INTRODUCTION

Microbial fuel cells(MFCs)can generate currents through the oxidation of organic substrates by exoelectrogenic microorganisms,and have previously been constructed using soil(Ringelberg et al.,2011;Wang et al.,2015).The current of soil MFCs was correlated with temperature(Deng et al.,2014),which is a vital factor significantly in fluencing microbial activity.When different concentrations of heavy metals were added to soil,electrical signals including the quantity of electrons and start-up time generated by soil exoelectrogenic bacteria were significantly correlated with soil dehydrogenase activity(Deng et al.,2015;Jiang et al.,2015),which is a sensitive indicator of microbial activity.In another bioelectrochemical system,the cathodic characteristics detected as electrical signals were correlated with the microbial growth and bio film development on the working electrodes when the electrochemical probes were directly inserted into the soil(Cristiani et al.,2008).Thus,electrical signals may potentially indicate overall soil microbial activity.Both soil microbial activity and electrical signals were previously found to be affected by common factors,including soil organic matter content and microbial community(Dunaj et al.,2012;Jiang et al.,2016).However,several other factors,such as soil conductivity and the content of electron shuttles,may specifically affect electrical signals,weakening their correlation with microbial activity(Zhou et al.,2014).Therefore,it is worth investigating whether electrical signals correlate with soil microbial activities across different soils.

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The red soil region in China has been subjected to severe erosion,primarily as a result of deforestation.Over the last three decades,the region has been revegetated with typical vegetation.Both soil microbial activities and physicochemical properties differ among the unplanted eroded soil and re-vegetated soil with different vegetation types(Deng et al.,2010;Zhang et al.,2010;Yin et al.,2014).Re-vegetated red soil also contains a relative abundance of iron and humus,which may act as electron shuttles and have specific effects on electrical signals(Roden et al.,2010;Zhou et al.,2014).Thus,re-vegetated red soils provide an ideal opportunity to assess the correlation between soil microbial activity and electrical signals under different chemical properties.

Nearly all exoelectrogenic bacteria and soil microorganisms thrive on soil organic substrates and favor neutral environments(Liu et al.,2005).The activity of exoelectrogenic bacteria may represent that of the overall microbial community.We hypothesized that the electrical signals of MFCs would significantly correlate with the microbial activity in re-vegetated red soils.To verify this hypothesis,we investigated the electrical signals of severely eroded red soil and those restored with Pinus massoniana,Lespedeza bicolor,Castanea mollissima,and Pennisetum purpureum using MFC operation.Soil dehydrogenase activity was measured simultaneously as a reference method to test the reliability of the electrical signals.The relationships between electrical signals and dehydrogenase activity or soil chemical properties were set up by using regression models.After MFC operation,the anodic bacterial community was analyzed using high-throughput Illumina sequencing(Daghio et al.,2015).The aim of this study was to verify whether a significant correlation exited between electrical signals and microbial activity in the re-vegetated red soils and unplanted eroded red soil.

MATERIALS AND METHODS

Site description and soil sampling

The sampling site was located at the Red Soil Ecological Experiment Station,Chinese Academy of Sciences(116°55′30′′E,28°15′20′′N)in Yingtan,Jiangxi Province,China.Soils are composed mainly of Quaternary red clay.The average annual precipitation is 1785 mm,and the average annual temperature is 17.8°C.Deforestation during the 1960s led to severe degradation in many areas,and native species were planted on the eroded areas in 1987.Some severely eroded areas were left unplanted as references.

Routine methods were applied for the analysis of soil physicochemical properties(Page et al.,1982).Briefly,soil pH was measured at a soil∶water ratio of 1∶2.5(weight∶volume)and soil electrical conductivity(EC)at 1∶5(weight∶volume);soil organic carbon(OC)was determined by K2CrO4oxidation and total nitrogen(TN)by Kjeldahl digestion;soil dissolved organic carbon(DOC)was extracted using 0.5 mol L−1 K2SO4,and total humic carbon(THC)by 0.1 mol L−1Na4P2O7and 0.1 mol L−1NaOH and measured using an automated TOC analyzer(TOC-L,Shimadzu,Kyoto,Japan);soil total dissolved iron(DFeT)was extracted using 0.2 mol L−1H2C2O4-(NH4)2C2O4(Mc-Keague and Day,1966)and measured by the Ferro-Zine colorimetric method(To et al.,1999).Soil dehydrogenase activity(DHA)was determined from the conversion of triphenyltetrazolium chloride to triphenylformazan according to Casida et al.(1964).All measurements were conducted prior to MFC operation.

