Powerful Pigments: Carotenoids and PNS Bacteria

Updated: Oct 6, 2021

Pigments are organic molecules of unbelievable power. Bacteria, archaea, algae, plants and other photoautotrophs create pigments to capture photons, thereby deriving metabolic energy from light. Without these miraculous compounds, light would remain forever elusive to biology. There would be no photosynthesis and very little life as we recognize it.

Different pigments absorb different spectra of light. Some pigments are common, such as the near-universal chlorophyll a which makes most land plants green. Other pigments are more rare and are of profound physiological value to a wide variety of organisms. Carotenoids are a set of pigments that not only enable photosynthesis, but also (1) protect cells from UV radiation, (2) reduce cell damage by free radicals, (3) assist in cell-to-cell signaling and (4) manifest coloration. They are crucial to the long-term health and coloration of nearly all aquatic life. However, fish and invertebrates cannot synthesize carotenoids from base molecules and thus must assimilate them through their diet. Reef aquaria in particular are ravenous consumers of carotenoids, which is why a constant and robust supply is necessary to realize their greatest potential impact on animal health and coloration.

Widely used in aquaculture, Rhodopseudomonas palustris is a novel biofiltration agent. In order to collect light, it synthesizes the carotenoid pigments lycopene, canthaxanthin, beta-carotene and astaxanthin. R. palustris systematically eliminates excess deposits of nitrogenous wastes, phosphates and waste carbons, converting them to less and less toxic forms through the processes of nitrification, denitrification, anammox, photosynthesis and heterotrophy. Robust colonies of R. palustris continuously release daughter cells into the reef display and refugium. These cells, along with their carotenoid pigments, can and will be consumed by any of the reef’s filter-feeders, micrograzers and/or substrate sifters. Thus, reef aquarists have the opportunity to craft the ecology of their biofilter to improve their aquarium’s water quality while boosting animal nutrition and coloration.

How exactly do carotenoids capture light energy? And why are carotenoids so valuable in the nutritional health of fish, humans and invertebrates alike? The answers to these mysteries are hidden within the unique chemical structure of carotenoids.

The bonds that tie

Carotenoids are composed of polyene hydrocarbon chains which are sometimes terminated by functional rings. Carotenoids are chained together by double bonds. These double bonds can energetically interact with each other through atomic conjugation—basically, allowing them to bounce electrons back and forth at a highly efficient rate. The more conjugated double bonds associated with a carotenoid, the greater its potential to exchange and disperse electrons. Carotenoids conduct two forms of energy transfer. The first is singlet-singlet where the polyene hydrocarbon tail enables lower-state light absorption for direct transport from the carotenoid molecules into chlorophyll a for photosynthesis.

Carotenoids also conduct higher state triplet-triplet energy transfer allowing them to act as a sort of electric ‘buffer.’ This higher state energy transfer is why carotenoids are such efficacious photoprotectants and antioxidizing agents. Complex metabolic functions, such as respiration and photosynthesis, can produce ‘waste’ electrons known as free radicals. Free radicals can wreak metabolic havoc throughout an organism. Carotenoids are deployed by cells to gobble up the stray electrons. The antioxidizing capacity of carotenoids is perhaps their most important contribution to the systemic health of fish, humans and invertebrates alike.

Carotenoids are valuable in most higher heterotrophs due to their amazing ability to regulate oxygen metabolism. Because carotenoids have such a high degree of double bonding, they easily interact with lipid molecules; far more oxygen can be transported through lipids than in water. Thus, many organisms embed carotenoids within the lipid bilayer of their cells to act as oxygen superhighways. This lipid architecture vastly improves cellular oxygenation, stimulates mitochondrial activity and is protected from free radicals by the presence of the carotenoids themselves. This creates a positive-feedback loop of stability for both autotrophs which need to discharge waste oxygen as well as heterotrophs that require it.

Carotenoids can also stimulate the formation of intracellular lipid droplets, which become incredibly dense energy and oxygen reserves. The presence of such Golden Fat reserves assist in stress resilience during unfavorable environmental conditions or disease. Flores-Ramirez et al 2007 demonstrated a marketed increase in carotenoid synthesis and tissue accumulation in colonies of Pocillopora capitata exposed to temperature stress. The carotenoids canthaxanthin and beta-carotene increase UV-stress tolerance in Corallium rubrum. The bacteria and algae typically associated with Sarcophyton corals are noted beta-carotene producers.

