Measuring Microbial Communities' Biomass
To sample microbial communities’ biomass there are two approaches. The two approaches include several different methods. These approaches are either direct or indirect sampling techniques [1]. Directing sampling involved counting while indirect sampling involves the use of chemicals [1].
Direct
Agar Plates
Outlined by Jones et al (1948) the use of agar plates was used to count organisms in microscopic fields [3]. Soil samples are first taken at random and sifted through a sieve and then weighed out [3,5]. Following this the sample is then put into a crucible with sterile distilled water and ground up with a glass rod [3,5]. The sample is then washed with sterile distilled water with the suspended matter poured into a flask [3,5]. The soil suspension is then made up to 50ml with 1.5% being agar [3,5]. Once this is done the flask is shaken and left to rest for a short period. Once rested using a pipette the samples are taken and put on a slide [3]. Once the slide is prepared it is then put into sterile distilled water [3,5]. Once dried the sample can then be put under a microscope to count organisms [3,5].
Extractable DNA
Torsvik et al (1990) used the extractable DNA to determine the identities of organisms in soil samples [1,13]. Six 30g soil samples were first prepared. Following this samples were washed with 2% sodium hexametaphosphate to increase the yield of DNA [7]. This 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
Chloroform Fumigation and Incubation (CFI)
The Chloroform fumigation and incubation method (CFI) is used to determine organic C biomass in soil microbials. Chloroform (CHCl2) vapor is used to fumigate soil [1,9]. Once the soil is fumigated the CHCl3 vapor is removed then the soil is incubated [9]. Evolved CO2 levels are then measured for both fumigated and then unfumigated soil to calculate biomass [1,9]. Using the expression B=F/kc (B=soil biomass C, F= carbon dioxide carbon evolved by fumigated soil minus CO2 evolved by nonfumigated soil, and kc= fraction of biomass mineralized to CO2 during the incubation) biomass can be calculated where kc is a constant [1]. Voroney and Paul (1984) then furthered this technique to include labile nitrogen and measured the fraction of biomass nitrogen (kn) 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. Like CFI in CFE soil samples are fumigated using CHCl3, but instead of being incubated 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])/kc [1,2,8].
Substrate-Induced-Respiration (SIR)
The SIR technique involves adding a substrate such as glucose to stimulate respiration [1,4]. Glucose os often the most used substrate due to microorganisms being able to readily utilize it as a carbon source [4]. Depending on the soils physical and chemical properties the amount of glucose 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