论文部分内容阅读
Abstract Fly ash from a municipal solid waste incineration plant in Haikou was taken as the research object. The biological toxicity of its leachate was determined by luminescent bacteria method. The leaching toxicity of the leachate was determined by atomic fluorescence spectrometry. The results showed that the leaching with Aspergillus niger had good removal effect on As and Hg， and the leached fly ash and leachate could meet the safe discharge standard. 37 ?? was the best temperature for A. niger leaching. The concentrations of As and Hg in fly ash leachate were reduced to 0.438 and 0.053 g/L， respectively after 5 d of leaching by A. niger at 37 ??. The luminous intensity of leachate increased with the increase of the number of days. Therefore， the leaching with A. niger can effectively remove As and Hg in fly ash from municipal solid waste incineration， and the leachate can also meet the safe discharge standard.
Key words MSWI fly ash； Heavy metals； Aspergillus niger； Bioleaching
In recent years， with the rapid development of China??s economy and the continuous expansion of urbanization scale， hazardous wastes including heavy metals in MSWI fly ash have been embedded underground continuously and become the sources of underground water and soil pollution. According to China Statistical Yearbook 2016， the amount of harmlessly??treated garbage in China reached 180 130 000 t in 2015， and has grown at a rate of 10% basically in recent years [1]. Data show that about 2/3 of large and medium size cities in China are surround by garbage [2-5]， and 1/4 of cities have got no occasions suitable for stacking garbage [6]. A series of eco??environment protection and livelihood issues brought by massive city domestic garbage have attracted more and more attention from government and the public [7-8]. The disposal methods for domestic rubbish include landfill， composting， sorting treatment and incineration. China has 677 municipal solid waste disposal facilities， including 547 refuse landfills which have an actual handling capacity of about 1.0??108 t/a， 109 incineration plants which have an actual handling capacity of about 2.6??107 t/a， and 21 refuse composting plants which have an actual handling capacity of about 4.27??106 t/a [9]. It could be seen that the application of landfill and incineration grows continuously， while the application of composting shrinks. Waste incineration has the advantages of high speed and small occupied area. This method could sterilize garbage through high temperature， and reduce its volume by 90% and quantity by 75%， thereby effectively relieving the contradiction between municipal solid waste and shortage of land resource [10]. Furthermore， the afterheat of incineration also could be used for supplying heat and generating electricity， thereby achieving the purpose of recycling resource [11-12]. However， the fly ash produced during municipal solid waste incineration contains various toxic heavy metals including Cd， Pb and Zn at higher leaching concentrations， belonging to hazardous wastes [13]， so it should be subjected to harmless disposal before the final disposal. Materials and Methods
Experimental materials
Source of fly ash Fly ash samples were collected from a municipal solid waste incineration plant according to provisions in Identification Standards for Hazardous Wastes??Identification of Leaching Toxicity （HJ/T 298??2007）. The collected fly ash was quartered to the amount required by the experiment.
Tested strains Freeze??dried powder of Aspergillus niger （A. niger CMCC（F）98003） was purchased from Guangdong Huankai Microbial Sci. & Tech. Co.， Ltd. Freeze??dried powder of photobacterium phosphoreum T3 race was provided by Institute of Soil Science， Chinese Academy of Sciences.
Instruments and reagents AFS??930 atomic florescence spectrophotometer （Beijing Titan Instruments Co.， Ltd.）； FE20 pH meter （Mettler??Toledo Instruments （Shanghai） Co.， Ltd.）； FZ??10 turnover type vibrator （Changsha Nuoda Instrument Co.， Ltd.）； SHZ??D （?ó） circulating water multipurpose vacuum pump （Henan Yuhua Instrument Co.， Ltd.）； THZ??92B vapor??bathing constant temperature vibrator （Shanghai Boxun Industrial Limited Company）； 5810 table type high??speed large??capacity centrifuge （eppendorf）； DXY??3 intelligent biological toxicity testing instrument （Institute of Soil Science， Chinese Academy of Sciences）.
As stock solution （1 000 ??g/ml Shanghai Aladdin Bio??Chem Technology Co.， Ltd.）； Hg stock solution （1 000 ??g/ml Shanghai Aladdin Bio??Chem Technology Co.， Ltd.）； 1% sodium borohydride solution； 5% thiourea and ascorbic acid solution； concentrated sulfuric acid （analytical pure）； concentrated nitric acid （analytical pure）.
Experimental conditions The test conditions of the atomic fluorescence spectrophotometer are shown in Table 1.
