Wen DANG, Chunhua GAO, Qiang ZHANG, Jianhua LI, Chaodong LU, Dongsheng JIN,Jinjing LU

1. School of Biological Engineering, Shanxi University, Taiyuan 030006, China;

2. Institute of Agricultural Environment & Resources, Shanxi Academy of Agricultural Sciences, Taiyuan 030006, China

Soil microorganism is the important component of soil ecosystem, which plays an irreplaceable role on animal and plant debris decomposition, nutrient transformation and circulation, soil structure and fertility keeping[1-3].Within a particular geographic area,the composition,structure and function of different microbial community showed a variety of differences in the same environment.Therefore, soil microbial diversity reflects the basic condition of the soil to some extent. Soil microbial diversity mainly includes species, genetic,community structure and functional diversity[4]. Currently, there are many kinds of methods used to explore microbial diversity. According to different objects of diversity research, it is roughly divided into plate dilution culture method, electron microscopy method and staining method that mainly on the microbial diversity;PLFA(phospholipid fatty acid analysis) that mainly on the community structural diversity; DG-GE (denaturing gradient gel electrophoresis), RFLP (restriction fragment length polymorphism),RAPD(random amplified polymorphic DNA marker)that mainly on the genetic diversity; Biolog microplate method and chloroform fumigation extraction method that mainly on the metabolic function diversity and activity and so on[5-7].All of these methods have advantages and disadvantages, but biolog method is favored by many researchers for itssimple operation, high sensitivity,strong resolution, no need to analysis pure microbial, rich data, directly reflect the overall activity of microbial population and clear the characteristic of microbial functional diversity[8]. Biolog microplate assay method was developed by the United States BIOLOG company in 1989, and it was initially applied to pure microbial identification. In 1991, Garland and Mills firstly applied this method to study the soil microbial communities, and indicated that the Biolog carbon source utilization method was an advanced method used to study the soil microbial community structure and diversity in different environment[9]. Due to different kinds of microorganisms and different microbial populations had different carbon sources utilization ability, it results in different carbon source utilization patterns that could characterize the differences of microbial communities[8]. Now Biolog technique was widely used in the analysis of microbial diversity. Jia xia’s research indicated that the culture time had a significant effect on the analysis results of microbial diversity index of Biolog Eco microplate, six kind of carbon sources utilization and PCA[10].There are many preprocessing methods used in the process of Biolog microplate research,but there were no comparative study about the preprocessing method. In this study, the results of three kinds of common preprocessing methods were compared,in order to select an optimal preprocessing method to analyze the soil in same area, and provide reference for future scientific experiments.

Materials and Methods

Soil and experimental treatments

Tested soil was collected from mining reclamation test base of Institute of Agricultural Environment and Resources, Academy of Agricultural Sciences,Luojianggou Village,Changzhi City, Shanxi Province. The corn planting area was treated by different bio-organic fertilizer, and the planting time was May 14thand the sampling time was July 8th in 2013. 0-20 cm of soil sample was collected after the topsoil (1-2 cm) and weeds were removed,then put into aseptic bags and took back to the laboratory.Stored at 4℃. The stones, roots and other debris of the rest soil sample were removed after air-dried, and sieved by 2 mm of sieve.Then put into sealed bags in order to determine the physical and chemical properties. In this test, two treatments were selected to screen preprocessing method. The basic physical and chemical properties of the tested soil was as follows: 0.037%of total nitrogen,0.051%of total phosphorus,1.78%of total potassium,7.71 mg/kg of available phosphorus, 142 mg/kg of rapidly available potassium,32.9 mg/kg of available nitrogen, 3.93 g/kg of organic matter,pH 8.29.

Test principle

Biolog EcoPlate has 96 micropores(8×12),and each 32 micropores was considered as a duplicate. Repeated 3 times. Added water to the control micropore.The other 31 micropores contained a different kind of organic carbon source and the same content of tetrazole purple dye. Under a certain temperature condition, the microorganism solution was inoculated into the micropores and cultured, then the electron generated by microbial respiration transferred and result in tetrazole purple dye turned purple. The shade of color reflected the ability of microorganism using corresponding carbon. OD value of Eco plant was measured at 590 nm wavelength by Biolog microplate reader system.Completed the data acquisition and storage. 31 kinds of single carbon in Eco plate could be divided into six categories: sugars and their derivatives(ten kinds), carboxylic acids (seven kinds), amino acids (six kinds), polymers (four kinds), polyamines (two kinds) and aromatic compounds (two kinds)[11-13].

Biolog Eco analysis

The main steps of the three kinds of different preprocessing methods selected in this study were same. Bacterium solution diluted by 1000 times and added to 96 micropores of Biolog Eco microplates by 8 channel pipetting device. Each micropore added 150 μl.Then the Biolog Eco microplate cultured at suitable temperature, and the data were collected at 24, 48, 72, 96,120, 144 and 168 h respectively by ELx808 microplate reader system. Analyzed the data which the scan wavelength was 590 nm.

