Difference between revisions of "Team:British Columbia/Project/Consortia"

Abstract

This year, our team aims to make processing and utilization of renewable biomass feedstocks cheaper and more efficient. For this, we decided to design a microbial consortia to separated metabolic processes between two members - Caulobacter and E.coli. As microbial consortia consisting of these two bacteria has never been described before, we needed to determine conditions in which this bacteria are able grow together. Next we needed to track dynamics of each member to ensure that one bacteria will not over-compete another. And last, as we defined the growth condition, we could start co-culturing Caulobacter displaying cellulases with E.coli producing β-carotene to confirm that our consortia can be efficient for direct transformation of lignocellulosic biomass in useful products.

Key Achievements

Introduction

Microorganisms live in complex microbial communities in the wild, in which individual species with specialized phenotypes interact and cooperate with each other to perform complex metabolic functions. Following nature's examples, there is an increasing trend in using microbial communities for biotechnological application due to their robustness and the ability to perform complex metabolic tasks through the division of labor(Shong, Jimenez Diaz, and Collins 2012). Construction of synthetic microbial communities allows to compartmentalize and optimize metabolic functions in different hosts. The goal of our project is to design a stable, robust microbial community for the production of valuable compounds from lignocellulosic biomass. The metabolic processes are split between biomass-degrading bacteria and the production bacteria, which transforms the degradation products into valuable products. For the first part, we engineered Caulobacter displaying functional biomass-transforming enzymes that act on cellulose. For the second part, we chose E.coli producing β-carotene as a proof of concept because it is colored, making it easily detectable. Now we need to confirm that these two bacteria can be co-cultured together to generate a stable consortia for consolidated bioprocessing.

Results

The first step in the development of successful co-culture conditions is to select defined media that would support the growth of both E.coli and C.crescentus and allow optimization of cellulose degradation to occur. Several alternatives were investigated to determine which produced best growth conditions for both cultures. Initially, M9 minimal media was tested with and without trace elements. C.crescentus p4A723 cultures inoculated into this media did not grow successfully as no growth was observed after 4 days of incubation. M16 minimal media was also considered as a candidate for the co-culture experiments, however, a complicated recipe made it a poor choice for our cultures. M2 minimal media (Presley et al. 2014) permitted successful growth for both E.coli and C.crescentus, although slow growth rates for both species was a disadvantage for this media. Due to its accommodation of both species, M2 minimal media was chosen for our cultures.

To determine the compatibility and co-culture dynamics between E.coli and C.crescentus, an experiment was attempted to determine each species relative prevalence in culture as a function of time. E.coli expressing green fluorescence protein (GFP) and C.crescentus expressing red fluorescence protein (RFP), seen in Figure 1, were inoculated at an initial OD600 of 0.01 with feed stock of 0.2wt% or 11.1mM glucose in 96-well plate and the plate was incubated at 30C in a Tecan platereader. The optical density at 600nm and fluorescence (617/673 and 390/500) was measured over 60 hours and the results were plotted as a function of time. Because there is no species dependence with glucose as a substrate, any negative impacts caused by co-culture should be evident with this method, and the compatibility of these species can be evaluated. Due to difficulty resolving wavelengths between the RFP and GFP, only basic observations can be made using this data.

Figure 1: Plated C.crescentus expressing RFP and E.coli expressing GFP.

As predicted by the developed consortia model and in the individual species cultures, the initial growth rate of the E. coli in the co-culture was significantly higher than the growth rate of C.crescentus. As seen in Figure 2, at longer culture times, the growth rate of E.coli drops significantly while the growth of C. crescentus climbs steadily. The decline in E. coli growth rate, while C. crescentus growth rate is affected minimally, possibly indicates that the glucose feedstock is not completely consumed and that oxygen could be limiting in microwells at relatively low culture densities for E. coli.