Soil property measurements

The soils were collected aseptically from five habitat classes covering the restoration scenarios∶eroded soils restored with Pinus massoniana(PM),Lespedeza bicolor(LB),Castanea mollissima(CM),and Pennisetum purpureum(PP),and unplanted eroded soil(ES).Three similar stands approximately 100 m apart from each other were chosen for each soil.For each stand,the surface soil(0–20 cm)was collected from three adjacent plots and combined to yield a composite sample.Fifteen soil samples were collected in total( five habitat classes×three stands).After removing the surface litter,the soils were sieved through a 2-mm diameter mesh,and the aliquots were stored at 4°C for microbial properties determination and MFC operation or air-dried for physicochemical analysis.

This study validated that the start-up time of the MFCs was significantly correlated with soil DHA based on 15 different re-vegetated and eroded soils.These findings are supported by previous studies that used soil sets with varying toxicity levels(Deng et al.,2015).Although some synthetic activity may be neglected,the DHA reflects the vast majority of the metabolic activity of soil microorganisms and is referred to as one of the most sensitive indicators for overall soil microbial activity(Monkiedje et al.,2002).The significant correlation between the start-up time and DHA might demonstrate a potential indication of soil microbial activity by the electrical signals generated by MFCs with re-vegetated red soils.

MFCs setup and operation

Significant differences between means(n=3)were determined by one-way analysis of variance at a level of P ＜ 0.05 using Tukey’s honestly significant difference test.Correlation and regression analyses(n=15)were conducted between electrical signals and soil physicochemical and microbial properties.All statistical tests were conducted using SPSS software version 18.0.

During vegetative restoration,plant litter and root exudates constitute the major sources of soil OC,nitrogen,and humic carbon(Deng et al.,2010).With the inclusion of soil exoelectrogenic bacteria,plant litter and root exudates enhance the activity of the soil microbial community.This might result in correlations between the start-up time and DHA,OC,and DOC.However,the peak voltage and coulomb quantity were not correlated with soil microbial activity and physicochemical properties.This may be explained by the addition of glucose,which is the substrate or electron donor in MFCs(Pant et al.,2010).To accelerate power generation,glucose has been added into soil as an organic substrate(Chae et al.,2009).This might mask the differences in carbon substrate contents among soil samples,leading to the generation of the same levels of peak voltage and coulomb quantity.With the exception of the correlation between start-up time and THC,the electrical signals were not correlated with THC and DFeT,which may affect power generation in MFCs(Roden et al.,2010;Zhou et al.,2014).Moreover,as THC was significantly correlated with OC and DHA(data not shown),it was impossible to ascertain the in fluence of THC on the start-up time among other soil properties.These results suggested that soil electron mediator content may not affect the electrical signals of MFCs with large additions of substrates.

MFC anodic bacterial community analysis

After the MFC operation,the anodes from the 15 MFCs(not including the three sterilized control MFCs)were collected and rinsed as previously described(Deng et al.,2015;Jiang et al.,2015).The genomic DNA was immediately extracted from the anodes using the Fast DNA SPIN kit for soil(BIO101,MP Biomedicals,Carlsbad,USA)following the manufacturer’s instructions.The extracted DNA samples were quantified using a Nanodrop ND-1000(NanoDrop Technologies,Wilmington,USA).

Polymerase chain reaction amplification and Illumina MiSeq sequencing were conducted at Personalbio(Shanghai Personal Biotechnology Co.Ltd.,Shanghai,China).The V4 hypervariable region of the 16S rRNA gene was amplified using universal primers 515F(5′-GTG CCA GCM GCC GCG GTA A-3′)and 907R(5′-CCG TCA ATT CMT TTR AGT TT-3′)(Weisburg et al.,1991),followed by quantification of the individual reactions and product purification.Sequencing was conducted on an Illumina MiSeq platform using 2×250 bp paired-end flow cells and reagent cartridges.The data generated from the Illumina sequencing were analyzed by Mothur(Schloss et al.,2009)using the MiSeq standard operating procedure(Kozich et al.,2013).The raw data were all deposited in the NCBI Sequence Read Archive database under the accession number SRP071044.

After MFC operation,the anodic bacterial community was analyzed based on the 16S rRNA gene V4 region by Illumina sequencing.Qualified sequences were assigned to known phyla,classes,and genera to identify the phylogenetic diversity of the bacterial communities.Nine identified phyla were detected,with Firmi-cutes and Proteobacteria being the most abundant(Fig.2a).Focusing on the two dominant phyla,Bacilli and Clostridia(Firmicutes)constituted the major classes in most anode samples.In some soil samples,such as PM2 and ES3, α-and γ-Proteobacteria comprised another two dominant classes(Fig.2b).At the genus level of all qualified sequences,Clostridium was the most abundant,followed by Sporolactobacillus,Thermus,and Acinetobacter(Fig.2c).

