Culturing experiments to determine the nutrient requirement for exopolymer production in HTCC 2155

By Connie LeePI: Stephen GiovannoniMentor: Amy CarterDepartment of Microbiology , OSUHHMI Summer Program 2011Culturing experiments to determine the nutrient requirement for exopolymer production in Lentisphaera araneosaGood morning everyone, my name is Connie Lee. The head of the lab is Dr. Stephen Giovannoni in the Microbiology Department. My mentor is Amy Carter, Faculty Research Assistantin the lab. Today I describe my research, which is culturing experiments to determine the nutrient requirement for exopolymer production in Lentisphaera araneosa. 1SignificanceMarine transparent exopolysaccharides (TEP)Marine snowRecycle organic compoundsMovement of organic matterMore efficient culture mediumFoundation for further investigationsSymbiotic relationshipEcological rolePotential use in industries

I am interested in this research because it helps us better understand the production of marine exopolysaccharides in Lentisphaera araneosa.

Marine transparent exopolysaccharide is an essential component of marine snow, which is the flocculent organic matter that commonly forms in the marine water column. Marine snow plays an important role in the recycling of organic carbon particles and the movement of organic matter from the surface to the deep ocean.It has been thought that marine TEP forms from dissolved organic matters excreted by microscopic plants that drift in marine community. Lentisphaera was the first reported marine bacterium that produces TEP. Thus, Lentisphaera is potentially a source of marine TEP and may play a role in recycling in low nutrient marine environments

Another goal of this research is to improve the current medium for Lentisphaeras growth. I seek for a more efficient recipe for its cultivation. The result of this research builds a foundation for further investigation on the characteristics of exopolymer, which may enable researchers a better grasp of its symbiotic relationship with other organisms and its ecological role in the marine community. It can also have a potential use in industries, such as additives in food production.

2Lentisphaera araneosaIsolated using High Throughput Microbial CultivationObligate aerobeChemoheterotrophFacultative oligotrophFound in surface and mesopelagic zones

The strain that I work with is Lentisphaera araneosa. Lentisphaeara was isolated using the High Throughput Microbial Cultivation method. Here is a picture of the bacterium. It is a marine bacterium that requires oxygen for growth, and uses organic compounds for energy and growth. It prefers growing in low nutrient conditions. They are found in surface and mesopelagic zones, which is 0 to 1000 meters below the ocean surface, in both the Pacific and Atlantic oceans. 3Transparent exopolysaccharidesProduces extracellular polysaccharide (EPS)Rhamnose, galactose, mannose, glucoseProduces transparent exopolysaccharides (TEP)

Lentisphaera has the unusual property of producing a net-like matrix of secreted transparent exopolysaccharide. The transparent material that the arrows point at in this picture is EPS produced by Lentisphaera. The major components of the extracellular polysaccharide (EPS) are rhamnose, galactose, mannose, and glucose. Extracellular polysaccharide is the main precursor of TEP formation. This next picture is the TEP produced by Lentisphaera. The little dots are the bacteria, they are connected by TEP, like a matrix. TEP is composed of various sugars. The exact composition of TEP in Lentisphaera is not yet known, but the focus of this experiment is the requirements for TEP production, not the composition of TEP.4Cell GrowthGrows in medium composed of various carbon sourcesIn low nutrient heterotrophic mediumIn artificial seawaterOptimum growth temperature at 16-20 degree CelsiusFast growth rate

Lentisphaera grows in a combination of various carbons sources. Here is the list of the components that we currently add in. There are glycine, methionine, serine, pyruvate, taurine, oxaloacetate, and glucose. It grows in two types of medium, low nutrient heterotrophic medium and artificial seawater. Its optimum growth temperature is between 16-20 degree Celsius. Comparing to other marine bacteria, its growth rate is fast.

ASW: has 15 different compounds + nanopure waterit mimics natural seawater, providing sources of nitrogen, phosphorus, iron, zinc, sodium, magnesium. LNHM: seawater collected + NH2Cl, K2HPO4, FeCl, vitamin (chloramine, Dipotassium phosphate, Iron (III) chloride)

5Here are the growth curves of Lentisphaera. It reaches its exponential phase in 1 to 2 days. It reaches stationary phase within 3 days. The generation time (or doubling time) is about 1 day. In the previous cultivations, the bacteria have been able to grow to about 2.5x10E7 cell/mL in low nutrient heterotrophic media with mixed carbon compounds. However, it did not produce TEP. Thus, in my research, I investigate the nutrient requirement and condition that allows TEP production.

Lentisphaera araneosa 22nd reinoculation growth curve (in LNHM)Lentisphaera araneosa 22nd reinoculation growth curve (in ASW)

6Comparing the growth curves of Lentisphaera araneosa in the two different seawater media, Lentisphaera grows better in artificial seawater than in low nutrient heterotrophic medium. In ASW, Lentisphaeras maximum cell density can reach about 5x107 cells/mL, in LNHM, Lentisphaera only grows to 2.5x107 cells/mL. It is about twice as much. Growth of Lentisphaera araneosa in different seawater

7HypothesisThe unusual property of TEP production in Lentisphaera araneosa is triggered by general nutrient input and growth condition.For my research, my hypothesis is that the production of TEP in Lentisphaera is triggered by general nutrient input and growth condition.8PredictionBy manipulating the nutrient input and growth condition, the production of TEP in Lentisphaera araneosa is affected.My prediction is that by manipulating the nutrient input, the production of TEP in Lentisphaera araneosa is affected. The degree of the effects of the different nutrient composition is not known, and it is the focus of my research. This experiment gives glues on how Lentisphaera araneosa uses nutrients in its metabolism, which may build foundation for further inquiries.9Experimental proceduresInoculate fresh culturesCultures in different mediaMonitor cell growthMeasure TEP production by viscosity

During my experiment, I inoculate fresh cultures every week. To test my hypothesis, I culture Lentisphaera in different media.

