Analysis of Microbial Abundance within Perennial and Annual Crop Systems

Abstract

In this project, soil systems of different crops were analyzed. The microbial community structure and composition were analyzed here. One particular fungus that was analyzed was mycorrhiza. This particular fungus is important because it aids in aggregate formation. The hyphae networks create a web that clumps the soil together. Within these aggregates, soil organic carbon can be stored. The more soil organic carbon stored in the soil, the less that is emitted into the atmosphere. Which in turn decreases the amount of greenhouse gases. The crops that were analyzed were broken down into two different groups, perennial and annual crops. The perennial crops were big bluestem, miscanthus, and switchgrass. The annual crops were corn (rotated with soy beans), corn (continuous), sweet sorghum, grain sorghum, and photosensitive sorghum. Four samples of each crop were collected from four different replicate plots from the KSU North Farm (Fall 2013) from a long term experiment comparing annuals and perennials which began in 2007. The samples were collected at three different depths (0-5 cm, 5-15 cm, 15-30 cm). This is to test how depth affects amount of microbial abundance. Once the soil samples are collected, the Bligh and Dyer method of extraction (1959) is used to extract the lipids from the soil. The end product are fatty acid methyl esters (FAMEs) which will be analyzed with gas chromatography (GC) with an internal standard (19:0). The biomarkers revealed by GC were analyzed to show absolute abundance and relative abundance. Relative abundance of PLFAs within the soil was very helpful in determining the composition of the microbial community. By analyzing these charts, it was concluded that the original hypothesis was correct. The perennial crops had more mycorrhiza biomarkers than the annuals, which could be a result of the greater biomass. It was also concluded that microbial abundance decreased with depth.

Introduction

Crop systems play a major role on their respective soil systems. When considering certain crops such as perennial crops, it is expected that this crop system will differ from that of an annual crop system. Key differences are within the root biomass, microbial biomass, and aggregate distribution. These differences also account for how much soil organic carbon each crop system contains. The more soil organic carbon that is stored in the soil, the less there is in the atmosphere. Crop systems sequester CO2 gas out of the atmosphere and store it as soil organic carbon through photosynthesis (Lal). And as mentioned earlier, each of those differences between the crop systems influences how much soil organic carbon is stored within the soil. For example, perennial crop systems tend to have more root biomass (roots) which provide more carbon inputs into the soil. Microbial biomass is important as well because it accounts for the organisms that aid in the formation of aggregates. A specific fungi called mycorrhiza have shown to play an important role in the formation of the aggregate, which physically contain and protect the soil organic carbon.

The key factor to all of this is microbial abundance within the soil. In order to determine what is in the soil, the phospholipid fatty acid (PLFA) markers will be analyzed. For example, the fungi, mycorrhiza have a specific PLFA biomarker that is unique to that group. And by extracting those biomarkers from samples of soil, it can be determined whether or not that fungi is present in that crop system.

Also, it is important to analyze the microbial abundance at different depths in the soil. When looking at annual crop systems, it can be expect that most of the microbial abundance will be near the surface. And this because annual crop systems don’t grow all year long. They are seasonal and die off in their off season. However, perennial crop systems grow all year long. Therefore it is important for their roots to reach deep into the soil and take in nutrients. This in turn will increase microbial abundance deeper within the soil.

Experimental Method

Samples used in the procedure were taken from four different plots. For this project, eight different crops were chosen to be analyzed which are grain sorghum, photosensitive sorghum, sweet sorghum, corn (rotated), corn (continuous), big bluestem, miscanthus, and switchgrass. Each of these crops were taken from each plot to ensure that results were not biased.

Soil Probe Soil sample

Samples are also taken at three different depths within the soil. Samples were taken from 0 to 5 centimeters, 5 to 15 centimeters, and 15 to 30 centimeters. This is done so that a comparison can be made between the annual and perennial crop systems. And to take samples at the different depths, a soil probe is used. Once the soil has been collected, it is grounded up into a fine powder and is ready for the PLFA extraction step.

The lipids were extracted following the method of Bligh and Dyer (1959) as modified by White et al (1979) (Zelles). The extraction step can be broken down into three different phases. Typically, these three phases are take about three days, one day for each phase. In each phase, solvents such as chloroform, acetone, methanol, and hexane were used to separate the PLFAs, NFLAs, and glycolipid fatty acids (GLFAs) from the soil. During phase one, a phosphate buffer mixed with chloroform and methanol is used to separate all the lipids from the soil. At this point, NLFAs, PLFAs, and glycolipids are mixed together. After three hours of venting, the portion containing the lipids is extracted out of the soil and is placed in a dark room at room temperature. Phase two begins the following day. Phase two is when the different lipids are separated (Fig. 1). To do so, silicic acid chromatography is used. 500 mg pre-conditioned disposable silica gel columns were used for the chromatography. Chloroform is used to extract the NLFAs, acetone is used to extract the glycolipids (not collected), and the PLFAs are extracted by methanol. This concludes phase two. In phase three, the collected extractions are methylated (KOH saponification and methylation) to create fatty acid methyl esters (FAMEs).

