et al., 1982). This assay has been utilized to show that poor growth conditions stimulate
the formation of uronic acid containing exopolymers (Uhlinger and White, 1983) and
their role in sediment stability (Nowell et al., 1985).
The nutritional status of biofilms can be estimated by monitoring the proportions of
specific endogenous storage compounds relative to the cellular biomass. The nutritional
status of microeukaryotes (algae, fungi, or protozoa) in biofilms can be monitored by
measuring the ratio of triglyceride glycerol to the cellular biomass (Gehron and White,
1982). Certain bacteria form the endogenous lipid poly-beta-hydroxy alkanoate (PHA)
under conditions of nutritional stress (Nickels et al., 1979). A more sensitive assay based
on GLC of the components of the polymer showed the presence of 3-OH acids longer than
4-carbons in these polymers (Findlay and White, 1983b). The ratio of the rate of
formation of phospholipid fatty acids to PHA has been shown to be an extraordinarily
sensitive measure of the nutrient environment in the bacterial niche (Findlay and White,
1984 ; Findlay et ai, 1985). This ratio of incorporation can be utilized to measure the
“disturbance artifact” involved in the application of labeled precursors to highly stratified
environments such as sediments (Findlay et al., 1985).
The analyses described above all involve the isolation of signature components of
microbial biofilms. Since each of the components are isolated, the incorporation of
labeled isotopes from precursors can be utilized to provide rates of synthesis or turnover.
This has proved useful in the quantitative description of the effects of predation on
detrital microbial biofilms (King et ai, 1977 ; Morrison and White, 1980). Analysis of
signatures by GC/ MS makes possible the utilization of mass labeled precursors that are
non-radioactive, have specific activities approaching 100 %, include isotopic marker for
nitrogen, and can be efficiently detected using the selective ion mode. The high specific
activity makes possible the assay of critical reactions using substrate concentrations in the
biofilms that are just above the natural levels. This is not possible with radioactive
precursors. Improvements in analytical techniques have increased the sensitivity of this
analysis. Utilizing a chiral derivative and fused silica capillary GLC with chemical
ionization and negative ion detection of selected ions, it proved possible to detect 8pg
(90 femtomoles) of D-alanine from the bacterial cell wall (the equivalent of 103 bacteria)
the size of E. coli (Tunlid et al., 1985). In this analysis it proved possible to reproducibly
detect a 1 % enrichment of 15 N-D-alanine in the 14 N-D-alanine.
Non-destructive infrared analysis
The analysis of biofilms based on the isolation of chemical signatures is a destructive
analysis and cannot be readily automated or utilized to give real time monitoring of
biofilms. The possibility of utilizing a non-destructive technique to monitor the chemistry
of living biofilms is now available in the Fourier transforming infrared spectrometers
(FT/IR).
The infrared portion of the spectrum is extraordinarily rich in information regarding the
vibrational and rotational motions of atoms in molecules. Not only specific infrared
absorption be assigned to particular types of covalent bonds but the modifications of
these bonds by the local electronic environment can be detected in the details of the
spectra (Bellamy, 1958 ; Parker, 1971). The infrared spectrum of a compound has long
been accepted as one of the best mondestructive absolute proofs of identity in organic
chemistry.
One of the problems restricting the application of infrared spectroscopy has been that the
atomic interactions sensed in the infrared portion of the spectrum are at relatively low
212
the formation of uronic acid containing exopolymers (Uhlinger and White, 1983) and
their role in sediment stability (Nowell et al., 1985).
The nutritional status of biofilms can be estimated by monitoring the proportions of
specific endogenous storage compounds relative to the cellular biomass. The nutritional
status of microeukaryotes (algae, fungi, or protozoa) in biofilms can be monitored by
measuring the ratio of triglyceride glycerol to the cellular biomass (Gehron and White,
1982). Certain bacteria form the endogenous lipid poly-beta-hydroxy alkanoate (PHA)
under conditions of nutritional stress (Nickels et al., 1979). A more sensitive assay based
on GLC of the components of the polymer showed the presence of 3-OH acids longer than
4-carbons in these polymers (Findlay and White, 1983b). The ratio of the rate of
formation of phospholipid fatty acids to PHA has been shown to be an extraordinarily
sensitive measure of the nutrient environment in the bacterial niche (Findlay and White,
1984 ; Findlay et ai, 1985). This ratio of incorporation can be utilized to measure the
“disturbance artifact” involved in the application of labeled precursors to highly stratified
environments such as sediments (Findlay et al., 1985).
The analyses described above all involve the isolation of signature components of
microbial biofilms. Since each of the components are isolated, the incorporation of
labeled isotopes from precursors can be utilized to provide rates of synthesis or turnover.
This has proved useful in the quantitative description of the effects of predation on
detrital microbial biofilms (King et ai, 1977 ; Morrison and White, 1980). Analysis of
signatures by GC/ MS makes possible the utilization of mass labeled precursors that are
non-radioactive, have specific activities approaching 100 %, include isotopic marker for
nitrogen, and can be efficiently detected using the selective ion mode. The high specific
activity makes possible the assay of critical reactions using substrate concentrations in the
biofilms that are just above the natural levels. This is not possible with radioactive
precursors. Improvements in analytical techniques have increased the sensitivity of this
analysis. Utilizing a chiral derivative and fused silica capillary GLC with chemical
ionization and negative ion detection of selected ions, it proved possible to detect 8pg
(90 femtomoles) of D-alanine from the bacterial cell wall (the equivalent of 103 bacteria)
the size of E. coli (Tunlid et al., 1985). In this analysis it proved possible to reproducibly
detect a 1 % enrichment of 15 N-D-alanine in the 14 N-D-alanine.
Non-destructive infrared analysis
The analysis of biofilms based on the isolation of chemical signatures is a destructive
analysis and cannot be readily automated or utilized to give real time monitoring of
biofilms. The possibility of utilizing a non-destructive technique to monitor the chemistry
of living biofilms is now available in the Fourier transforming infrared spectrometers
(FT/IR).
The infrared portion of the spectrum is extraordinarily rich in information regarding the
vibrational and rotational motions of atoms in molecules. Not only specific infrared
absorption be assigned to particular types of covalent bonds but the modifications of
these bonds by the local electronic environment can be detected in the details of the
spectra (Bellamy, 1958 ; Parker, 1971). The infrared spectrum of a compound has long
been accepted as one of the best mondestructive absolute proofs of identity in organic
chemistry.
One of the problems restricting the application of infrared spectroscopy has been that the
atomic interactions sensed in the infrared portion of the spectrum are at relatively low
212