Currently, Lentisphaera grows in a medium composed of these carbon compounds.

At the beginning of my experiment, I knock out each component of the current recipe one by one to observe the effect of its removal on cell growth and TEP production. Instead of growing the bacteria in low nutrient heterotrophic medium, I grow them in artificial seawater since Lentisphaera has a better growth in artificial seawater. I incubate the cultures under 16 degree Celsius. I break down the mixed carbon components individually to determine the specific combination of nutrient(s) required for TEP production in Lentisphaera araneosa.

During the experiment, I also monitor and record the cell growth with Guava EasyCyte flow cytometer.

Production of TEP is measured by testing the viscosity of the media. This is a picture of the glass viscometer that I use in my experiment. I measure the time it takes for the culture to flow over a distance. Multiplying the measured time with the viscosity constant, I obtain the kinematic viscosity of the culture in centistokes, which is mm2/s. Using the data, I examine the relative viscosities of the cultures to see the effects of different treatments on Lentisphaeras TEP production.

10Oxygen requirementMajority of the cultures in the lab are cultured with 5:1 ratio (oxygen:medium volume) to allow enough oxygen for bacteria to reach maximum cell density. I tested the oxygen requirement for Lentisphaera araneosa, growing 100mL, 150mL, and 200mL in 250mL flasks. Although the 100mL flask reaches a slightly higher cell density, but there is not that much difference between different volume ratio, concluding that the oxygen:medium ratio does not affect Lentisphaeras growth that much . Considering the amount of samples needed for viscosity measurement, I grow Lentisphaera with 500mL medium in 1L bottles.11Positive controlsTwo positive controlsBoth in artificial seawater

Before starting my experiment, I did two positive controls. I tested two medium to see if TEP production occurs with these two lists of nutrients. One is the current one, the other is the medium used in the past for Lentisphaera (read it aloud), and both are in artificial seawater. 12Here are the growth curves of the positive controls. Comparing the two, Lentisphaera grows to a higher cell density with the current medium.13Viscosity of positive controlsHere is the kinematic viscosity of the positive controls. In general, the viscosity of both positive controls has increased over time, meaning both positive controls produce TEP. The viscosity of both positive controls decreased at the end of the stationary phase, it might be because the cells are dead. Since Dr. Cho, one of the professors in our lab, has observed TEP production in Lentisphaera before, Dr. Cho helped me looked at the cultures, and we observed that both flasks have bubbles staying in the medium after swirling it. This is a sign of TEP production. Thus, I continued my experiment. (Water is about 0.8802 cSt)14Experiment schemeGlycineMethionineSerinePyruvateTaurineOAAGlucose2155.22.#12155.22.#2X2155.22.#3X2155.22.#4X2155.22.#5X2155.22.#6X2155.22.#7X2155.22.#8XHere is a scheme on the experiment I conducted. Due to limited amount of 1L bottles in the lab, I only tested one of the media. And because the current medium gives a better growth, I decided to test the current medium first. Bottle #1 has every single nutrient in it. For the other bottles, I knocked out each component of the list one by one to test the effects of its removal on Lentisphaeras growth and TEP production.15Growth curveComparing the results, bottles 2, 4, 6, 8 have better growth than bottle 1. Meaning that the bottles without glycine, serine, taurine, or glucose give better growth than the one with everything in it. And without methionine and oxaloacetate, Lentisphaera could not grow well comparing to the others. 16ViscosityGenerally, the viscosities of all treatments increased over time, meaning TEP is produced. Bottle #8, the bottle without glucose, which had the highest cell density among all, also has the highest viscosity among all. Bottle #4, the bottle without serine, has the second highest viscosity. These two treatments (without serine and glucose) still allow both high cell growth and TEP production just like the control group. This may lead to a new nutrient recipe for Lentisphaeras cultivation.17ConclusionTEP production positiveLate exponential and stationary phaseCultures without serine or without glucoseDoes produce TEP, at late exponential and stationary phaseCultures without serine or without glucose are possible new nutrient recipes, as they give high cell densities while give high TEP production. Need further investigation on it.18Future workCulturing at different concentration and different temperatureFurther investigation on the effects of different carbon compounds based on Lentisphaera araneosas genome sequences.Effects of nitrogen, phosphorus, and sulfur on Lentisphaera araneosas growth and TEP productionAfter determining the required nutrients for its growth, I add in different concentrations of the new mixed carbon recipes into the cultures. I incubate the cultures under 16, 20, and 25 degree Celsius to observe the effects.

Afterwards, with the genome information, I add a combination of different carbon compounds into multiple cultures with a control group to test the effect of different treatment on Lentisphaera.

I also test the effects of nitrogen, phosphorus and sulfur on Lentisphaeraalter sulfur, it has a lot of sulfatase

19AcknowledgementDr. Stephen GiovannoniAmy CarterThe Stephen Giovannoni LabKevin VerginDr. Jang-Cheon ChoPaul CariniDr. Yanlin ZhaoDr. Kevin AhernThe Howard Hughes Medical InstituteURISCOregon State University Honors CollegeCripps scholarshipsHere is my acknowledgement page. Thank you.20