From Waste to Nutrition: Fatty Acid Profile of PNS Bacteria

Purple non-sulfur bacteria (PNSB) are potent and multifaceted biofiltration agents. Due to their Swiss army knife metabolism, PNSB such as Rhodopseudomonas palustris can switch between nitrification, denitrification, photosynthesis, heterotrophy and any mix thereof. This allows PNSB to aggressively consume organic wastes, nitrogenous wastes, phosphates and sulfides--therefore depriving pest algae and opportunistic bacteria the opportunity to grow.

However, there is yet another important benefit from this aggressive nutrient consumption in that PNSB metabolize these compounds back into valuable proteins and lipids and packages them in stabilized cells. Corals, sponges and other micrograzers are adept at capturing bacteria for sustenance and will feast upon excess Rhodopseudomonas palustris cells as they are “sloughed off’’ from biofilms. By having robust PNSB colonies, aquarists recapture dissolved wastes and reclaim them in the form of microbial lipids and proteins. Understanding the fatty acid profile of Rhodopseudomonas palustris during its various metabolic faculties is crucial to quantifying this nutritional return.

Fatty acids are the building blocks of lipids (fats) and are of profound importance to the metabolisms of all organisms. In a highly generalized sense, fatty acids function as high-capacity energy storage reserves for cells but have infinite nuanced functions such as dictating membrane fluidity, reducing inflammation and regulating hormone concentrations. Some fatty acids such as 5-aminolevulinic acid (ALA) is an intermediate metabolite utilized for the biosynthesis of complex biomolecules such as hemes, chlorophyll, and vitamin B12. Because of this diverse array of functions, all fatty acids are not nearly created equal and their structure greatly dictates their function and relative metabolic worth.

Fatty acids are composed of long chains of carbon and hydrogen atoms connected by various single and double bonds. When a fatty acid has no double bonds it is described as saturated. Saturated fats are relatively metabolically inferior because they are associated with inflammation and store less energy than highly unsaturated or polyunsaturated fatty acids. HUFAs and PUFAs possess double bonds and store exponentially more energy than saturated fatty acids. Because of this, they act as cleaner-burning fuel and can be catabolized without inducing inflammation. Because of this, polyunsaturated fatty acids such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are absolutely essential to early embryonic development as well as the long-term health of both invertebrates and vertebrates alike. This dietary need is most dramatically pronounced in larval marine organisms which, as a whole, begin life poorly-developed and with relatively sparse maternal lipid reserves. Thus, marine larvae MUST consume polyunsaturated fatty acids through the diet within a matter of hours/days post-hatch or they will not survive.

Polyunsaturated fatty acids are vital to the health of any reef aquarium. Many terrestrial and freshwater organisms have evolved enzymes which can elongate and desaturate lesser fatty acids into essential PUFAs such as EPA and DHA. Most marine organisms lack these enzymes and thus MUST receive minimal levels of EPA and DHA through the diet. In many organisms, increasing dietary PUFA levels is met with appreciable return in the form of improved color, increased survival, increased body weight, increased growth and increased disease resistance. For example, it is a common practice in seahorse farms to ‘gut-load’ Artemia with PUFA-rich algae oil prior to feeding. Woods et al 2003 demonstrated faster growth in seahorse juveniles fed Artemia enriched with the highest levels of DHA. The same can be said in the clownfish industry, where greater dietary DHA levels are associated with higher broodstock fecundity, egg viability and early larval survival. Diaz-Jimenez et al 2019 demonstrated that peppermint shrimp (Lysmata wurdemanni) produce significantly more viable eggs when fed a diet of 8% lipid versus 7%. DHA has been observed as vital to the construction of chromatophores---pigment cells which produce and maintain the vibrant color of various fish species.

The value of PUFAs translates beyond faster growth and greater reproductive success, for they have a nuanced role in the regulation of various immune systems. For example, Zuo et al 2012 demonstrated an association between dietary DHA and increased nonspecific immunity to marine ich (Cryptocaryon irritans) in yellow croaker (Larimichthys crocea). Such a study offers potential avenues to improve the innate immunity of aquarium fish, through diet, thus granting them increased immunity against the industry’s many pathogens.

