ena-STUDY-MAX PLANCK INSTITUTE FOR MARINE MICROBIOLOGY-27-09-2016-09:42:53:702-492
Chlorophyll degradation by aerobic marine bacterium
Chlorophylls are the most important pigments for the capture of solar light energy for photosynthesis. In particular, they constitute the basis of oxygenic photosynthesis in green plants and cyanobacteria, i.e. of organic primary production on Earth, and thus belong to the most ubiquitous and abundant natural organic compounds. Their annual global production amounts to approx. 1012 kg. Chlorophylls are tetrapyrrols, the biosynthesis of which from simple building blocks is understood in much detail. Biodegradation of chlorophylls has been documented so far only to the level of linear tetrapyrrols, a well-studied reaction sequence in living plants. There is little knowledge about degradation of free chlorophylls from dead photosynthetic organisms in natural habitats. In marine environments, the fate of chlorophylls from phytodetritus is essentially unknown.The main aim of the present study was (a) to investigate whether chemotrophic marine bacteria can degrade free chlorophyll by its utilization as growth substrate, (b) to attempt the isolation of such bacteria, with subsequent characterization, (c) to document chlorophyll utilization in quantitative degradation experiments, and (d) to gain first insights into the degradation pathway on the basis of genomic and chemical analyses.Chlorophyll a, the most abundant chlorophyll, was chosen for all experiments. It was prepared from the cyanobacterium Arthrospira sp. (Spirulina) by acetone extraction, dioxane precipitation and subsequent purification via a CaCO3 column, as described by Mikhail Tswett more than 100 years ago. Purity and identity of obtained chlorophyll a was verified by chemical and spectrophotometric analysis.Chlorophyll a was supplied as the sole potential growth substrate for the enrichment of aerobic marine bacteria. Incubation indeed led to growth of marine bacteria growing as a biofilm on the insoluble substrate which was gradually consumed. Six chlorophyll a-degrading strains were isolated. Analysis of the 16S rRNA gene indicated that the strains may represent a new genus affiliating with the BD1-7 clade of Gammaproteobacteria. One strain, KOV_DT_Chl was selected for more detailed studies. The estimated doubling time on chlorophyll was 2 days. Apart from chlorophyll a, the strain could use acetate, propionate, fumarate, succinate, alanine, aspartate, glutamate, glutamine, and glucose. No growth was observed with chlorophyll a-related compounds including Mg-chlorophyllin, methyl-pheophorbide a, protoheme IX, pyrrol-2-carboxylate, and phytol. Growth was observed between 8 and 31 °C and within an NaCl concentration range of 10 to 50 g l-1.During chlorophyll a utilization, the ratio between formed CO2 and consumed O2 was in accordance with the stoichiometry expected for complete oxidation. Furthermore, there was no need for ammonium as a nitrogen source, indicating that nitrogen from pyrrols is assimilated. These observations suggest that strain KOV_DT_Chl is able to mineralize the tetrapyrrol macrocycle. The metabolic fate of the bound phytol chain could not be clarified; even though free phytol was not used as a growth substrate, it was not detected as a product in chlorophyll a incubations.An analysis of the genome revealed genes for structural homologs of pheophytinase, pheophorbide a oxygenase, and red chlorophyll catabolite reductase. These enzymes are responsible for chlorophyll a breakdown to linear tetrapyrrols in higher plants; hence the initial steps in chlorophyll a degradation in the presently isolated bacteria may be as in green plants.
Chlorophylls are the most important pigments for the capture of solar light energy for photosynthesis. In particular, they constitute the basis of oxygenic photosynthesis in green plants and cyanobacteria, i.e. of organic primary production on Earth, and thus belong to the most ubiquitous and abundant natural organic compounds. Their annual global production amounts to approx. 1012 kg. Chlorophylls are tetrapyrrols, the biosynthesis of which from simple building blocks is understood in much detail. Biodegradation of chlorophylls has been documented so far only to the level of linear tetrapyrrols, a well-studied reaction sequence in living plants. There is little knowledge about degradation of free chlorophylls from dead photosynthetic organisms in natural habitats. In marine environments, the fate of chlorophylls from phytodetritus is essentially unknown.The main aim of the present study was (a) to investigate whether chemotrophic marine bacteria can degrade free chlorophyll by its utilization as growth substrate, (b) to attempt the isolation of such bacteria, with subsequent characterization, (c) to document chlorophyll utilization in quantitative degradation experiments, and (d) to gain first insights into the degradation pathway on the basis of genomic and chemical analyses.Chlorophyll a, the most abundant chlorophyll, was chosen for all experiments. It was prepared from the cyanobacterium Arthrospira sp. (Spirulina) by acetone extraction, dioxane precipitation and subsequent purification via a CaCO3 column, as described by Mikhail Tswett more than 100 years ago. Purity and identity of obtained chlorophyll a was verified by chemical and spectrophotometric analysis.Chlorophyll a was supplied as the sole potential growth substrate for the enrichment of aerobic marine bacteria. Incubation indeed led to growth of marine bacteria growing as a biofilm on the insoluble substrate which was gradually consumed. Six chlorophyll a-degrading strains were isolated. Analysis of the 16S rRNA gene indicated that the strains may represent a new genus affiliating with the BD1-7 clade of Gammaproteobacteria. One strain, KOV_DT_Chl was selected for more detailed studies. The estimated doubling time on chlorophyll was 2 days. Apart from chlorophyll a, the strain could use acetate, propionate, fumarate, succinate, alanine, aspartate, glutamate, glutamine, and glucose. No growth was observed with chlorophyll a-related compounds including Mg-chlorophyllin, methyl-pheophorbide a, protoheme IX, pyrrol-2-carboxylate, and phytol. Growth was observed between 8 and 31 °C and within an NaCl concentration range of 10 to 50 g l-1.During chlorophyll a utilization, the ratio between formed CO2 and consumed O2 was in accordance with the stoichiometry expected for complete oxidation. Furthermore, there was no need for ammonium as a nitrogen source, indicating that nitrogen from pyrrols is assimilated. These observations suggest that strain KOV_DT_Chl is able to mineralize the tetrapyrrol macrocycle. The metabolic fate of the bound phytol chain could not be clarified; even though free phytol was not used as a growth substrate, it was not detected as a product in chlorophyll a incubations.An analysis of the genome revealed genes for structural homologs of pheophytinase, pheophorbide a oxygenase, and red chlorophyll catabolite reductase. These enzymes are responsible for chlorophyll a breakdown to linear tetrapyrrols in higher plants; hence the initial steps in chlorophyll a degradation in the presently isolated bacteria may be as in green plants.