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.
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.
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: Growth of C.crescentus and E.coli and their co-culture in M2 minimal media with 0.2% glucose over 60 hours.Finally, we attempted to prove the functional prototype of our consortia.Check the results in Proof section!
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.
- 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.
Check out other parts of our project below!