To better assess dynamics of each bacteria in consortia we are currently growing the co-culture for 3 days and plate every 12 hours on PYE-agar media to count CFU of C.crescentus and E. coli. That would allow us to separate the bacteria in consortia and get insight into their ratio over time

Figure 2: Green fluorescent protein and red fluorescent protein detection as a function of time in a C.crescentus and E.coli mixed culture.

As a part of our design, we have developed a functional prototype of the proposed consortium. In the prototype co-culture, cellulases expressed on C.crescentus surface degrade cellulose to serve as the substrate for E.coli which is producing β-carotene. Due to the slow rate of cellulose degradation by expressed cellulases, shown in our modelling section, culture lengths for the prototype experiment were three days or longer. E.coli and C.crescentus containing the recombinant plasmids were inoculated in M2 media with 0.2% cellulose as the sole carbon source. C.crescentus strains expressing Endo5A and Gluc1C cellulase enzymes were cultured alongside E.coli to determine the best candidates for a consortium. As a control, C.crescentus without cellulase expression genes were seeded with E.coli using it as a negative control for cellulose degradation. Over the course of three days, samples were taken and plated. These plated cultures can be identified based on either species unique morphology.

A measurement of OD600 indicates that the growth has occurred in several conditions with cellulose as the only substrate. Cultures containing C.crescentus expressing Endo5A cellulases seem to be most prolific. For these cultures, based on plate count data, E.coli growth has increased compared to cultures grown without cellulase expressing cells. In the cultures containing C.crescentus expressing Gluc1C, the ratio of E.coli to C.crescentus is nearly equal to the control, while in cultures containing C.crescentus expressing Endo5A, have a dominant C.crescentus population. This result likely indicates that there are diffusion limitations for glucose in the conditions with Endo5A expressing C.crescentus. A culture with mixed Endo5A and Gluc1C expressing C.crescentus seems to have increased C.crescentus growth, possibly due to the Endo5A expressing cells proliferating more rapidly. A marked increase in E.coli growth relative to the control cultures without expressed cellulases indicate the potential for our system.

To confirm that the E.coli densities were higher in the co-cultures containing Endo5A and Gluc1C cellulases, samples from each culture were plated after 6 days of growth on PYE and culture at 37 degrees Celsius. These conditions do not permit C.crescentus growth, and as a result, the relative cell density grown from each sample is directly proportional to the E.coli density in each culture. When the colony forming units were counted, there was nearly a four-fold increase in the number of E.coli existing in cultures containing C.crescentus with Endo5A expression. In the cultures containing C.crescentus only expressing Gluc1C, there was no clear difference in E.coli growth compared to the control cultures. The densities of E.coli in each culture can be seen in Figure 3.

Figure 3: E.coli densities in the different mixed culture systems

Conclusion

This experiment validates the design, showing the possibility for E.coli growth and product formation supported by cheap cellulose feed stock. The co-culture growth of both species indicates, in the absence of substrate limitation, E.coli and C.crescentus are compatible and there is no unexpected negative impact on community growth: a successful co-culture between E.coli and C.crescentus is possible.

The co-culture growth of both species indicates, in the absence of substrate limitation, E.coli and C.crescentus are compatible and there is no unexpected negative impact on community growth: a successful co-culture between E.coli and C.crescentus is possible. Another result from this experiment is the indication that the dissolved oxygen concentration must be increased in order to successfully culture and sustain a high density of E.coli using expressed cellulases on the C.crescentus surface to degrade cellulose for substrate.

References

Presley, Gerald N. et al. “Extracellular Gluco-Oligosaccharide Degradation by Caulobacter Crescentus.” Microbiology (United Kingdom) 160.PART 3 (2014): 635–645.

Shong, Jasmine, Manuel Rafael Jimenez Diaz, and Cynthia H. Collins. “Towards Synthetic Microbial Consortia for Bioprocessing.” Current Opinion in Biotechnology 23.5 (2012): 798–802.