Statistical analysis

The dual chamber MFC reactors were used as previously described(Deng et al.,2015).The area of each carbon felt electrode was 16 cm2(4 cm×4 cm).The anode chamber was packed with 150 g(dry weight)soil amended with 4%(weight∶weight)glucose and filled with 100 mL distilled water to keep the soil saturated.Soil from one stand was packed into one MFC,thereby resulting in 15 MFCs( five habitat classes×three stands).The cathode chamber was filled with 50 mmol L−1potassium ferricyanide dissolved in 50 mmol L−1 phosphate buffer solution.A cation exchange membrane was used to separate the two chambers.The external load was 1000 Ω with the electrodes connected using titanium wire.The MFCs were operated at constant 30°C in an incubator for 228 h.The voltage of the MFCs was recorded every 10 min using a data acquisition module.To con firm that the voltage had originated from microbial processes rather than chemical processes,three MFCs,packed with sterilized soils from three stands of P.massoniana by chloroform fumigation,were operated as controls under the same conditions as mentioned above.

RESULTS

Electrical signals and their relationships with soil properties

The voltages of the MFCs packed with sterilized soil were consistently below 10 mV during the operation(data not shown),indicating that the voltage had originated mainly from microbial processes.The voltage curves generated from the MFCs with soil samples are illustrated in Fig.1.The electrical signals extracted from the voltage curves included start-up time,peak voltage,and coulomb quantity(Table I).The start-up time of ES was significantly longer than that of CM and PP,but there was no significant difference between ES and PM or LB.There was also no significant diffe-rence in the peak voltage and coulomb quantity.Properties reflecting the physicochemical and microbial status of the soil samples are also shown in Table I.The OC,DOC,and EC of ES were significantly lower(P＜0.05),while soil pH was significantly higher than those of the re-vegetated soils.The DHA of ES was not detected and was assumed to be below the detection limit.There was no significant difference in DHA among the re-vegetated soils.

TABLE I Electrical signals of microbial fuel cells operated with and physicochemical and microbial propertiesa)of the unplanted eroded soil(ES)and the soils restored with Pinus massoniana(PM),Lespedeza bicolor(LB),Castanea mollissima(CM),and Pennisetum purpureum(PP)

a)EC=electrical conductivity;OC=organic carbon;DOC=dissolved organic carbon;TN=total nitrogen;THC=total humic carbon;DFeT=total dissolved iron;DHA=dehydrogenase activity;TFP=trifunctional protein. b)Means±standard deviations(n=3). c)Means followed by the same letter(s)within each column are not significantly different at P ＜ 0.05. d)Not detected.

SoilStart-up Peak CoulombpH EC OC DOC TN THC DFeT DHA time voltage quantity mg kg−1 mg TFP kg−1d−1 PM 3.80± 273.07± 62.72± 4.30± 40.40± 16.87± 218.44± 0.70± 9.13± 103.00± 10.93±1.60b)abc) 239.6a 63.34a 0.08c 2.15ab 6.98a 66.70c 0.20ab 6.96ab 25.31a 1.12a LB 2.83± 198.40± 36.13± 4.13± 59.00± 28.85± 396.87± 1.05± 16.92± 125.61± 15.17±2.49ab 228.2a 38.41a 0.08c 14.05a 4.49a 55.30a 0.22a 3.56a 26.99a 6.00a CM 1.28± 373.93± 73.72± 4.16± 48.10± 20.01± 343.44± 0.47± 12.29± 99.10± 15.33±0.36b 91.8a 27.08a 0.12c 6.41ab 6.23a 27.40ab 0.23bc 4.56ab 23.43a 6.93a PP 0.73± 432.77± 103.90± 4.51± 31.83± 19.57± 264.70± 0.71± 11.61± 123.98± 10.74±0.14b 27.5a 20.03a 0.02b 1.45b 2.88a 14.70bc 0.06ab 1.28ab 14.78a 3.21a ES 5.10± 329.87± 35.17± 4.85± 8.74± 2.14± 81.95± 0.17± 1.76± 103.07± NDd)1.01a 65.7a 3.64a 0.01a 0.43c 0.29b 3.25d 0.02c 0.12b 1.37a d mV C µS cm−1 g kg−1 mg kg−1g kg−1

Table II indicates the Pearson correlation coeffi-cients(r)between the electrical signals and soil properties.The start-up time was negatively correlated with DHA(P＜0.01)and also negatively correlated with OC,DOC,and THC(P＜0.05).The peak voltage and coulomb quantity were correlated with none of the soil properties,including THC and DFeT,which may act as specific factors of electrical signals.