Experiment methods
Determination of initial As and Hg concentrations At first， 5-10 g of the fly ash sample was accurately weighed and placed in a vessel with a cover. The fly ash was dried to constant weight at 105 ??， as shown in Table 2. According to formula， water content in the fly ash sample was about 3%， suggesting that 100 g of the fly ash contains about 3 ml of water.
An initial concentration leaching experiment of fly ash sample was carried out with reference to Solid Waste??Extraction Procedure for Leaching Toxicity??Sulfuric Acid & Nitric Acid Method （HJ/T 299??2007）. A leaching agent was prepared from 2 ml of concentrated sulfuric acid and 1 ml of concentrated nitric acid at a volume ratio of 2?? 1， and another 1 ml of concentrated nitric acid was diluted to 1%. Four 2 L plastic bottles matching with a turnover type vibrator were marked to be I， II， III and IV， respectively. Each of the bottle was added with 997 ml of deionized water and 1-2 drops of the leaching agent， and the obtained solution was adjusted with the 1% diluted nitric acid to a pH value of 3.2??0.05， giving the sulfuric acid??nitric acid leaching solution. I was set as the blank group free of the addition of fly ash， and other three plastic bottles were added with 100 g of fly ash sample， respectively. In order to avoid leakage during the turning and vibrating process， the plastic bottles were sealed with sealing film at first and tightened with a cover. The plastic bottles were vibrated in the turnover type vibrator and shaken for 24 h at （30??2） r/min. During vibration， the bottles were check for 2 times to see whether leakage or power cut happens. Then， the leaching solutions were all vacuum filtered with the circulating water multipurpose vacuum pump. For each filtrate， 10 ml was reserved for later determination. Bioleaching A. niger was activated on PDA slant for 3 times and then preserved on slant as solid strain. In a 150 ml triangular flask， 50 ml of potato glucose liquid medium was prepared and inoculated under sterile condition with one ring of the solid strain， which was cultured at 37 ?? in a shaker. The inoculated strain was observed once every 8 h， and A. niger mycelial pellets were observed 24 h later， which meant that the liquid strain was obtained.
Twelve potato glucose liquid medium samples were prepared， each of which contained 48 ml of potato glucose liquid medium. Then， 1 ml of the liquid strain and 1 g of the fly ash were inoculated to the liquid medium， giving 50 ml of sample solution. The prepared medium was all preserved at low temperature in a refrigerator. A temperature gradient （27， 32， 37 and 42 ??） was set as the first variable， and time was the second variable. Each temperature had three replicates， which were cultured in a constant??temperature shaker. Then， 5 ml of leachate was taken every 24 h， for 5 times in total， and 60 samples were obtained in total. The samples were centrifuged at a high speed， obtaining supernatants which were preserved at a low temperature for later determination.
Testing of As and Hg concentrations in leachate The leaching toxicity of As and Hg was detected by standard curve method. The concentrations of the As standard solutions were set to be 0.00， 1.00， 2.00， 5.00 and 10.00 ??g/L， and the Hg standard solutions were 0.00， 0.10， 0.20， 0.50 and 1.00 ??g/L. During the preparation of As standard solutions， 15 ml of 5% thiourea??vitamin C solution was added into each of them， while the Hg standard solutions were not added with the solution. During the determination of As concentration， sodium borohydride solution was added into concentrated hydrochloric acid， to reduce As3+ in the solutions into AsH and Hg2+ into simple Hg [14-15]. Ar gas was selected as the carrier gas which was introduced into an atomizer， to allow atomization of heavy metals in Ar??H2 flame. The two heavy metals were activated through the excitation by a hollow cathode lamp， thereby emitting fluorescence. Because As concentration and Hg concentration have positive linear relation with respective fluorescence intensity， As and Hg concentrations could be calculated according to the linear relation.
Testing of biotoxicity in leachate An ampoule bottle was opened and added with sterilized 2.5% NaCl solution to revive T3 strain， and about 2 min later， the ampoule bottle was observed to be shining in the shade. Testing tubes were added with 2 ml of sterilized 3% NaCl solution and 10 ??l of activated luminous T3 liquid. The luminous intensity of a blank liquid was tested after shaken under the magnification??1， and the liquid was usable when the luminous intensity was higher than 600 mv. Testing was carried out 5 min after the revival of the luminous liquid [16]. Because the prepared samples had color which influenced the testing of the biotoxicity， the samples were diluted. In this experiment， 10 ??l of each sample was taken and diluted with 1 990 ??l of 3% NaCl solution， i.e.， the samples were diluted by 200 times， to greatly reduce the influence of sample color on luminous quantity. Analysis method Curves were drawn in Excel 2013， and significance of difference was analyzed by DPS 15.0 Duncan??s new multiple range method.