Carotenoids are capable of synergistic modality, meaning they can form complexes with other molecules. These carotenoid complexes can be organism-specific and exhibit a wide array of physiological uses. Most recognized are the role carotenoid complexes play in vitamin A synthesis, boosting tissue oxygenation and activating prohormones, proenzymes and vitamin D3. They can also act as signaling agents, triggering the production of plant hormones such as abscisic acid.

Some carotenoids contain oxygen molecules. These are known as xanthophylls and are yellow in appearance because they absorb blue light within (400-470nm). Carotenoids without oxygen are carotenes and range from orange to red absorbing UV-A and UV-B light (290-400nm). However, once carotenoids become part of a lipid-complex, their color manifests in unique ways. Direct examples of this include the bold colors of flamingos, tangs and cichlids alike. Carotenoid complexes also help support delicate tissue structures in livers/intestines, dye the flesh of krill deep red, etc. all while suppressing mutagenesis and enhancing mechanisms of immunity.

Growing colors

Carotenoids are essential for realizing the natural coloration of reef fish, coral and invertebrates. Since the early days of the freshwater aquarium trade, it has been recognized that fish fed minimal levels of carotenoids will become pale and unattractive over time. This is because carotenoids are essential in both the formation and metabolism of chromatophores or ‘color cells.’ Chromatophores are extremely diverse in structure and functionality, and the coloration of beautiful fish can be attributed to six general cell types: Melanophores, xanthophores, erythrophores, iridophores and cyanophores.

These cell types are mosaically interlayered throughout the skin, muscle, integument and organs of the fish, collectively reflecting back the unique coloration of that particular individual. Chromatophores stockpile carotenoids and other pigments which allow them to manipulate light. Carotenes are most commonly found in erythrophores, whereas xanthopyhlls are most commonly found in xanthophores. Carotenoids consumed by fish also contribute to the animals' coloration: Tunaxanthin (gold), lutein (greenish-yellow), beta-carotene (orange), doradexanthins (yellow), zeaxanthin (yellow-orange) and astaxanthin (red). It is the collage of these species-specific complexes which are responsible for the jaw-dropping colors of the reef. Without a nutritional surplus of carotenoids, marine organisms are physically incapable of achieving the splendorous coloration of the wild...and of our dreams.

Fish and other marine organisms cannot synthesize carotenoids themselves and must consume them through their diets. This is difficult as carotenoids cannot be transported through plasma and are not absorbed nearly as much as other nutritious elements. Carotenoids must be absorbed when associated with lipid plasma proteins, or in other words, assimilated into the fat of the consumed organism. Because of this, many synthetic carotenoids are expensive, wasteful and incredibly impractical for feed formulation. Therefore, the most practical route for achieving maximum levels of carotenoids in an aquarium is by having robust colonies of autotrophs, such as Rhodopseudomonas or Rhodospirillum, to actively produce them.

The carotenoids produced by a PNS bacteria-rich biofilter are either directly consumed/assimilated by corals and other filter-feeders (clams, sponges, scallops, sabellids etc.) or by micrograzers such as copepods, rotifers and polychaete worms. Many of these clean-up crew organisms can metabolize unique carotenoid complexes which benefit the fish and inverts which feast upon them. A shining example of this is the popular harpacticoid copepod Tigriopus californicus. Weaver et al 2018 demonstrated the ability of T. californicus to convert the yellow carotenoids zeaxanthin and lutein to the more valuable red pigment astaxanthin. Robust PNS biofilters can act as localized superproducers of carotenoids—flushing their reef aquarium ecosystem with dietary pigmentation.

What makes PNS bacteria so practical and so extraordinary is that they are able to convert toxic nitrogenous wastes, phosphates and excess organic carbons into precious carotenoid pigments. It has long been recognized that Rhodopseudomonas palustris is capable of synthesizing valuable carotenoids such as canaxantin, beta-carotene and astaxanthin. The coming years will see more and more PNS bacterial strains and culture techniques selected for carotenoid production. For example, Kuo et al 2012 reported 348% increase in total carotenoid production in Rhodopseudomonas palustris cultures illuminated with blue LED’s versus white incandescent bulbs. Giraud et al 2018 demonstrated how PNS bacteria cultures exposed to anaerobic conditions exhibited increased conversion of lycopene to beta-carotene and canthaxanthin. Lopez-Romero 2020 achieved carotenoid concentrations of 8.8mg/g cell when PNS cultures were exposed to high levels of nitrates and low levels of light.

