Processes 2018, 6,38
Impacts of sample treatment were also investigated with E. coli, including freezing of the sample
and washing of the cell pellet prior to treatment. Fresh and frozen samples from the same culture were
assayed and not found to be significantly different (Figure A2b). Downs and Wilfinger [53] reported
washing samples with a cell wash solution (150 mM NaCl, 15 mM citrate, 3 mM EDTA, pH 7.0 with
HCl) before performing the lysis step. In the current work, washing the sample with cell wash solution
resulted in lower DNA recovery as compared to not washing (Figure A2b) and may indicate cell loss
or lysis during the washing process. Extracellular DNA was not expected to comprise a significant
proportion of total DNA in the planktonic, exponentially growing samples; however, this may not be
the case for all samples, such as biofilm or natural environmental samples.
The DNA percentage of dry biomass obtained for E. coli is lower than the value of 3.1% reported in
Neidhardt et al. [32], which could be due to differences in methods used or E. coli strains (K-12 vs. B/r),
while the percentage obtained for Synechococcus 7002 is similar to the results reported in Vu et al. [46]
measured with the diphenylamine method. Differences in growth rate or growth phase may contribute
to differences in measured percentages. No literature comparison was available for A. acidocaldarius.
6. Lipid
6.1. Literature Review
Lipids are essential to cellular membranes and are carbon and energy storage molecules.
Measurement of total lipid is commonly performed gravimetrically, with an absorbance-based
sulfo-phospho-vanillin assay [57], or using gas chromatography. A common gravimetric method is
the Bligh and Dyer chloroform–methanol extraction [58]. Other solvents have been used to mitigate
the hazards of chloroform, and a variety of modifications to the original Bligh and Dyer procedure
exist [16]. There is debate regarding the performance of these different methods, and methods may
vary depending on downstream applications. Much research has been done on lipid extraction from
biofuel-producing organisms such as algae, and recent testing and comparison of methods have shown
microwave extraction with GC analysis to provide optimal results [16]. However, cyanobacteria
synthesize predominantly diacylglycerol lipids as opposed to the triacylglycerol lipids of algae [59].
Cyanobacterial lipids are also located throughout the cytoplasm in the thylakoid membranes rather
than in granular pockets as in algae. Many lipids are also associated with protein and photosynthetic
components through hydrogen bonding. Different methods have been tested for the cyanobacterium
Synechocystis sp. PCC 6803, and the traditional Bligh and Dyer and Folch methods were found to
produce optimal results [59]. Different cell disruption methods have also been tested for Synechocystis sp.
PCC 6803, and microwave extraction and autoclaving were found to be the most efficient disruption
methods [60]. The traditional Bligh and Dyer method was selected for the current study for analysis of
total cell lipid [58].
6.2. Procedure (After Bligh and Dyer, 1959)
6.2.1. Reagents
•
Cell pellet (10 mg dry biomass, fresh or frozen, washed with carbon-free media).
•
Chloroform.
•
Methanol.
•
Water.
6.2.2. Assay
(1) Re-suspend cell pellet to 0.6 mL total volume with water in a 15-mL polypropylene centrifuge tube.
(2) Sequentially add chloroform (0.75 mL) and methanol (1.5 mL) (vortexing between additions is
not necessary).
(3) Vortex 15 min.
163
Impacts of sample treatment were also investigated with E. coli, including freezing of the sample
and washing of the cell pellet prior to treatment. Fresh and frozen samples from the same culture were
assayed and not found to be significantly different (Figure A2b). Downs and Wilfinger [53] reported
washing samples with a cell wash solution (150 mM NaCl, 15 mM citrate, 3 mM EDTA, pH 7.0 with
HCl) before performing the lysis step. In the current work, washing the sample with cell wash solution
resulted in lower DNA recovery as compared to not washing (Figure A2b) and may indicate cell loss
or lysis during the washing process. Extracellular DNA was not expected to comprise a significant
proportion of total DNA in the planktonic, exponentially growing samples; however, this may not be
the case for all samples, such as biofilm or natural environmental samples.
The DNA percentage of dry biomass obtained for E. coli is lower than the value of 3.1% reported in
Neidhardt et al. [32], which could be due to differences in methods used or E. coli strains (K-12 vs. B/r),
while the percentage obtained for Synechococcus 7002 is similar to the results reported in Vu et al. [46]
measured with the diphenylamine method. Differences in growth rate or growth phase may contribute
to differences in measured percentages. No literature comparison was available for A. acidocaldarius.
6. Lipid
6.1. Literature Review
Lipids are essential to cellular membranes and are carbon and energy storage molecules.
Measurement of total lipid is commonly performed gravimetrically, with an absorbance-based
sulfo-phospho-vanillin assay [57], or using gas chromatography. A common gravimetric method is
the Bligh and Dyer chloroform–methanol extraction [58]. Other solvents have been used to mitigate
the hazards of chloroform, and a variety of modifications to the original Bligh and Dyer procedure
exist [16]. There is debate regarding the performance of these different methods, and methods may
vary depending on downstream applications. Much research has been done on lipid extraction from
biofuel-producing organisms such as algae, and recent testing and comparison of methods have shown
microwave extraction with GC analysis to provide optimal results [16]. However, cyanobacteria
synthesize predominantly diacylglycerol lipids as opposed to the triacylglycerol lipids of algae [59].
Cyanobacterial lipids are also located throughout the cytoplasm in the thylakoid membranes rather
than in granular pockets as in algae. Many lipids are also associated with protein and photosynthetic
components through hydrogen bonding. Different methods have been tested for the cyanobacterium
Synechocystis sp. PCC 6803, and the traditional Bligh and Dyer and Folch methods were found to
produce optimal results [59]. Different cell disruption methods have also been tested for Synechocystis sp.
PCC 6803, and microwave extraction and autoclaving were found to be the most efficient disruption
methods [60]. The traditional Bligh and Dyer method was selected for the current study for analysis of
total cell lipid [58].
6.2. Procedure (After Bligh and Dyer, 1959)
6.2.1. Reagents
•
Cell pellet (10 mg dry biomass, fresh or frozen, washed with carbon-free media).
•
Chloroform.
•
Methanol.
•
Water.
6.2.2. Assay
(1) Re-suspend cell pellet to 0.6 mL total volume with water in a 15-mL polypropylene centrifuge tube.
(2) Sequentially add chloroform (0.75 mL) and methanol (1.5 mL) (vortexing between additions is
not necessary).
(3) Vortex 15 min.
163