Results and Analysis
Plotting of standard curve
The standard curves of two heavy metals were plotted with mass concentration of standard solution as X??axle and fluorescence values measured with the atomic fluorescence spectrophotometer as Y??axle， as shown in Fig. 1 and Fig. 2. The standard curve of As was I=60.597??C+15.118 （R2=0.997 9）， and that of Hg was I=535.13??C+16.472 （R2=0.998 6）.
Agricultural Biotechnology 2018As and Hg concentrations in leachate
As concentration analysis It could be seen from Fig. 3 that with the prolong of culture time， the As concentrations in the leachates showed a trend of increasing at first and decreasing then， and all reached a peak value on the second day under various temperatures. The leaching process of As from solid fly ash by A. niger proceeds with the accompany of the growth of A. niger which has certain hysteresis effect in the fly ash solution [17]. After the second day， the As concentrations in the leachates decreased to different degrees over time， which was because that the mycelia of A. niger had certain adsorption effect on heavy metals [18]. The comparison of the four curves at the four culture temperatures showed that the As concentration in the 37 ?? leachate was lower than those in other leachates in the whole process， and exhibited the lowest value at the end of the leaching especially. Therefore， 37 ?? was the optimal temperature for leaching of metal As in fly ash by A. niger.
Hg concentration analysis It could be seen from Fig. 4 that with the prolonging of culture time， the Hg concentrations in the leachates showed a trend of increasing at first and decreasing then， and all reached a peak value on the second day under various temperatures. The leaching process of Hg from solid fly ash by A. niger proceeds with the accompany of the mycelia growth of A. niger which has certain hysteresis effect in the fly ash solution [17]. After the second day， the Hg concentrations in the leachates decreased to different degrees over time， which was because that the mycelia of A. niger had certain adsorption effect on heavy metals [18]. The comparison of the four curves at the four culture temperatures showed that the Hg concentration in the 37 ?? leachate was lower than those in other leachates mostly， and exhibited the lowest value at the end of the leaching especially. Therefore， 37 ?? was the optimal temperature for leaching of metal Hg in fly ash by A. niger. Evaluation of leaching effect The heavy metal contents in fly ash leached at 37 ?? with the best leaching effect were compared with the standards in Standard for Pollution Control on the Landfill Site of Municipal Solid Waste （GB 16889??2008）， and the results showed that the As and Hg concentrations in fly ash leached by A. niger all did not exceed standards. The comparison results are shown in Table 3.
The heavy metal contents in the final leachate obtained at 37 ?? which had the best leaching effect were compared with the standards in Standard for Pollution Control on the Landfill Site of Municipal Solid Waste （GB 16889??2008）， and the results showed that the As and Hg concentrations in the leachates obtained after leaching by A. niger did not exceed standards. The comparison results are shown in Table 3.
Biotoxicity of leachate
Fly ash was leached with A. niger at 27， 32， 37 and 42 ?? for 5 d. The luminous intensities of various groups of leachates were tested by luminous bacteria method. The data were processed with DPS 15.0， and the significance of differences was analyzed between different days under the same temperature. The results are shown in Table 4.
Different uppercase letters indicate a very significant difference （P??0.01）， and different lowercase letters indicate a significant difference（P??0.05）.
The luminous intensities in samples of each day and each temperature were compared， and the dynamic change of the luminous intensity is shown in Fig. 5.
As shown in Fig. 5， the luminous intensities in samples increased with the number of days increasing， indicating that the concentrations of heavy metals including As and Hg in fly ash leachate decreased over time. On the first day of leaching， heavy metals in solid fly ash had not been dissolved into liquid under various temperatures， so the heavy metal concentrations in leachates were not high， and the inhibition of the final leachates on luminous bacteria was not significant. On the second day of leaching， the leachates at the various culture temperatures all remarkably inhibited the luminous quantity of luminous bacteria， suggesting that the heavy metals in solid fly ash had been dissolved in the leachates to the largest extent， while the growth of A. niger showed a lag effect due to the inhibition caused by heavy metal toxicity. On the third day， the luminous quantity increased gradually over time， suggesting that with the growth of A. niger， heavy metals in leachates were gradually adsorbed， and the toxicity of the leachates was reduced gradually， i.e.， A. niger well leached toxic heavy metals in fly ash. Luminous quantity was compared between different temperatures， and the results showed that within 5 d in the leaching process， the luminous quantity of bacteria at 37 ?? were commonly higher than those at other temperatures， suggesting that A. niger had a better leaching effect on fly ash at 37 ??. Conclusions
The MSWI fly ash from some waste incineration plant in Haikou City was detected， and conclusions were made as below.