Once the FAMEs have been evaporated, they are ran through a gas chromatography (GC) mass spectrometer. The FAMEs are ran through the GC with nonadecanoic acid methyl ester (19:0) which serves as an internal standard. The peaks revealed by the GC can be compared to the internal standard in order to quantify how much of each biomarker is present in the sample.

Results and Discussion

The FAMEs analyzed by the GC were separated into two different categories. One set of the data was dedicated to the PLFAs from the soil samples, and the next set was for NLFAs from the same soil samples. PLFA and NLFA FAMEs were separated in the silicic acid phase of the extraction. Using the peak from the internal standard (19:0), the sample peaks were able to be quantified by comparing it to the internal standard. Using this comparison, the abundance of PLFA, expressed in nmol g-1 of soil (Fig. 1), was determined. Using this data, relative abundance of microbial groups was also plotted (Fig. 3). In addition to the differences in crop type, the effect of depth was also analyzed. The amount of PLFAs in each depth, expressed in nmol g-1, was also plotted (Fig. 2). The biomarkers were broken down into their respective microbial groups.

By looking at the chart indicating how much PLFA was present per gram of soil (Fig. 1), it is easy notice that there a lot Gram (-) and Gram (+). This indicates an abundance of bacteria in the soil. Which is expected because bacteria are essential in soil systems. Another thing to note from this chart is the differences between the perennials and the annual crops. The first three crops (switchgrass, big bluestem, and miscanthus) generally lead each category in amount of PLFA. It is especially important to the note the differences in the AMF and fungi categories. This is important because these are the biomarkers that are indications of the fungi, mycorrhiza.

Fig. 1 Fig. 1

An obvious trend can be noticed in the chart analyzing amount of PLFA in each depth (Fig. 2). Levels of PLFA are significantly higher near the surface (p < 0.05). The data also shows that Gram (-) and Gram (+) bacterial groups are the highest in abundance. This confirms the conclusions made from Fig. 1.

Fig. 2 Fig. 2

It is important to note that absolute abundance is not an accurate representation of the microbial community composition. Absolute abundance only expresses amounts of biomarkers based on weight. This isn’t an accurate representation because certain biomarkers are heavier than others. Therefore, it is important to analyze the relative abundance (Fig. 3) within each crop. This determines the composition of each microbial community. And it is important to note that within the perennial crops, AMF and fungal microbial groups are more prominent than those of the annual crops. This is an indication of more mycorrhiza in the perennials.

Fig. 3Fig. 3

Another way to compare the crops relatively, a fungal (AMF and fungi) to bacterial (Gram (-), Gram (+), and actinomycetes) ratio has been calculated (Fig. 4). This another visual to show the microbial community structure. In this case, the fungal groups are associated with the mycorrhiza, and the perennials have the higher ratios. This confirms the conclusions made from Fig. 3.

Fig. 4 Fig. 4

Along with PLFAs, the NLFAs were also analyzed. However, the interesting fact about the NLFA biomarkers is that they are only found in eukaryotes (Chamberlain). Therefore, prokaryotes such as bacteria will not have the NLFA biomarkers. This serves as a better representation for the AMF and other fungal biomarkers (Fig. 5). However, when looking at absolute abundance of PLFAs, miscanthus is significantly greater (p < 0.05) than all the other crops in the AMF and fungal microbial groups. But PLFA biomarkers for AMF and fungal groups can also be shared among other bacteria. By analyzing the NLFAs for those biomarkers, it can be determined with confidence that those biomarkers are from fungal groups and not bacteria. It is also important to note that there is a greater amount of NLFAs compared to PLFAs. This is because NLFAs are involved in storage structures within microbes and the PLFAs are not.

Fig. 5 Fig. 5

Conclusions

The results confirm that the original hypothesis is correct. It was hypothesized that the perennial crops would have more of the fungi, mycorrhiza. This was confirmed by amount of AMF and fungal microbial groups in the perennials. This can be observed in the relative abundances (Fig. 3). Fungal and AMF microbial groups are indications of mycorrhiza. Mycorrhiza play an important role in aggregate formation, therefore it can be concluded that perennial crops produce more aggregates than annuals. This means that perennial crops sequester more CO2 gas and store it as soil organic carbon. It was also hypothesized that microbial biomass would decrease with depth. And PLFA amount per gram of soil by depth (Fig. 2) significantly decreased with depth (p < 0.05). The most abundant depth for PLFA was near the surface.

About Me

  • University of Maryland College Park
  • Mechanical Engineering, Class of 2017
  • Home town: Silver Spring, MD
  • Hobbies: basketball, volleyball, biking, hiking, bowling, and having fun

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Acknowledgements

  • Andrew McGowan
  • Dr. Charles Rice
  • Dr. Amber Campbell Hibbs

This material is based upon work supported by National Science Foundation No. EPS-0903806 and the State of Kansas.

References

  • Lal, R. Soil Carbon Sequestration To Mitigate Climate Change. Geoderma , 1-22.
  • Chamberlain, P. M. The fat that matters: Soil food web analysis using fatty acids and their carbon stable isotope signature. Soil Biology and Biochemistry, 1898-1910.
  • Zelles, L. (1999). Fatty acid patterns of phospholipids and lipopolysaccharides in the characterisation of microbial communities in soil: a review. Biology and Fertility of Soils, 29(2), 111-129.