Team:Nevada/Project/Co-Cult

Introduction

      Co-culturing multiple bacterial strains is inherently complicated. The two species must co-exist while at the same time competing for resources. Over time the growth rate of two co-cultured species will establish equilibrium. However, conditions will not be optimal for either individual species. In our case, we want to grow the E. coli in a culture that is being provided with glucose from the cyanobacterium, Synechocystis PCC6803. It is clear that E. coli growth will be limited by the productivity of the cyanobacterium. Therefore, we need to develop a system that will optimize Synechocystis growth. One of the major considerations in cyanobacteria growth is the availability of sufficient light to optimize rates of photosynthesis. This is particularly important in our system since we are also depending on photosynthesis for the production of glucose to feed E. coli. While it is possible to grow E. coli and cyanobacteria in the same culture vessel (Niederholtmeyer et al., 2010) Applied and Environmental Microbiology 76: 3462-3466), the photosynthetic efficiency of the system will be limited by light blockage caused by E. coli. For this reason, we have developed an apparatus that physically partitions the two bacterial species from each other, while still allowing for the free exchange of growth medium. In this way, we can ensure high photosynthetic rates for Synechocystis and total accessibility by E. coli to the cyanobacterial produced glucose.

Choice of Growth Medium

      BG-11 must be used for culturing Synechocystis. However, this medium does not provide adequate nourishment to E. coli. Thus, this project must determine what additives to BG-11 will foster growth of both organisms. The supplemented media must allow for growth of E. coli comparable to LB but must not inhibit Synechocystis with toxic levels of solutes.

      Initial tests will determine if BG-11 has an inherently sufficient nitrogen supply for E. coli. Previous research has shown that NH₄⁺ is more effective than nitrates and nitrites when used by E. coli as a nitrogen source (Strevett, K.A., and Chen, G., 2003). Therefor, testing will be done with the growth of NEB 10-ß cells in BG-11 with NH₄Cl as an additional source of nitrogen. The growth in this media will be compared with that of BG-11 alone. It must then be shown that Synechocystis PCC6803 can tolerate the levels of ammonium chloride required. If ammonium chloride cannot be used, nitrites and nitrates may then be used as a backup option.

Apparatus Components

Culture Vessels
    The actual chambers are made from modified chromatography columns. They are Pyrex glass tubes with removable plastics caps. Each chamber has a double seal to prevent leaking. Both of these are supported by a ring stand with clamps.

Transfer Pump
      One main thing required to have an effective apparatus is a pump to transfer one bacteria solution to the other chamber into a permeable membrane. It needs to be able to transfer enough volume to fill the dialysis tube completely but not create too much pressure that the tubing would burst. An external pump was chosen to reduce the number of components that would touch the bacteria and lesson the chances of contamination. It also reduces the amount of parts to be cleaned. On our apparatus the E. coli is gravity fed to the transfer pump inlet, then pumped through clear vinyl tubing to the dialysis tube and returned back to the E. coli chamber. It pumps approximately 25 gallons/hr.

Semipermeable Membrane
      The dialysis tubing allows regulated bacteria interaction, meaning glucose from the Cyanobacteria can transfer over to the E. coli but the two bacteria never contact. Something was needed to give the dialysis tubing rigidity and also allow effective flow of E. coli through the inside of the tubing. The dialysis tubing also needed something to seal the ends to prevent contamination. A borosilicate tube was taken and had bubbled ends installed for the sealing surface to the dialysis tubing. The glass tubing then had small holes placed in it with a wall in the middle. This made one end an inlet where the media would enter the glass tube flow out of the small holes into the dialysis tubing filling it on its way to the other end. From there it would exit through the other set of small holes and return to the vinyl tubing and the second chamber. To seal the dialysis tubing to the glass a piece of shrink tube was placed over the dialysis tubing compressing it against the glass. Next a rubber o-ring was placed over the shrink tube to provide an extra seal.

Oxygenation
      For proper growth, each chamber needs a constant flow of oxygen. A four-channel variable aquarium oxygen pump, inside the apparatus base, oxygenates the Cyanobacteria and E. coli. Each chamber is fed from the bottom through two lines, check valves were installed to prevent the bacteria from draining down the tubing into the oxygen pump. On the top of the base is the adjustment knob to control the oxygen flow. Hand blown glass bubblers, made from borosilicate glass, were attached to the top of each chamber to allow proper venting without releasing the bacteria out.

Illumination
     In order for photosynthesis to take place some sort of artificial light is necessary. Two T5 14W fluorescent bulbs, each connected to a slider that allows them to be independently adjusted to vary the amount of light as needed in the Cyanobacteria chamber.

Controller
      The base is constructed of 6 panels of 6061 aluminum tig(GTAW) welded together. Its dimensions are approximately sixteen inches long, fourteen inches wide, and three inches tall. All electrical switches and wiring are inside with the oxygen pump and rings stand base to keep bacteria out and make it easier to clean.