Low levels of blue LED light in an anaerobic zone with excess nitrate? Sounds a bit like the shallow subsurface of the sand bed in a reef tank or refugium, doesn’t it? These are promising findings for those aiming to successfully utilize PNS bacteria as both an aquarium biofiltration agent as well as a Pigment Production Powerhouse!

Literature Cited

Ako, H., Tamaru, C. S., Asano, L., Yuen, B., & Yamamoto, M. (2000). Achieving natural coloration in fish under culture. Spawning and maturation of aquatic species. United States-Japan cooperative program in natural resources technical report, (28).

Azira, Z., Hafizah, N., Rahman, M. M., Kamaruzzaman, B. Y., & Faizul, N. (2014). Carotenoid Contents in Anoxygenic Phototrophic Purple Bacteria, Marichromatium Sp. and Rhodopseudomonas Sp. of Tropical Environment, Malaysia. Oriental Journal of Chemistry, 30(2).

Caramujo, M. J., De Carvalho, C. C., Silva, S. J., & Carman, K. R. (2012). Dietary carotenoids regulate astaxanthin content of copepods and modulate their susceptibility to UV light and copper toxicity. Marine drugs, 10(5), 998-1018.

Chatzifotis, S., Pavlidis, M., Jimeno, C. D., Vardanis, G., Sterioti, A., & Divanach, P. (2005). The effect of different carotenoid sources on skin coloration of cultured red porgy (Pagrus pagrus). Aquaculture Research, 36(15), 1517-1525.

Corol, D. I., Dorobantu, I. I., Toma, N., & Nitu, R. (2002). Diversity of biological functions of carotenoids. Romanian Biotechnological Letters, 8, 1067-1074.

Cvejic, J., Tambutté, S., Lotto, S., Mikov, M., Slacanin, I., & Allemand, D. (2007). Determination of canthaxanthin in the red coral (Corallium rubrum) from Marseille by HPLC combined with UV and MS detection. Marine Biology, 152(4), 855-862.

Das, A. P., & Biswas, S. P. (2016). Carotenoids and pigmentation in ornamental fish. Journal of Aquaculture and Marine Biology, 4(4), 00093.

Flores-Ramírez, L. A., & Liñán-Cabello, M. A. (2007). Relationships among thermal stress, bleaching and oxidative damage in the hermatypic coral, Pocillopora capitata. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, 146(1-2), 194-202.

Fujii, R. (1993). Cytophysiology of fish chromatophores. International review of cytology, 143, 191-255.

Galasso, C., Corinaldesi, C., & Sansone, C. (2017). Carotenoids from marine organisms: Biological functions and industrial applications. Antioxidants, 6(4), 96.

García-Chavarría, M., & Lara-Flores, M. (2013). The use of carotenoid in aquaculture. Research Journal of Fisheries and Hydrobiology, 8(2), 38-49.

Giraud, E., Hannibal, L., Chaintreuil, C., Fardoux, J., & Verméglio, A. (2018). Synthesis of carotenoids of industrial interest in the photosynthetic bacterium Rhodopseudomonas palustris: bioengineering and growth conditions. In Microbial Carotenoids (pp. 211-220). Humana Press, New York, NY.

Goda, M., Ohata, M., Ikoma, H., Fujiyoshi, Y., Sugimoto, M., & Fujii, R. (2011). Integumental reddish‐violet coloration owing to novel dichromatic chromatophores in the teleost fish, Pseudochromis diadema. Pigment cell & melanoma research, 24(4), 614-617.

Goda, M., Fujiyoshi, Y., Sugimoto, M., & Fujii, R. (2013). Novel dichromatic chromatophores in the integument of the mandarin fish Synchiropus splendidus. The Biological Bulletin, 224(1), 14-17.

Hill, G. E. (1999). Is there an immunological cost to carotenoid-based ornamental coloration?. The American Naturalist, 154(5), 589-595.

