Cu into Oxi-Cu,and the treatment amended with PR had higher degrees of conversion.The Res-fraction accounted for large proportions of Cd,Cr and Pb,and co-composting increased the percentages of Res-Cd,Res-Cr and Res-Pb (Fig.7).

Compared to the CK treatment,the PR treatment decreased the BF of Zn,Cu,Cd and Pb by 247.41,41.84,176.25 and 13.44%,respectively (Table 2).Regarding the variation of BF-HM,the PR obviously decreased the bioavailability of Cu,followed by Zn and Cd,with BF reduced from 73.78 to 33.30%,98.37 to 85.62% and 21.06 to 6.17%,respectively.Since Cr and Pb mainly existed as Res-Cr and Res-Pb,the addition of PR had little effect on BF-Cr and BF-Pb.In summary,the addition of PR into composting helps to passivate Cu,but this was not evident for Cr or Pb.

3.5.The correlations of environmental parameters and HM-fractions

pH showed positive correlations with Oxi-Zn,Res-Zn,Oxi-Cu,Res-Cu and Res-Cd,and a negative correlation with Exc-Zn,which indicated that elevated pH was conducive to passivation of Zn,Cu and Cd during composting (Fig.8).EC was positively related with Red-Cu and Exc-Cd,but negatively related with Res-Cr,Red-Pb and Oxi-Pb.There was a negative relationship between MC and Res-Zn as well as Exc-Cr.Temperature correlated positively with Exc-Cu and Oxi-Cr,but negatively with Red-Cd and Oxi-Cd.A positive correlation between C:N ratio and Exc-Cu as well as Oxi-Cr,and a negative correlation between C:N ratio and Red-Cd as well as Oxi-Cd were observed,suggesting that a lower C:N ratio promotes the passivation of Cu during composting.

To further understand the bacterially-controlled passivation mechanisms of HMs,the interactions among physico-chemical parameters,bacterial community and BFHM were established.Path analysis results demonstrated that the C:N ratio and EC inactivated Zn by regulating the activity of Bacteroidetes and Deinococcus-Thermus(Table 3).pH and temperature achieved Cu-passivation by affecting the activity of Bacteroidetes.Proteobacteria,regulated by MC and the C:N ratio,were the primary bacteria to passivate Cd.pH,MC and the C:N ratio together controlled the passivation of Cr by regulating the activity of Bacteroidetes.Bacteroidetes and Deinococcus-Thermus,regulated by pH and the C:N ratio,were the dominant factors explaining the bioavailability of Pb.

Fig.5 Dynamic changes in bacterial compositions during the composting process.CK and PR represent non-amended treatment and phosphate rock-amended treatment,respectively.Lowercase letters represent the significant differences in the bacterial compositions between the CK and PR treatments on the corresponding composting day (n=3).Bars are SD.

Fig.6 Logarithmic-converted heatmap of bacterial communities during the composting process.CK and PR represent non-amended treatment and phosphate rock-amended treatment,respectively.

Fig.7 Dynamic changes (from day 1 to day 52) in heavy metal fractions during composting.CK and PR represent non-amended treatment and phosphate rock-amended treatment,respectively.

Table 2 Change of bioavailability factor (%) for heavy metals during composting1)

4.Discussion

PR has performed well with respect to HM-passivation during aerobic composting.Compared to the CK treatment,BF-Zn,BF-Cu,BF-Cd and BF-Pb decreased by 247.41,41.84,176.25 and 13.44% in the PR treatment,respectively.This result showed a similar tendency with Wanget al.(2013),who proved that adding 20% PR into the compost was the most effective treatment tested for mitigating metal toxicity.The difference in BF-HM reduction observed may be attributed to the passivation mechanism of metal cations with additives.As previously mentioned,Cu and Pb can form compounds with phosphates,Cu10(PO4)6F2and Pb10(PO4)6F2,thereby reducing their bioavailability through co-precipitation (Caoet al.2004).Zn shows an affinity with Fe and Mn oxides,while Cu is associated with organic matter (Luet al.2015),which is similar to our results that Exc-Zn and Red-Zn were the main Znfractions throughout the composting process,and the primary Cu-fraction was Oxi-Cu in the maturation phase.Additionally,Cu and Pb adsorption onto PR is exothermic and more likely to be spontaneous,while Zn sorption is endothermic and needs external energy (Caoet al.2004).This conclusion is consistent with our results that BF-Cu and BF-Pb in the end-product are 40.08 and 12.75% lower than the feedstock during co-composting amended with PR.Previous research has also demonstrated that PR accelerates the rapid increase of temperature (Liet al.2020),and facilitates both the decomposition of organic matter and the release of organic acids (Zhouet al.2018).These factors further promote HM-passivation through the reactions between metal cations and long-chain organic fractions,such as carbohydrates,simple peptides and phenols,during humus production that mainly happened in the maturation phase (Amiret al.2010;Wanget al.2013).This is in line with our results that the PR treatment had a higher extreme temperature than the CK treatment,and that bioavailable HM-fractions (Exc-fraction and Red-fraction)were transformed into non-bioavailable HM-fractions (Oxifraction and Res-fraction) between 25–35 days,which was especially evident for Zn and Cu.