Compared with national standards， the As and Hg concentrations in the fly ash leached with A. niger did not exceed national standards； and compared with the discharge standard of sewage， the As and Hg concentrations in the leachates obtained after leaching of fly ash with A. niger did not exceed national standards.
37 ?? was the optimal temperature for leaching of As and Hg in fly ash with A. niger. After five days of leaching with A. niger at 37 ??， the As and Hg concentrations in fly ash leachate were both reduced to the minimum value. Specifically， As concentration was reduced to 0.438 ??g/L， and Hg concentration was reduced to 0.053 ??g/L.
The detection results of biotoxicity showed that over time， the luminous quantity of samples also increased gradually， i.e.， the luminous bacteria had very strong bioactivity， with strong ability of producing ATP and luciferase， which meant that the biotoxicity of the samples to luminous bacteria was lowered gradually. The biotoxicity of samples on luminous bacteria was also reduced gradually. It indicated that the leaching with A. niger could cause the reduction of toxic heavy metals?? concentrations.
References
[1] LI H. Municipal waste： Current situation and way out[J]. ecological economy， 2014， 30（4）： 10-13.
[2] MA XX. Current situation and development countermeasure of municipal solid water incineration[J]. Technology Wind， 2014（16）： 214-216.
[3] WANG LQ， LI XM， ZHU FH. Current situation of municipal solid wastes disposal and development proposals in China[J]. Environmental Pollution Control， 2015（2）： 106-109.
[4] MAO YN， WANG XH， ZHAO DQ， et al. Comprehensive evaluation of different municipal solid waste （MSW） incineration technologies[J]. Environmental Pollution Control， 2015（2）： 32-37， 41.
[5] YANG Z. Current situation and prospects for municipal waste??to??energy power generation technology[J]. China Resources Comprehensive Utilization， 2016（12）： 33-35.
[6] CHEN HB， WANG Y， HUANG JX， et al. Several measures to strengthen waste classification organization and management in residential area[J]. Environmental Sanitation Engineering， 2016（2）： 64-66.
[7] GAN J. How to avoid NIMBY conflict of municipal solid waste incineration power plant： A case study of Hangzhou Jiufeng project[J]. Environmental Sanitation Engineering， 2016（4）： 87-88. [8] XIE XH. Preliminary study on hazard and disposal of municipal solid waste[J]. Low Carbon World， 2016， （3）： 7-8.
[9] Committee of Urban Domestic Refuse Treatment， China Association of Environmental Protection Industry. China development report on disposal industries of urban domestic refuse in 2012[J]. China Environmental Protection Industry， 2013（3）： 20-26.
[10] ZHANG Q， XU HY. Current situation and development proposals of household waste incineration disposal technique[J]. Environmental Engineering， 2012， 30（2）： 79-82.
[11] LU RL. Summarizing on solidification technology of solid waste incineration[J]. Jiangsu Construction， 2012（1）： 104-106.
[12] State Environmental Protection Administration. GWKB3??2000 Standard for pollution control on the municipal solid waste incineration[S]. Beijing： China Environmental Science Press， 2000.
[13] MANGIALARDI T. Disposal of MSWI fly ash through a combined washing??immobilisation process[J]. J Hazard Mater， 2003， 98（1P2P3）： 225-240.
[14] RAO BQ. Experiment method discussion on arsenic detection in water by AFS??930 atomic fluorescence spectrometer[J]. Guangdong Chemical Industry， 2013， 12（40）： 193-194.
[15] CHEN YJ， LUO HY. Simultaneous mensuration of arsenic and mercury in water using AFS??930 atomic fluorescence spectrometer[J]. Guangdong Chemical Industry， 2005， 32（7）： 61-62.
[16] State Department of Environmental Conservation， State Bureau of Technical Supervision. GB/T 15441??1995， Water Quality??Determination of the acute toxicity??Luminescent bacteria test [S]. Beijing： China Standards Press， 1995： 618-624.
