Determination of Cell Biochemicals

Microbial biomass can also be measured by determination of specific cell biochemical constituents such as ATP, DNA, RNA, proteins, phospholipids, bacterial cell wall components, or photosynthetic pigments (Sutton, 2002).

a.        ATP

Adenosine triphosphate has often been used to determine live microbial biomass in environmental samples, using a ratio of C/ATP = 250 for aquatic samples. However, the ATP content of cells varies with the growth rate and metabolic state of microorganisms and nutrient limitation. A better measure is the total adenylate pool AT (AT = ATP + ADP + AMP) because it does not change greatly with changes in metabolic activities of the microorganisms. The adenylate energy charge (EC) ratio provides information on growth potential of naturally occurring microbial populations.

An EC of 0.5–0.6 indicates senescence of the microbial population, whereas an EC of 0.8–0.9 indicates active microbial growth. Adenosine triphosphate determination has been applied to wastewater treatment, disinfection control, and pollution assessment.

b. Thymidine and Leucine Incorporation into Cells

Bacterial biomass and production can also be estimated by measuring the incorporation of tritiated thymidine into DNA or radioactive leucine into bacterial proteins (bacterial biomass comprises 60 percent proteins) (Kirchman and Ducklow, 1993).

c. Lipid Biomarkers

Cell lipids can be classified into neutral lipids (NL), glycolipids (GL), and polar lipids (PL). They serve as storage material (e.g., poly-b-hydroxybutyrate), electron acceptors in the electron transport chain in respiration (e.g., quinones), and components of membranes (e.g., phospholipids) or outer membranes of gram-negative bacteria (e.g., lipopolysaccharides) (Tunlid, 2002).

Lipid biomarkers give the following information:

Microbial biomass. Lipid biomarkers can help in the determination of microbial biomass. For example, phospholipid analysis can determine either the phospholipidbound phosphate (PLP) or the ester-linked phospholipid fatty acids (PLFA), which serve as biomarkers for estimating microbial biomass and community composition.

Suggested conversion factors are 190 mmol P/g C, 100mmol P/g C, and 50 mmol P/g C for aerobic bacteria, anaerobic bacteria, and eukaryotes, respectively. Phospholipid analysis is unfortunately complex and requires sophisticated equipment. Ergosterol can serve as a biomarker for living fungal biomass.

Community composition. For example, detection of signature PLFAs indicates the presence of specific groups of microorganisms in an environmental sample. Phytanylether lipids indicate the presence of archaea.

Metabolic activity of the microbial community. This is accomplished by measuring the uptake of 14C-labeled substrates into lipid biomarkers such as PLFAs or poly-b-hydroxybutyrate.

d. Bacteria Cell Wall Components

Cell wall components such as muramic acid and lipopolysaccharide can serve as biomarkers for estimating bacterial biomass.

e. Molecular Techniques for the Determination of Cell Viability/Activity

Both mRNA and rRNA are well correlated with cell viability. However, due to its shorter half-life in the environment, mRNA is generally preferred over rRNA for indicating cell viability. Reverse transcriptase-PCR (RT-PCR) can also give an indication of cell viability. It consists of transcribing a target RNA sequence into a complimentary DNA (cDNA) sequence, which is then amplified using PCR. Some of these methods have been used to monitor the viability of bacterial pathogens and protozoan parasites (Keer and Birch, 2003).