Autotrophs are the only organisms which synthesize fatty acids from inorganic molecules such as nitrates, phosphates and sulfides. Through photosynthesis and chemosynthesis, autotrophs such as Rhodopseudomonas palustris convert metabolic waste into the invaluable metabolic currency of life. Understanding the fatty acid metabolism of PNSB is paramount to utilizing it as a biofeed agent. The enzyme pyruvate phosphate dikinase (PPDK) plays an integral role in carbon utilization, CO2 assimilation, glycolysis and gluconeogenesis within the Rhodopseudomonas palustris cell. Hu et al 2012 demonstrated how PPDK expression levels were significantly higher in PNSB cultures grown photoheterotrophically than those grown under dark anaerobic conditions. The result was a greater overall extracellular lipid accumulation in the illuminated cultures. Choorit et al 2011 demonstrated that PNS produced more ALA under illuminated conditions than dark. Such studies suggest that partially illuminated anaerobic biofilters may be an effective mechanism to ensure maximum nutritional return.

Although many terrestrial organisms have evolved to possess enzymes which allow them to elongate and desaturate saturated fatty acids into essential PUFAs, many marine organisms cannot. This is why both commercial marine aquaculture and reef aquaria depend on feeds rich in dietary PUFAs. Traditionally, these PUFAs are provided through processed fish oil sourced through commercial forage fisheries. Increased human population and demand from these industries has made these fisheries growingly unsustainable. Because of this, direct cultivation of fatty acid-producing species is the pressing ultimatum of the future.

Rhodopseudomonas palustris has been formally investigated as a potential biofeed species. Kim et al 2000 investigated the application of PNS as a feed agent for aquaculture. When grown through anaerobic fermentation, it produced predominantly the monounsaturated fatty acid oleic acid (18:1). Loo et al 2013 demonstrated that Rhodopseudomonas palustris can be cultivated on waste PUFAs (palm oil effluent). The resulting PNS had significantly higher levels of the polyunsaturated acids DHA and EPA. The enriched PNS was then fed to rotifers (Brachionus plicatilis) which were then able to satisfy the nutritional requirement of marbled goby larvae (Oxyeleotris marmorata). This study suggests that PNS cultures will be enriched by the quality fatty acids of the reef waste they consume.

In the reef aquarium, free-floating Rhodopseudomonas palustris are directly preyed on by various micrograzers such as sponges, sabellid worms, clams and many corals. Conlan et al 2017 demonstrated an association between increasing bacterial DHA and ARA concentrations in seawater with increased polyp growth in Acropora hyacinthus, A. loripes, A. millepora, and A. tenuis. Teece et al 2011 observed similar findings in the scleractinian coral species Montastraea faveolata and Porites astreoides. Gut-analysis of these corals suggested that these corals receive the majority of their saturated fatty acids from their zooxanthellae symbiont, but that heterotrophic foraging was vital to them obtaining essential PUFAs. These studies hint at the numerous potential benefits that photosynthetic corals may receive from lipid-rich bacterial grazing.

This benefit is even greater in actively feeding corals such as gorgonians. Many heterotrophic corals, sea fans and marine sponge species are obligate bacteria-foragers and because of this are not particularly common in the hobby. Xue et al 2009 demonstrated success aquaculturing the marine sponge Hymeniacidon perlevis on a diet that includes Rhodopseudomonas. Such species may benefit tremendously from a diet of PNSB enriched with reef waste. Even less understood is the realization that many marine sponges and corals conduct symbiotic relationships with lipid-producing bacteria. Through such considerations, biofilters with ever-improving fatty acid profiles will likely make a whole new slough of non-photosynthetic corals, sponges, tunicates and other previously difficult-to-keep micrograzers available to the reef hobby. Additionally, zooplanktivores benefit from the increased number and nutritional quality of copepods, which anecdotally feed intensely on these bacteria and may pass PNSB-synthesized fatty acids up the food chain.

