Measuring Microbial Communities' Biomass

To analyze the biomass of a microbial community, there are two types of approaches incorporating several different methods. These approaches include the use of either direct or indirect sampling techniques [1]. Directing sampling involves counting numerical values, while indirect sampling involves the use of chemicals [1].

Direct Approaches

Agar Plates

Top view of soil microorganisms nutrient agar in plate [14]

Outlined by Jones et al (1948) the use of agar plates was used to count organisms present in microscopic fields [3]. To begin, random samples of soil are obtained and sifted through a sieve before being weighed out [3,5]. Following this, the sample is placed into a crucible with sterile distilled water and ground up with a glass rod [3,5]. Next, the sample is washed with sterile distilled water, and any remaining suspended matter is poured into a separate flask [3,5]. The soil suspension is then made up to 50ml with 1.5% being agar [3,5]. The sample flask is then shaken and left to rest for a short period. Once rested, a pipette is used to remove the sample solution and place drops onto a glass slide [3]. The prepared slide is then put into sterile distilled water [3,5]. Once dried, the sample can be placed under a microscope to count organisms [3,5].

Extractable DNA

Torsvik et al (1990) used extractable DNA to determine the identities of organisms within soil samples [1,13]. In this study, six 30g soil samples were prepared. These samples were then were washed with 2% sodium hexametaphosphate to increase the yield of DNA [7]. This step allows for the extraction of naked DNA adsorbs to colloids [7]. The suspensions are then stored in a refrigerator and the pellets were stored in isopropanol [7]. Pellets are then centrifuged, suspended in a buffer, and then homogenized to lyse the soil bacteria [7]. Following this the volume is adjusted to 25ml using a buffer and then incubated for one hour [7]. Then 2 mg of proteinase K ml-1 was added and then incubated for another hour [6]. The suspension is then heated to 60oC, sodium dodecyl sulfate was added, and then incubated for five minutes [7]. The lysate was then, KCl was added, refrigerated overnight, and then centrifuged [7]. The supernatants were pooled and purified on a hydroxyapatite (HAP) column [7]. DNA from the pooled fractions were then concentrated by cetylpyridinium bromide precipitation to purify the DNA [7].

Signature Lipid Biomarkers (SLB)

This technique involves the measurement of ester-linked polar lipid fatty acids and steroids to find microbial biomass and community structure [1,10,12]. A common biomarker used for this technique are phospholipids fatty acids (PFLAs) [1,10,12]. The total number of PFLAs provides quantitative measure of viable or potentially viable biomass [11,12]. When a cell dies the cellular enzymes hydrolyze and release a phosphate group. The remaining lipid is then compared to the ratio of PFLAs to the remaining lipid [12]. This provides evidence of viable and non-viable microbes [12].

Indirect Approaches

Chloroform Fumigation and Incubation (CFI)

1. Soil samples are exposed to chloroform fumigation and extraction. (a).Biomass is assumed to be extracted with equal and complete efficiency 2. A fraction of the soil samples are are incubated. 3. New DNA is present from incubation 4. Relationship between DNA and microbial biomass carbon content of the community.[6]

The Chloroform fumigation and incubation method (CFI) is used to determine organic C biomass within soil microbials. Chloroform (CHCl₂) vapor is used to fumigate soil [1,9]. Once the soil is fumigated, the CHCl₃ vapor is removed before incubating the soil [9]. Evolved CO₂ levels are then measured for both fumigated and then unfumigated soil to calculate biomass [1,9]. Using the expression B=F/k_c (B=soil biomass C, F= carbon dioxide carbon evolved by fumigated soil minus CO₂ evolved by nonfumigated soil, and k_c= fraction of biomass mineralized to CO₂ during the incubation) biomass can be calculated where kc is a constant [1]. Voroney and Paul (1984) furthered this technique to include labile nitrogen, measuring the fraction of biomass nitrogen (k_n) mineralized to inorganic nitrogen [9].

Chloroform Fumigation and Extraction (CFE)

When soil pH reaches levels below 5.0 CFI, is not well suited to measure microbial biomass [1]. Vance et al (1987) modified the original CFI technique to create the Chloroform fumigation and extraction technique. Similar to CFI in CFE, soil samples are fumigated using CHCl₃; though, instead of incubation, samples are extracted using .5M potassium sulfate (K₂SO₄ [1,2,8]. The filtrate of both fumigated and nonfumigated samples are analyzed for total organic carbon (TOC) [1,2,8]. Microbial biomass is then calculated by (TOC [fumigated]-TOC [nonfumigated])/k_c [1,2,8].