Fig.8 Ordination biplots of redundancy analyses (RDA) between fractions and bioavailability factors of heavy metals as well as chemical parameters.MC,EC and C:N represent moisture content,electrical conductivity and carbon-to-nitrogen ratio,respectively.Exc,Red,Oxi and Res represent exchangeable heavy metals,reducible heavy metals,oxidizable heavy metals and residual heavy metals,respectively.BF represents the bioavailability factor of heavy metals.

During the composting process,as Firmicutes decreased,Proteobacteria,Bacteroidetes,Gemmatimonadetes and Deinococcus-Thermus increased,suggesting that Firmicutes dominated the mesophilic and thermophilic phases,while Proteobacteria,Bacteroidetes,Gemmatimonadetes and Deinococcus-Thermus jointly controlled the maturation phase.A similar finding,that Firmicutes,Proteobacteria and Bacteroidetes were the three most abundant bacterial phyla within aerobic composting,was reported in a national-level survey (Wanget al.2020).As expected,the addition of PR brought forward the variation in primary bacterial phyla in this study.This could be because composting stimulates the activation of non-available phosphorus in the PR and provides more nutrients for bacterial growth (Zhenget al.2020).Microbially-driven mineralization and humification could also strengthen HM-passivation (Li Ret al.2019;Robledoet al.2020).In this study,Bacteroidetes dominated BF-Zn and BF-Cu,confirming the well-established observation that Bacteroidetes has a significant influence on the degradation of cellulose and lignocellulose into short fatty acids,which in turn provide energy for bacteria and can complex with metal cations to form stable compounds (Li Jet al.2019).Proteobacteria,associated with carbon and nitrogen cycling (Li Jet al.2019),were an important factor controlling Cd-passivation.This result was consistent with previous findings which proved that nitrogen-rich manure mixed with carbon-rich straw can produce a favorable environment for Proteobacteria metabolism (Ericksonet al.2009).This may result in metal cations released from unstable organic matter being adsorbed by PR,in agreement with the observation that Res-Cd was the main Cd-fraction in this study.In summary,these results may provide a reference for the use ofHM-passivation techniques in aerobic composting.

Table 3 Path analysis results among bioavailability factor (BF) of heavy metals,physico-chemical parameters and bacterial communities

5.Conclusion

In this study,the addition of PR had a significant influence on HM-passivation and bacterial community composition during composting.Compared to the CK,the BF of Zn,Cu,Cd and Pb was reduced by 247.41,41.84,176.25 and 13.44% in the PR treatment,respectively.In terms of bacterial compositions,as Firmicutes decreased,Proteobacteria,Bacteroidetes,Deinococcus-Thermus and Gemmatimonadetes increased in all treatments,while PR significantly changed the primary bacterial phyla in the thermophilic phase.Bacteroidetes were the primary bacteria controlling the passivation of Zn,Cu and Cr,while Deinococcus-Thermus mainly regulated the bioavailability of Zn and Pb,and Proteobacteria only dominated the transformations among Cd-fractions.

Acknowledgements

This research was financially supported by the National Key Research and Development Program of China(2018YFD0500205) and the Strategic Priority Research Program of the Chinese Academy Sciences (XDA23070502).

Declaration of competing interest

The authors declare that they have no conflict of interest.