The future of sustainable aquaculture feeds begins with, but doesn’t end with, the mass-cultivation of planktonic bacteria; we must also reduce the need to extraneously feed by converting wastes to nutrition. It is in this focus that Rhodopseudomonas palustris excels. Its versatile metabolism allows it to access both organic and inorganic wastes that are inaccessible to many traditional aquarium detritivores. Phongjarus et al 2018 demonstrated the potential of Rhodopseudomonas palustris to photoheterotrophically digest palm and soybean oils under anaerobic and microaerobic conditions. Indeed, Rhodopseudomonas palustris is capable of waste lipid-reutilization in anaerobic conditions where it is also denitrifying. Through these various metabolic services, this microbe reduces levels of nitrogenous wastes and fatty acid wastes, all the while depriving opportunistic bacteria and algae that would otherwise thrive off them.

PNSB convert wastes into proteins and fatty acids that would otherwise need to be dumped into the surrounding environment with a water change. Therefore, through its ability to synthesize fatty acids, PNSB are a potential tool for the reef aquarist eager to reduce the need for water changes and fully maximize the feed input into their system. By embracing the power of PNSB, both commercial aquaculture and the reef aquarium hobby in general might attain a higher degree of success and sustainability.

Literature Consulted

Bharathi, S., Antony, C., Cbt, R., Arumugam, U., Ahilan, B., & Aanand, S. (2019). Functional feed additives used in fish feeds. International Journal of Fisheries and Aquatic Studies, 7(3), 44-52.

Carlozzi, P., Pintucci, C., Piccardi, R., Buccioni, A., Minieri, S., & Lambardi, M. (2010). Green energy from Rhodopseudomonas palustris grown at low to high irradiance values, under fed-batch operational conditions. Biotechnology letters, 32(4), 477-481.

Choorit, W., Saikeur, A., Chodok, P., Prasertsan, P., & Kantachote, D. (2011). Production of biomass and extracellular 5-aminolevulinic acid by Rhodopseudomonas palustris KG31 under light and dark conditions using volatile fatty acid. Journal of bioscience and bioengineering, 111(6), 658-664.

Conlan, J. A., Humphrey, C. A., Severati, A., & Francis, D. S. (2017). Influence of different feeding regimes on the survival, growth, and biochemical composition of Acropora coral recruits. PloS one, 12(11), e0188568.

Conlan, J. A., Bay, L. K., Severati, A., Humphrey, C., & Francis, D. S. (2018). Comparing the capacity of five different dietary treatments to optimise growth and nutritional composition in two scleractinian corals. PloS one, 13(11), e0207956.

Darias, M. J., Andree, K. B., Boglino, A., Fernández, I., Estévez, A., & Gisbert, E. (2013). Coordinated regulation of chromatophore differentiation and melanogenesis during the ontogeny of skin pigmentation of Solea senegalensis (Kaup, 1858). PLoS One, 8(5), e63005.

Díaz‐Jiménez, L., Hernández‐Vergara, M. P., & Pérez‐Rostro, C. I. (2019). Protein and lipid requirement for the growth and reproduction of the peppermint shrimp Lysmata wurdemanni. Aquaculture Research, 50(8), 2281-2288.

Di Lorenzo, F., Palmigiano, A., Al Bitar‐Nehme, S., Sturiale, L., Duda, K. A., Gully, D., ... & Silipo, A. (2017). The lipid A from Rhodopseudomonas palustris strain BisA53 LPS possesses a unique structure and low immunostimulant properties. Chemistry–A European Journal, 23(15), 3637-3647.

Faulkner, D. J., Unson, M. D., & Bewley, C. A. (1994). The chemistry of some sponges and their symbionts. Pure and applied chemistry, 66(10-11), 1983-1990.