Regression analysis was then performed to assess the relationships between the start-up time and soil properties with significant correlations(Table III).The linear model had both the highest coefficient of determination(R2)and the lowest Akaike information criterion(indicating the superiority of statistical model fitting)values of the three models.

Fig.1 Voltages of microbial fuel cells operated with the unplanted eroded soil(ES)and the soils restored with Pinus massoniana(PM),Lespedeza bicolor(LB),Castanea mollissima(CM),and Pennisetum purpureum(PP).Three similar stands(1–3)approximately 100 m apart from each other were chosen for each soil.

Dominant anodic bacterial community groups of MFCs

Start-up time,peak voltage,and coulomb quantity were selected as the electrical signals.Peak voltage and coulomb quantity were defined as the maximum voltage and total charge transferred during MFC operation,respectively.Coulomb quantity was calculated as previously described(Deng et al.,2015).Start-up time was defined as the time span from the beginning of operation until 10 mV was exceeded,set as a criterion because the voltages of the MFCs with fumigated soil had all been found to be consistently below 10 mV(Jiang et al.,2015).

R a应根据工程地质勘察报告提供的每一个勘察孔的土层分布分别计算，待所有孔R a的确定后，取最小值作为下一步设计用R a，该最小值可取整，也可留少量富余，但不能太大，否则会使计算的土层压缩模量不准确，进而影响复合地基的变形计算。

TABLE II Pearson correlation coefficients between electrical signals of microbial fuel cells and soil physicochemical and microbial propertiesa)(n=15)

*,**Significant at P＜0.05 and P＜0.01,respectively. a)EC=electrical conductivity;OC=organic carbon;DOC=dissolved organic carbon;TN=total nitrogen;THC=total humic carbon;DFeT=total dissolved iron;DHA=dehydrogenase activity.

Electrical signal pH EC OC DOC TN THC DFeT DHA Start-up time 0.363 −0.407 −0.609* −0.535* −0.381 −0.581* −0.227 −0.652**Peak voltage 0.225 −0.390 −0.101 0.001 −0.210 −0.154 −0.368 −0.098 Coulomb quantity −0.023 −0.770 0.187 0.193 0.099 0.094 −0.208 0.213

TABLE III Regression analyses between start-up time(ST)of the microbial fuel cells and soil dehydrogenase activity(DHA),organic carbon(OC),dissolved organic carbon(DOC),and total humic carbon(THC)

a)Akaike information criterion.

Relationship Model type Equation R2 AICa) P value in F-test ST-DHA Linear DHA=16.4576−2.1919ST 0.381 56.60 ＜0.01 Polynomial DHA=16.5365−2.2731ST−0.0126ST2 0.329 60.42 ＜0.05 Exponential DHA= −241.0713+257.5894e−0.000 875ST 0.329 60.42 ＜ 0.001 ST-OC Linear OC=25.5761−2.9421ST 0.324 68.84 ＜0.05 Polynomial OC=24.7605−2.1024ST−0.1305ST2 0.267 72.63 ＞0.05 Exponential OC= −746.1923+771.8016e−0.003 9ST 0.266 72.66 ＜ 0.001 ST-DOC Linear DOC=346.0921−30.9287ST 0.232 145.16 ＜0.05 Polynomial DOC=325.6955−9.9292ST−3.2631ST2 0.173 148.88 ＞0.05 Exponential DOC= −100869.832+101215.9496e−0.000 305 9ST 0.168 148.98 ＜ 0.001 ST-THC Linear THC=15.1968−1.7662ST 0.286 55.72 ＜0.05 Polynomial THC=15.1283−1.6957ST−0.011ST2 0.227 59.54 ＞0.05 Exponential THC= −1036.008+1051.214e−0.001 69ST 0.227 59.54 ＜ 0.001

DISCUSSION

第三，优化教学内容，实行理论教学和实践教学并重。不断拓展校外实训基地的建设，并形成制度化，建立旅游专业校外实训企业档案制，加强学校与各实训企业的合作与联系，定期总结经验，吸取教训，形成长期稳定合作。可以邀请企业专家到校给学生教授实践课程和进行就业指导，形成学校教育和企业培训间的“无缝链接”，极大提高学生的创新思维，并为他们未来就业创业奠定良好的实践基础。