[17] YANG J， WANG QH， WANG Q， et al. Influence of fly ash concentrations on the growth of Aspergillus niger and the bioleaching efficiency of heavy metals[J]. Environmental Science， 2008， 29（3）： 825-830.
[18] LI B， LI HJ， JIAN L， et al. Effect of Aspergillus Niger on bioleaching of Haikou municipal refuse incineration fly ash[J]. Environmental Science and Management， 2016， 41（5）： 77-80.
Key words MSWI fly ash； Heavy metals； Aspergillus niger； Bioleaching
In recent years， with the rapid development of China??s economy and the continuous expansion of urbanization scale， hazardous wastes including heavy metals in MSWI fly ash have been embedded underground continuously and become the sources of underground water and soil pollution. According to China Statistical Yearbook 2016， the amount of harmlessly??treated garbage in China reached 180 130 000 t in 2015， and has grown at a rate of 10% basically in recent years [1]. Data show that about 2/3 of large and medium size cities in China are surround by garbage [2-5]， and 1/4 of cities have got no occasions suitable for stacking garbage [6]. A series of eco??environment protection and livelihood issues brought by massive city domestic garbage have attracted more and more attention from government and the public [7-8]. The disposal methods for domestic rubbish include landfill， composting， sorting treatment and incineration. China has 677 municipal solid waste disposal facilities， including 547 refuse landfills which have an actual handling capacity of about 1.0??108 t/a， 109 incineration plants which have an actual handling capacity of about 2.6??107 t/a， and 21 refuse composting plants which have an actual handling capacity of about 4.27??106 t/a [9]. It could be seen that the application of landfill and incineration grows continuously， while the application of composting shrinks. Waste incineration has the advantages of high speed and small occupied area. This method could sterilize garbage through high temperature， and reduce its volume by 90% and quantity by 75%， thereby effectively relieving the contradiction between municipal solid waste and shortage of land resource [10]. Furthermore， the afterheat of incineration also could be used for supplying heat and generating electricity， thereby achieving the purpose of recycling resource [11-12]. However， the fly ash produced during municipal solid waste incineration contains various toxic heavy metals including Cd， Pb and Zn at higher leaching concentrations， belonging to hazardous wastes [13]， so it should be subjected to harmless disposal before the final disposal. Materials and Methods
Experimental materials
Source of fly ash Fly ash samples were collected from a municipal solid waste incineration plant according to provisions in Identification Standards for Hazardous Wastes??Identification of Leaching Toxicity （HJ/T 298??2007）. The collected fly ash was quartered to the amount required by the experiment.
Tested strains Freeze??dried powder of Aspergillus niger （A. niger CMCC（F）98003） was purchased from Guangdong Huankai Microbial Sci. & Tech. Co.， Ltd. Freeze??dried powder of photobacterium phosphoreum T3 race was provided by Institute of Soil Science， Chinese Academy of Sciences.
Instruments and reagents AFS??930 atomic florescence spectrophotometer （Beijing Titan Instruments Co.， Ltd.）； FE20 pH meter （Mettler??Toledo Instruments （Shanghai） Co.， Ltd.）； FZ??10 turnover type vibrator （Changsha Nuoda Instrument Co.， Ltd.）； SHZ??D （?ó） circulating water multipurpose vacuum pump （Henan Yuhua Instrument Co.， Ltd.）； THZ??92B vapor??bathing constant temperature vibrator （Shanghai Boxun Industrial Limited Company）； 5810 table type high??speed large??capacity centrifuge （eppendorf）； DXY??3 intelligent biological toxicity testing instrument （Institute of Soil Science， Chinese Academy of Sciences）.
As stock solution （1 000 ??g/ml Shanghai Aladdin Bio??Chem Technology Co.， Ltd.）； Hg stock solution （1 000 ??g/ml Shanghai Aladdin Bio??Chem Technology Co.， Ltd.）； 1% sodium borohydride solution； 5% thiourea and ascorbic acid solution； concentrated sulfuric acid （analytical pure）； concentrated nitric acid （analytical pure）.
Experimental conditions The test conditions of the atomic fluorescence spectrophotometer are shown in Table 1.
Experiment methods
Determination of initial As and Hg concentrations At first， 5-10 g of the fly ash sample was accurately weighed and placed in a vessel with a cover. The fly ash was dried to constant weight at 105 ??， as shown in Table 2. According to formula， water content in the fly ash sample was about 3%， suggesting that 100 g of the fly ash contains about 3 ml of water.
