Measurement of chlorophylls a and b and bacteriochlorophyll a in organisms from hypereutrophic auxinic waters

Abstract

Sewage lagoons and wastewater ponds from industrialised swine and poultry farms are typically hypereutrophic, auxinic and dominated by purple non-sulphur bacteria and unicellular green algae both typically growing photoheterotrophically. To manage such ponds, it is essential to know the balance between oxygenic and anoxygenic photosynthesis. Typical spectrophotometric algorithms to estimate chlorophyll use 750 nm as a zero (A750 nm) but a 750 nm zero protocol is unsuitable where substantial amounts of bacteriochlorophylls are present. Algorithms were developed to estimate chlorophylls a and b (Chl a and Chl b) and bacteriochlorophyll a (BChl a) in solvent in ethanol, 7:2 acetone/ethanol and 90% acetone. The algorithms use an 850-nm absorbance zero (A850 nm) well outside the absorbance ranges of both chlorophylls and bacteriochlorophylls in solvent. There are many habitats where the presence of anoxygenic photosynthetic bacteria is unsuspected and so using a routine A750 nm zero effectively masks their presence and leads to underestimations of Chl a and Chl b. The in-solvent red peak of bacteriochlorophyll c is too close to that of Chl a for BChl c and Chl a to be resolved spectroscopically, but its presence can be easily identified from in vivo scans. The spectroscopic advantage of 90% acetone is negated by its poor quantitative extraction of pigments. Acetone/ethanol (7:2) is an excellent solvent spectroscopically and as an extractant.