Substrate-Induced-Respiration (SIR)

The SIR technique involves adding a substrate, such as glucose, to stimulate respiration [1,4]. Glucose is one of the most commonly used substrates due to microorganisms to its ability to be utilized as a carbon source [4]. Depending on the physical and chemical properties of a soil sample, the amount of substrate needed varies [4] Once the substrate is added, respiration rapidly increases and remains constant for several hours [4]. The initial maximum respiration is proportional to the size of the original soil microbial biomass [4]. SIR technique is useful for the "measurement of the contribution of bacterial and fungal biomass to substrate-induced CO₂ respiration through coupling with antibiotics" [4]. This has been deemed successful is arable grasslands, rhizosphere-rhizoplane soils, and in plant residue [4].

References

[1] Coleman, D.C., Crossley, D.A., Hendrix, P.F., 2007. Fundamentals of soil ecology, 2. ed., [Nachdr.]. ed. Elsevier/Academic Press, Amsterdam.

[2]Jenkinson, D.S., Powlson, D.S., 1976. The effects of biocidal treatments on metabolism in soil—V. Soil Biology and Biochemistry 8, 209–213. https://doi.org/10.1016/0038-0717(76)90005-5

[3] Jones, P.C.T., Mollison, J.E., Quenouille, m. H., 1948. A Technique for the Quantitative Estimation of Soil Micro-organisms 54–69. https://doi.org/10.1099/00221287-2-1-54

[4] Lin, Q., Brookes, P.C., 1999. An evaluation of the substrate-induced respiration method. Soil Biology and Biochemistry 31, 1969–1983. https://doi.org/10.1016/S0038-0717(99)00120-0

[5]Olsen, R.A., Bakken, L.R., 1987. Viability of soil bacteria: Optimization of plate-counting technique and comparison between total counts and plate counts within different size groups. Microb Ecol 13, 59–74. https://doi.org/10.1007/BF02014963

[6] Pold, G., Domeignoz-Horta, L.A., DeAngelis, K.M., 2019. Heavy and wet: evaluating the validity and implications of assumptions made when measuring growth efficiency using 18 O water (preprint). Microbiology. https://doi.org/10.1101/601138

[7] Torsvik, V., Goksøyr, J., Daae, F.L., 1990. High diversity in DNA of soil bacteria. Applied and Environmental Microbiology 56, 782–787. https://doi.org/10.1128/AEM.56.3.782-787.1990

[8] Vance, E.D., Brookes, P.C., Jenkinson, D.S., 1987. An extraction method for measuring soil microbial biomass C. Soil Biology and Biochemistry 19, 703–707. https://doi.org/10.1016/0038-0717(87)90052-6

[9] Voroney, R.P., Paul, E.A., 1984. Determination of kC and kNin situ for calibration of the chloroform fumigation-incubation method. Soil Biology and Biochemistry 16, 9–14. https://doi.org/10.1016/0038-0717(84)90117-2

[10] White, D., 1993. In situ measurement of microbial biomass, community structure and nutritional status. Phil. Trans. R. Soc. Lond. A 344, 59–67. https://doi.org/10.1098/rsta.1993.0075

[11] Willers, C., Jansen van Rensburg, P.J., Claassens, S., 2015. Microbial signature lipid biomarker analysis - an approach that is still preferred, even amid various method modifications. J Appl Microbiol 118, 1251–1263. https://doi.org/10.1111/jam.12798

[12] 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, 111–129. https://doi.org/10.1007/s003740050533

[13] Zhou, J., Bruns, M.A., Tiedje, J.M., 1996. DNA recovery from soils of diverse composition. Applied and environmental microbiology 62, 316–322. https://doi.org/10.1128/AEM.62.2.316-322.1996

[14] N.d. . Stock Photo- Top view soil microorganisms Nutrient agar in plate on black background. URL https://www.123rf.com/photo_122899146_top-view-soil-microorganisms-nutrient-agar-in-plate-on-black-background-.html