Preprocessing methods

Method A:Added fresh soil(equal to 10 g of dired soil)to 90 ml of streilied saline (0.85% Nacl solution) in erlenmeyer flask.Shook for 1 min, then ice bathed for 1 min. Repeated 3 times.Standing for 2 min. Added 5 ml of supernatant to 45 ml of sterile saline,and shook for a moment.Repeated the last step. Then obtained 1 ∶1 000 dilution solution.After that,the solution was inoculated to Biolog Eco microplate and cultured at 30 ℃[14].

Method B:Added fresh soil(equal to 10 g of dired soil) to 90ml of streilied saline (0.85% Nacl solution)in erlenmeyer flask. The rotating speed of shaking table was 250 r/min and shook for 30 min. Standing for 10 min. Then diluted to 1 000 times in turn. After that, the solution was inoculated to Biolog Eco microplate and cultured at 28 ℃[15-16].

Method C: 10 g of fresh soil was added to 100 ml of 0.05 mol/L sterile phosphate buffer (pH 7.0).The rotating speed of the shaking table was 180 r/min and shook for 20 min.Then diluted to 1 000 times.After that,the solution was inoculated to Biolog Eco microplate and cultured at 25 ℃[17].

Added 10 g of fresh soil to the control group in each method.

Data analysis

The average well color development (AWCD value)of Biolog Eco microplate indicates the carbon source metabolism strength of microbial communities,which is an important index of soil microbial activity and function diversity[16].

In this formula,C refers to the absorbance value of each well of 31 well,R refers to the absorbance value of control well.

Average absorbance value of six kinds of carbon sources. AWCD = Σ(C-R)/n,n refers to the total number of carbon sources[18].

Diversity indexes. AWCD value reflects the overall activity of soil microorganism. The diversity index reflects the detailed microbial community species composition and the individuals distribution, and the different aspects of microbial functional diversity[16]. Common indexes include McIntosh index U,Simpson index D, Shannon richness H, Shannon evenness index E and Carbon source utilization richness index S et al[19-21].

(1) McIntosh index is used to measure the homogeneity degree of the community.

(2) Simpson index D=1-ΣPi2reflects the most common species of the community, and was often used to assess the dominance degree of microbial community.

(3) Shannon richness reflects the species richness. H=-ΣPilnP. In this formula,Pirefers to the ratio of the relative absorbance value of i well and the total relative absorbance value of the whole wells.

(4)Shannonevenness E=H/Hmax=H/lnS, H refers to the Shannon index,S refers to the number of well that the color changed.

(5) Carbon source utilization richness index S equal to the total number of the used carbon sources.

All data were proceeded variance analysis and principal component analysis[22-23]by Excel,Sigmaplot,SPSS17.0 software, repeated three times and calculated the average value.

Results and Analysis

Effect of different preprocessing method on AWCD value

The results showed that the change of AWCD value that refers to the use ability of soil microorganism on single carbon source present the similar trend with microbial metabolism,which increased with time extended[24]and changed gently in later stage and finally stabilized (Fig.1). Because after inoculation culturing, the soil microorganism via 24 h of lag phase,gradually adapt to the Biolog microplate environment,and went into the logarithmic growth phase until 96 h, then slowly grew and gradually stabilized. But the rise speed of different preprocessing method was different.The results obviously indicated that method B’s overall variation trend of AWCD value of 2 treatments was superior to A and C’s,which showed that metabolism intensity of microorganism carbon source was maximum under this preprocessing(Fig.1). Variance analysis results indicated that AWCD value of three kinds of preprocessing methods had no significant differences at first and then method B had significant differences with A and C method at 120 h.It indicated that the method B changed fast, even on the final stable stage,method B’s value was significantly higher than other two methods (Table 1). So method B was the better preprocessing method.

Table 1 The change of AWCD value of soil microbial communities under different treatments with the change of culture time

Carbon source utilization ratio of microorganism under different preprocessing method

The results of 1# showed that the carbon source utilization ratio of saccharides and their derivatives was B＞A＞C,the carboxylic acids was B＞A＞C,the amino acids was A ＞B ＞C, the polymers was B＞A＞C,the polyamines was B＞C＞A, the aromatic compounds was A＞B＞C (Fig.2).Method A’s carbon source utilization ratio of amino acids and aromatic compounds was more than other 2 method’s, but the polyamines was less. Method C’s carbon source utilization ratio was all less. The results of 2# showed the same t trends that method B’s microbial utilization of six carbon sources was higher by variance analysis, and the result was relatively stable.