培育大学生文化自信，要坚持牢牢把握社会主义先进文化发展方向，坚持社会主义核心价值体系引领，并且处理好批判与继承、外来文化与本土文化的关系，坚持面向现代化、面向世界、面向未来四个原则，以古建筑传统文化的当代价值为载体，以高校思想政治教育为路径，提升高校大学生的文化自信。

After MFC operation,exoelectrogenic bacteria enrich on the anode,resulting in the anodic bacterial community significantly different from that of the soil(Wang et al.,2015).The taxonomic classification of anodic bacteria at the phylum level found that Firmicutes and Proteobacteria were dominant in the bacterial community.The exoelectrogenic bacteria detected in the soil mostly belonged to these two phyla(Ringelberg et al.,2011).The anodic bio films may be another source of inocula for MFCs,and the microbial community may vary substantially with different soils(Dunaj et al.,2012).At the class level,Bacilli and Clostridia were dominant in most soil samples and have commonly been found at the anodes of soil MFCs(Chae et al.,2009;Dunaj et al.,2012).Clostridium,Sporolactobacillus,Thermus,and Acinetobacter were dominant at the genus level.Clostridium can oxidize organic substrates and extracellularly transfer electrons to iron oxides or solid electrodes under anaerobic conditions(Park et al.,2001;Shah et al.,2014).Several strains belonging to Clostridium have been isolated from soils and identified as exoelectrogenic bacteria by the MFC operation(Jiang et al.,2016).Acinetobacter secretes an unidentified endogenous compound similar to pyrroloquinoline quinine and conducts extracellular electron transfer in MFCs(Rabaey et al.,2008;Freguia et al.,2010).In addition to being dominant in MFCs inoculated with soils,Clostridium and Acinetobacter were reported to constitute,at least in part,the anodic bacteria in MFCs using anaerobic sludge from wastewater treatment plants as inocula(Yu et al.,2012;Yusoffet al.,2013).These two genera might contain certain exoelectrogenic bacteria that contribute to Electricity generation.Considering that glucose is not a substrate available for typical anodic exoelectrogenic bacteria(Chae et al.,2009),the electrical signals might be generated through secondary anaerobic oxidation steps after the primary fermentation,with which Clostridium may also be associated.The exoelectrogenic role of Clostridium and Acinetobacter in the soil requires further verification including isolation and electrochemical identification.However,there is no evidence that Sporolactobacillus and Thermus possess an exoelectrogenic ability,and they are likely to be involved in the fermentation of glucose(Wang et al.,2011).

红军长征在雪山草地，由于执行了党的民族政策，注重从少数民族地区实际出发，团结帮助少数民族参与民族革命的解放战争中，不仅获得了少数民族中、下层群众的支持帮助，也削弱了上层势力同国民党勾结，减轻了红军的压力，是成功解决粮食问题的重要原因。

Fig.2 Taxonomic classification of bacterial DNA sequences from communities of the microbial fuel cell anode at the phylum level(a),the class level distribution of the most dominant phyla Firmicutes and Proteobacteria(b),and the genus level(c).The phyla,classes,or genera that constituted less than 1%of the total composition in all libraries were excluded.Three similar stands(1–3)approximately 100 m apart from each other were chosen for the unplanted eroded soil(ES)and the soils restored with Pinus massoniana(PM),Lespedeza bicolor(LB),Castanea mollissima(CM),and Pennisetum purpureum(PP).

CONCLUSIONS

This study verified that the start-up time of MFCs was significantly correlated with soil dehydrogenase activity over different soils.Electrical signals generated by MFCs may indicate soil microbial activity in re-vegetated red soils.Clostridium and Acinetobacter were the dominant bacterial genera on the MFC anode and may comprise exoelectrogenic bacteria.We suggest the start-up time as a potential indicator of soil microbial activity to be validated in other soils in addition to these re-vegetated red soils.

初步设计和概算获得批复后，应立即开始施工图设计。在图纸设计阶段，甲方基建部门应做好审图把关，对照初步设计文件审查，并核对图纸套用设计规范的准确性。后续的概算编制工作应重点审查概算和预算的差价，并及时调整相关工程的设计标准，以符合初步设计和概算批复要求。

ACKNOWLEDGEMENT

This study was supported by the Natural Science Foundation of China(No.41671250),the Jiangsu Provincial Natural Science Foundation of China(No.BK20171476),and the Outstanding Innovation Team in Colleges and Universities in Jiangsu Province,China.

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