Garcia, A. E. (1996). Role of lipids and vitamin A on pigmentation success of flatfish (Doctoral dissertation, 鹿児島大学).

Getha, K., & VIKINESWARY, S. (1998). Potential Use of the Phototrophic Bacterium, Rhodopseudomonas palustris, as an Aquaculture. Asian Fisheries Science, 10, 223-232.

Hu, C. W., Lin, M. H., Huang, H. C., Ku, W. C., Yi, T. H., Tsai, C. F., ... & Wu, S. H. (2012). Phosphoproteomic analysis of Rhodopseudomonas palustris reveals the role of pyruvate phosphate dikinase phosphorylation in lipid production. Journal of proteome research, 11(11), 5362-5375.

Imhoff, J. F., Hiraishi, A., & Süling, J. (2005). Anoxygenic phototrophic purple bacteria. In Bergey’s manual® of systematic bacteriology (pp. 119-132). Springer, Boston, MA.

Jian-bin, L. I., Wei, H. E., Da-lie, L. I., & Min, L. Y. U. (2014). Effect of different amount of Rhodopseudomonas palustris feed additive on Tilapia mossambica breeding. Journal of Southern Agriculture, 45(9).

Kim, J. K., & Lee, B. K. (2000). Mass production of Rhodopseudomonas palustris as diet for aquaculture. Aquacultural engineering, 23(4), 281-293.

Loo, P. L., Vikineswary, S., & Chong, V. C. (2013). Nutritional value and production of three species of purple non‐sulphur bacteria grown in palm oil mill effluent and their application in rotifer culture. Aquaculture Nutrition, 19(6), 895-907.

Loo, P. L. (2012). Phototrophic bacteria grown in palm oil mill effluent as feed for culturing rotifers and marble goby larvae/Loo Poh Leong (Doctoral dissertation, University of Malaya).

Mekjinda, N., & Ritchie, R. J. (2015). Breakdown of food waste by anaerobic fermentation and non-oxygen producing photosynthesis using a photosynthetic bacterium. Waste Management, 35, 199-206.

Phongjarus, N., Suvaphat, C., Srichai, N., & Ritchie, R. J. (2018). Photoheterotrophy of photosynthetic bacteria (Rhodopseudomonas palustris) growing on oil palm and soybean cooking oils. Environmental Technology & Innovation, 10, 290-304.

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.

Teece, M. A., Estes, B., Gelsleichter, E., & Lirman, D. (2011). Heterotrophic and autotrophic assimilation of fatty acids by two scleractinian corals, Montastraea faveolata and Porites astreoides. Limnology and Oceanography, 56(4), 1285-1296.

Varghese, B., Paulraj, R., Gopakumar, G., & Chakraborty, K. (2009). Dietary influence on the egg production and larval viability in true Sebae clownfish Amphiprion sebae Bleeker 1853. Asian Fisheries Science, 22(1), 7-2o.

Wood, B. J. B., Nichols, B. W., & James, A. T. (1965). The lipids and fatty acid metabolism of photosynthetic bacteria. Biochimica et Biophysica Acta (BBA)-Lipids and Lipid Metabolism, 106(2), 261-273.

Woods, C. M. (2003). Effects of varying Artemia enrichment on growth and survival of juvenile seahorses, Hippocampus abdominalis. Aquaculture, 220(1-4), 537-548.

Xue, L., & Zhang, W. (2009). Growth and survival of early juveniles of the marine sponge Hymeniacidon perlevis (Demospongiae) under controlled conditions. Marine Biotechnology, 11(5), 640-649.

Zuo, R., Ai, Q., Mai, K., Xu, W., Wang, J., Xu, H., ... & Zhang, Y. (2012). Effects of dietary docosahexaenoic to eicosapentaenoic acid ratio (DHA/EPA) on growth, nonspecific immunity, expression of some immune related genes and disease resistance of large yellow croaker (Larmichthys crocea) following natural infestation of parasites (Cryptocaryon irritans). Aquaculture, 334, 101-109.