Jalal, K. C. A., Zaima Azira, Z. A., Nor Hafizah, Z., Rahman, M. M., Kamaruzzaman, B. Y., & Noor Faizul, H. N. (2014). Carotenoid contents in anoxygenic phototrophic purple bacteria, Marichromatium sp. and Rhodopseudomonas sp. of tropical aquatic environment, Malaysia. Oriental Journal of Chemistry, 30(2), 607-613.

Kaur, R., & Shah, T. K. (2017). Role of feed additives in pigmentation of ornamental fishes. International Journal of Fisheries and Aquatic Studies, 5(2), 684-686.

Kuo, F. S., Chien, Y. H., & Chen, C. J. (2012). Effects of light sources on growth and carotenoid content of photosynthetic bacteria Rhodopseudomonas palustris. Bioresource Technology, 113, 315-318.

Kusmita, L., Mutiara, E. V., Nuryadi, H., Pratama, P. A., Wiguna, A. S., & Radjasa, O. K. (2017). Characterization of carotenoid pigments from bacterial symbionts of soft-coral Sarcophyton sp. from North Java Sea. International Aquatic Research, 9(1), 61-69.

Ligon, R. A., & McCartney, K. L. (2016). Biochemical regulation of pigment motility in vertebrate chromatophores: a review of physiological color change mechanisms. Current zoology, 62(3), 237-252.

Lopez-Romero, J., Salgado-Manjarrez, E., Torres, L., & Garcia-Peña, E. I. (2020). Enhanced carotenoid production by Rhodopseudomonas palustris ATCC 17001 under low light conditions. Journal of Biotechnology, 323, 159-165.

Maoka, T. (2011). Carotenoids in marine animals. Marine drugs, 9(2), 278-293.

Nakano, T., Tosa, M., & Takeuchi, M. (1995). Improvement of biochemical features in fish health by red yeast and synthetic astaxanthin. Journal of Agricultural and Food Chemistry, 43(6), 1570-1573.

Nakano, T. (2020). Stress in Fish and Application of Carotenoid for Aquafeed as an Antistress Supplement. Encyclopedia of Marine Biotechnology, 2999-3019.

Ramamoorthy, K., Bhuvaneswari, S., Sankar, G., & Sakkaravarthi, K. (2010). Proximate composition and carotenoid content of natural carotenoid sources and its colour enhancement on marine ornamental fish Amphiprion ocellaris (Cuveir, 1880). World Journal of Fish and Marine Sciences, 2(6), 545-550.

Reaksputi, R., Boonprab, K., Tunkijjanukij, S., & Salaenoi, J. (2019). Carotenoid production at various salinities in bacterium Rhodopseudomonas palustris. Agriculture and Natural Resources, 53(5), 500-505.

Sathyaruban, S., Uluwaduge, D. I., Yohi, S., & Kuganathan, S. (2021). Potential natural carotenoid sources for the colouration of ornamental fish: a review. Aquaculture International, 1-22.

Tacon, A. G. (1981). Speculative review of possible carotenoid function in fish. The Progressive Fish-Culturist, 43(4), 205-208.

Terao, J. (1989). Antioxidant activity of β‐carotene‐related carotenoids in solution. Lipids, 24(7), 659-661.

Wassef, E. A., Chatzifotis, S., Sakr, E. M., & Saleh, N. E. (2010). Effect of two natural carotenoid sources in diets for gilthead seabream, Sparus aurata, on growth and skin coloration. Journal of Applied Aquaculture, 22(3), 216-229.

Weaver, R. J., Cobine, P. A., & Hill, G. E. (2018). On the bioconversion of dietary carotenoids to astaxanthin in the marine copepod, Tigriopus californicus. Journal of Plankton Research, 40(2), 142-150.

Wingerter, K. The Physiology of Coloration in Fishes.

Xu, W., Ma, X., Yao, J., Wang, D., Li, W., Liu, L., ... & Wang, Y. (2021). Increasing coenzyme Q10 yield from Rhodopseudomonas palustris by expressing rate‐limiting enzymes and blocking carotenoid and hopanoid pathways. Letters in Applied Microbiology.

Yang, Y., Wu, L. N., Chen, J. F., Wu, X., Xia, J. H., Meng, Z. N., ... & Lin, H. R. (2020). Whole-genome sequencing of leopard coral grouper (Plectropomus leopardus) and exploration of regulation mechanism of skin color and adaptive evolution. Zoological research, 41(3), 328.

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