An initial concentration leaching experiment of fly ash sample was carried out with reference to Solid Waste??Extraction Procedure for Leaching Toxicity??Sulfuric Acid & Nitric Acid Method （HJ/T 299??2007）. A leaching agent was prepared from 2 ml of concentrated sulfuric acid and 1 ml of concentrated nitric acid at a volume ratio of 2?? 1， and another 1 ml of concentrated nitric acid was diluted to 1%. Four 2 L plastic bottles matching with a turnover type vibrator were marked to be I， II， III and IV， respectively. Each of the bottle was added with 997 ml of deionized water and 1-2 drops of the leaching agent， and the obtained solution was adjusted with the 1% diluted nitric acid to a pH value of 3.2??0.05， giving the sulfuric acid??nitric acid leaching solution. I was set as the blank group free of the addition of fly ash， and other three plastic bottles were added with 100 g of fly ash sample， respectively. In order to avoid leakage during the turning and vibrating process， the plastic bottles were sealed with sealing film at first and tightened with a cover. The plastic bottles were vibrated in the turnover type vibrator and shaken for 24 h at （30??2） r/min. During vibration， the bottles were check for 2 times to see whether leakage or power cut happens. Then， the leaching solutions were all vacuum filtered with the circulating water multipurpose vacuum pump. For each filtrate， 10 ml was reserved for later determination. Bioleaching A. niger was activated on PDA slant for 3 times and then preserved on slant as solid strain. In a 150 ml triangular flask， 50 ml of potato glucose liquid medium was prepared and inoculated under sterile condition with one ring of the solid strain， which was cultured at 37 ?? in a shaker. The inoculated strain was observed once every 8 h， and A. niger mycelial pellets were observed 24 h later， which meant that the liquid strain was obtained.
Twelve potato glucose liquid medium samples were prepared， each of which contained 48 ml of potato glucose liquid medium. Then， 1 ml of the liquid strain and 1 g of the fly ash were inoculated to the liquid medium， giving 50 ml of sample solution. The prepared medium was all preserved at low temperature in a refrigerator. A temperature gradient （27， 32， 37 and 42 ??） was set as the first variable， and time was the second variable. Each temperature had three replicates， which were cultured in a constant??temperature shaker. Then， 5 ml of leachate was taken every 24 h， for 5 times in total， and 60 samples were obtained in total. The samples were centrifuged at a high speed， obtaining supernatants which were preserved at a low temperature for later determination.
Testing of As and Hg concentrations in leachate The leaching toxicity of As and Hg was detected by standard curve method. The concentrations of the As standard solutions were set to be 0.00， 1.00， 2.00， 5.00 and 10.00 ??g/L， and the Hg standard solutions were 0.00， 0.10， 0.20， 0.50 and 1.00 ??g/L. During the preparation of As standard solutions， 15 ml of 5% thiourea??vitamin C solution was added into each of them， while the Hg standard solutions were not added with the solution. During the determination of As concentration， sodium borohydride solution was added into concentrated hydrochloric acid， to reduce As3+ in the solutions into AsH and Hg2+ into simple Hg [14-15]. Ar gas was selected as the carrier gas which was introduced into an atomizer， to allow atomization of heavy metals in Ar??H2 flame. The two heavy metals were activated through the excitation by a hollow cathode lamp， thereby emitting fluorescence. Because As concentration and Hg concentration have positive linear relation with respective fluorescence intensity， As and Hg concentrations could be calculated according to the linear relation.
Testing of biotoxicity in leachate An ampoule bottle was opened and added with sterilized 2.5% NaCl solution to revive T3 strain， and about 2 min later， the ampoule bottle was observed to be shining in the shade. Testing tubes were added with 2 ml of sterilized 3% NaCl solution and 10 ??l of activated luminous T3 liquid. The luminous intensity of a blank liquid was tested after shaken under the magnification??1， and the liquid was usable when the luminous intensity was higher than 600 mv. Testing was carried out 5 min after the revival of the luminous liquid [16]. Because the prepared samples had color which influenced the testing of the biotoxicity， the samples were diluted. In this experiment， 10 ??l of each sample was taken and diluted with 1 990 ??l of 3% NaCl solution， i.e.， the samples were diluted by 200 times， to greatly reduce the influence of sample color on luminous quantity. Analysis method Curves were drawn in Excel 2013， and significance of difference was analyzed by DPS 15.0 Duncan??s new multiple range method.