Changes of soil microbial diversity index under different preprocessing method

According to the change dynamics of AWCD value under different preprocessing method, the resultsshowed that AWCD value appeared inflection point after cultured for 96 h,so the data of 96h were selected to do diversity index analysis[25]. The larger the McIntosh index, the better the community uniformity; the greater the Simpson index,the better the community diversity; the higher the Shannon richness index, the more homogeneous the community and the more uniform individual distribution; the greater the carbon source utilization richness index S, the more available carbon source and the better diversity(Table 2). So the Simpson index,Shannon richness index, Shannon evenness index and Carbon source utilization richness index of 1# sample were B ＞C ＞A, except McIntosh index was A＞B＞C. The variance of each diversity index of method B was smaller.The variance analysis results showed that the Shannon evenness had no significant difference, while the other indexes had significant difference.The results of 2# sample was similar to 1#sample, only the Shannon evenness index of method B was lower than the other two methods, but the difference was not significant,while the other indexes were higher than the other two methods. It indicated that different preprocessing methods had significant impact on the results of soil microbial diversity index. In conclusion, method B’s diversity indexes were higher than method A and C’s, so it was an ideal preprocessing methods.

Principal component analysis method

Principal component analysis(PCA) can better describe the carbon source utilization of different soil microbial communities and their metabolic characteristics, it is one of the widely used multivariate analysis[24]. It expresses the complicated data by two-dimensional or three-dimensional icon, because similar distribution has similar position,the difference of communities can be intuitively seen. Load factor refers to greater absolute value has larger impact on the carbon source. Generally 0.8 of load factor or more had greater impact on soil microorganisms.Referred to Dong liguo’s research,the change rate of the data in 96 h was the maximum[2],so the absorbance value of 96 h were selected to conduct principal component analysis. Extracted 8 principal components from each treatments in microplates.

The contribution rate of the first principal component of 1 # was 23.69% , which the weight was the biggest. The contribution rate of the second principal component was 21.35%, and the other principal component was lesser. So the former two principal components were chose to analyze, PC1 was horizontal axis, and PC2 was vertical axis. The score of two principal components of different treatments plotted as coordinate, then the principal component analysis diagram of soil microbial carbon source utilization treated by different preprocessing methods was obtained. The results showed that different preprocessing had obvious distribution difference in PC axis(Fig.3).In PC1 axis,A and B were distributed in the positive direction. In PC2 axis, B was all distributed in the positive direction of the first and second principal component,A was distributed in the negative direction. The results of 2# sample had the same conclusion, which indicated that method B’s carbon source utilization ability was strong and method B was better than other two methods.The results of 1#sample indicated that the weight of carboxylic acids and amino acids in PC1 axis was larger,which was the major carbon source used for discrimination. Carbon sources related to PC2 was mainly amino acids and polyamines. PC1of 2# sample mainly distinguished by amino acids and polymers, and carboxylic acids was the carbon source relate to PC2,and the relationship was relatively large.

Table 2 The microbial diversity index of different preprocession at 96 h

Conclusions

Compared with the change of AWCD value, six kinds of carbon source utilization ability, five kinds of diversity indexes and principal component analysis, method B’s data were the best, method A and C were relatively poor, and A was bigger than C.Three methods were commonly used in all kinds of literatures,and were verified the correct methods. Compared with the three methods, although the overall trend of the conclusions obtained by these three methods were identical, there were differences be-tween the different methods. In addition, the reagent of method B was simple and easy to obtain, and the operation was easy.Seen from the data,that most of method B’s data were larger than A and C’s, the difference of 70% of diversity indexes reached significance level at 96 h,and the stability of the data was higher.Therefore,method B could be used as the effective method for similar soil analysis with the help of Biolog microplate automatic analysis system.

Discussion

Currently, there are many methods used to explore the microbial diversity. Because of the relatively simple operation of Biolog method, which can directly reflect the overall activity of microbial populations,it is commonly used in the analysis of functional diversity of microbial communities.However,the preprocessing methods were not the same in the process of experiments, and there was no research about the preprocessing method in the literatures. In order to screen the optimal preprocessing method, this study did the research of the effects of different methods on testing results. The results showed that all aspects of method B was superior to A and C.But this preprocessing method also had many problems, such as the culture time was short, the microplate has a certain selectivity that only the microorganisms took advantage of the carbon source could grow. However, it only represented a portion of the microbial community,while the various microorganisms cultured in microplates, the results of color was not simple addition of color caused by each various species, and these synergistic effects or antagonistic effects depended on cell density. There were many factors affected coloration, in addition to the preprocessing,inoculation density and incubation time were also the key factors, and the composition, quantity and activity of microbial populations,the number of interfering substances and other factors in nutrient solution would affect coloration of microplate,which would caused unstable testing results[26].

Therefore, it was necessary to combine with other methods and explore more rational data analysis methods,in order to deeply study the structure and function of microbial community and their relationships, so as to obtain more comprehensive results.

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