Results and Analysis
Plotting of standard curve
The standard curves of two heavy metals were plotted with mass concentration of standard solution as X??axle and fluorescence values measured with the atomic fluorescence spectrophotometer as Y??axle， as shown in Fig. 1 and Fig. 2. The standard curve of As was I=60.597??C+15.118 （R2=0.997 9）， and that of Hg was I=535.13??C+16.472 （R2=0.998 6）.
Agricultural Biotechnology 2018As and Hg concentrations in leachate
As concentration analysis It could be seen from Fig. 3 that with the prolong of culture time， the As concentrations in the leachates showed a trend of increasing at first and decreasing then， and all reached a peak value on the second day under various temperatures. The leaching process of As from solid fly ash by A. niger proceeds with the accompany of the growth of A. niger which has certain hysteresis effect in the fly ash solution [17]. After the second day， the As concentrations in the leachates decreased to different degrees over time， which was because that the mycelia of A. niger had certain adsorption effect on heavy metals [18]. The comparison of the four curves at the four culture temperatures showed that the As concentration in the 37 ?? leachate was lower than those in other leachates in the whole process， and exhibited the lowest value at the end of the leaching especially. Therefore， 37 ?? was the optimal temperature for leaching of metal As in fly ash by A. niger.
Hg concentration analysis It could be seen from Fig. 4 that with the prolonging of culture time， the Hg concentrations in the leachates showed a trend of increasing at first and decreasing then， and all reached a peak value on the second day under various temperatures. The leaching process of Hg from solid fly ash by A. niger proceeds with the accompany of the mycelia growth of A. niger which has certain hysteresis effect in the fly ash solution [17]. After the second day， the Hg concentrations in the leachates decreased to different degrees over time， which was because that the mycelia of A. niger had certain adsorption effect on heavy metals [18]. The comparison of the four curves at the four culture temperatures showed that the Hg concentration in the 37 ?? leachate was lower than those in other leachates mostly， and exhibited the lowest value at the end of the leaching especially. Therefore， 37 ?? was the optimal temperature for leaching of metal Hg in fly ash by A. niger. Evaluation of leaching effect The heavy metal contents in fly ash leached at 37 ?? with the best leaching effect were compared with the standards in Standard for Pollution Control on the Landfill Site of Municipal Solid Waste （GB 16889??2008）， and the results showed that the As and Hg concentrations in fly ash leached by A. niger all did not exceed standards. The comparison results are shown in Table 3.
The heavy metal contents in the final leachate obtained at 37 ?? which had the best leaching effect were compared with the standards in Standard for Pollution Control on the Landfill Site of Municipal Solid Waste （GB 16889??2008）， and the results showed that the As and Hg concentrations in the leachates obtained after leaching by A. niger did not exceed standards. The comparison results are shown in Table 3.
Biotoxicity of leachate
Fly ash was leached with A. niger at 27， 32， 37 and 42 ?? for 5 d. The luminous intensities of various groups of leachates were tested by luminous bacteria method. The data were processed with DPS 15.0， and the significance of differences was analyzed between different days under the same temperature. The results are shown in Table 4.
Different uppercase letters indicate a very significant difference （P??0.01）， and different lowercase letters indicate a significant difference（P??0.05）.
The luminous intensities in samples of each day and each temperature were compared， and the dynamic change of the luminous intensity is shown in Fig. 5.
As shown in Fig. 5， the luminous intensities in samples increased with the number of days increasing， indicating that the concentrations of heavy metals including As and Hg in fly ash leachate decreased over time. On the first day of leaching， heavy metals in solid fly ash had not been dissolved into liquid under various temperatures， so the heavy metal concentrations in leachates were not high， and the inhibition of the final leachates on luminous bacteria was not significant. On the second day of leaching， the leachates at the various culture temperatures all remarkably inhibited the luminous quantity of luminous bacteria， suggesting that the heavy metals in solid fly ash had been dissolved in the leachates to the largest extent， while the growth of A. niger showed a lag effect due to the inhibition caused by heavy metal toxicity. On the third day， the luminous quantity increased gradually over time， suggesting that with the growth of A. niger， heavy metals in leachates were gradually adsorbed， and the toxicity of the leachates was reduced gradually， i.e.， A. niger well leached toxic heavy metals in fly ash. Luminous quantity was compared between different temperatures， and the results showed that within 5 d in the leaching process， the luminous quantity of bacteria at 37 ?? were commonly higher than those at other temperatures， suggesting that A. niger had a better leaching effect on fly ash at 37 ??. Conclusions
The MSWI fly ash from some waste incineration plant in Haikou City was detected， and conclusions were made as below.
Compared with national standards， the As and Hg concentrations in the fly ash leached with A. niger did not exceed national standards； and compared with the discharge standard of sewage， the As and Hg concentrations in the leachates obtained after leaching of fly ash with A. niger did not exceed national standards.
37 ?? was the optimal temperature for leaching of As and Hg in fly ash with A. niger. After five days of leaching with A. niger at 37 ??， the As and Hg concentrations in fly ash leachate were both reduced to the minimum value. Specifically， As concentration was reduced to 0.438 ??g/L， and Hg concentration was reduced to 0.053 ??g/L.
The detection results of biotoxicity showed that over time， the luminous quantity of samples also increased gradually， i.e.， the luminous bacteria had very strong bioactivity， with strong ability of producing ATP and luciferase， which meant that the biotoxicity of the samples to luminous bacteria was lowered gradually. The biotoxicity of samples on luminous bacteria was also reduced gradually. It indicated that the leaching with A. niger could cause the reduction of toxic heavy metals?? concentrations.
References
[1] LI H. Municipal waste： Current situation and way out[J]. ecological economy， 2014， 30（4）： 10-13.
[2] MA XX. Current situation and development countermeasure of municipal solid water incineration[J]. Technology Wind， 2014（16）： 214-216.
[3] WANG LQ， LI XM， ZHU FH. Current situation of municipal solid wastes disposal and development proposals in China[J]. Environmental Pollution Control， 2015（2）： 106-109.
[4] MAO YN， WANG XH， ZHAO DQ， et al. Comprehensive evaluation of different municipal solid waste （MSW） incineration technologies[J]. Environmental Pollution Control， 2015（2）： 32-37， 41.
[5] YANG Z. Current situation and prospects for municipal waste??to??energy power generation technology[J]. China Resources Comprehensive Utilization， 2016（12）： 33-35.
[6] CHEN HB， WANG Y， HUANG JX， et al. Several measures to strengthen waste classification organization and management in residential area[J]. Environmental Sanitation Engineering， 2016（2）： 64-66.
[7] GAN J. How to avoid NIMBY conflict of municipal solid waste incineration power plant： A case study of Hangzhou Jiufeng project[J]. Environmental Sanitation Engineering， 2016（4）： 87-88. [8] XIE XH. Preliminary study on hazard and disposal of municipal solid waste[J]. Low Carbon World， 2016， （3）： 7-8.
[9] Committee of Urban Domestic Refuse Treatment， China Association of Environmental Protection Industry. China development report on disposal industries of urban domestic refuse in 2012[J]. China Environmental Protection Industry， 2013（3）： 20-26.
[10] ZHANG Q， XU HY. Current situation and development proposals of household waste incineration disposal technique[J]. Environmental Engineering， 2012， 30（2）： 79-82.
[11] LU RL. Summarizing on solidification technology of solid waste incineration[J]. Jiangsu Construction， 2012（1）： 104-106.
[12] State Environmental Protection Administration. GWKB3??2000 Standard for pollution control on the municipal solid waste incineration[S]. Beijing： China Environmental Science Press， 2000.
[13] MANGIALARDI T. Disposal of MSWI fly ash through a combined washing??immobilisation process[J]. J Hazard Mater， 2003， 98（1P2P3）： 225-240.
[14] RAO BQ. Experiment method discussion on arsenic detection in water by AFS??930 atomic fluorescence spectrometer[J]. Guangdong Chemical Industry， 2013， 12（40）： 193-194.
[15] CHEN YJ， LUO HY. Simultaneous mensuration of arsenic and mercury in water using AFS??930 atomic fluorescence spectrometer[J]. Guangdong Chemical Industry， 2005， 32（7）： 61-62.
[16] State Department of Environmental Conservation， State Bureau of Technical Supervision. GB/T 15441??1995， Water Quality??Determination of the acute toxicity??Luminescent bacteria test [S]. Beijing： China Standards Press， 1995： 618-624.
[17] YANG J， WANG QH， WANG Q， et al. Influence of fly ash concentrations on the growth of Aspergillus niger and the bioleaching efficiency of heavy metals[J]. Environmental Science， 2008， 29（3）： 825-830.
[18] LI B， LI HJ， JIAN L， et al. Effect of Aspergillus Niger on bioleaching of Haikou municipal refuse incineration fly ash[J]. Environmental Science and Management， 2016， 41（5）： 77-80.