Rhodopseudomonas palustris CGA009 Lignin Catabolism and Bioplastic Production
Polyhydroxybutyrate (PHB) is a biopolymer that has similar thermomechanical properties as petroleum-based plastics, and is produced by bacteria under certain stress conditions. Unfortunately, the commercial production and widespread adaptation of PHB is limited by higher productions costs. A renewable and cheaper carbon source, such as lignocellulosic biomass, could help reduce production costs. In this study, PHB was produced from the metabolically versatile Rhodopseudomonas palustris CGA009 when grown on lignin breakdown products. Genome-scale metabolic modeling was applied, and three overarching design strategies for the overproduction of PHB were generated. In the end, these design strategies can be used by all PHB-producing bacteria for enhanced PHB production.
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[00:00:01.290]Hi everyone, my name is Brandi Brown,
[00:00:02.730]and I'm a PhD candidate here in Bioengineering at UNL.
[00:00:05.880]We're really excited to share with you guys
[00:00:07.470]our study for R. palustris,
[00:00:09.080]which is a non-model bacterium
[00:00:11.110]for producing bioplastics from lignin,
[00:00:13.390]which is a big part of agricultural waste.
[00:00:18.810]So why lignin and why R. palustris?
[00:00:21.620]Typically when people think of agricultural waste,
[00:00:24.150]they think of the sugars which have been engineered
[00:00:26.250]and produced into a wide variety
[00:00:28.300]of high value products such as fuels.
[00:00:31.260]However, lignin is also a large portion
[00:00:33.450]of agricultural waste,
[00:00:35.030]can be upwards of 30% of it.
[00:00:37.130]But as you see here,
[00:00:37.963]it's a pretty complex structure of phenolic compounds,
[00:00:40.250]and it's really hard to break down
[00:00:42.110]and turn into high value products.
[00:00:44.100]And so unfortunately, it's typically burnt for energy.
[00:00:47.150]However, there are some microbes that can convert
[00:00:50.440]this lignin into high value products as well.
[00:00:54.460]This is why we focus on R. palustris in our lab,
[00:00:56.930]at the systems and synthetic biology lab at UNL.
[00:00:59.900]We are focusing on taking this really
[00:01:02.370]metabolically robust non-model bacterium
[00:01:05.690]called R. palustris.
[00:01:08.198]And utilizing it because it can take a wide variety
[00:01:09.900]of waste products and turn them into
[00:01:11.930]a variety of value-added products,
[00:01:14.310]such as hydrogen and bioplastics.
[00:01:16.670]And our lab is really interested in combining
[00:01:19.130]metabolic modeling with synthetic biology.
[00:01:24.100]And so, the first thing we did is to get
[00:01:25.860]a more holistic understanding
[00:01:27.140]or a good foundation of
[00:01:28.330]how this unique microbe is able to use
[00:01:30.890]different components of lignin
[00:01:32.820]and under what conditions.
[00:01:34.400]And so here you see that it was able to grow
[00:01:36.080]on a wide variety of lignin breakdown products
[00:01:38.540]or LBPs as we call them here under aerobic conditions.
[00:01:42.180]And this was the first
[00:01:44.620]case or the first reported case of
[00:01:46.630]many of these lignin breakdown products
[00:01:48.160]that had never been reported before.
[00:01:50.380]What's notable is that compared to acetate
[00:01:52.643]a lot of these lignin breakdown products,
[00:01:54.900]majority of them grow to a higher biomass.
[00:01:58.800]The same conditions for anaerobic as well,
[00:02:00.990]so without oxygen,
[00:02:03.350]this is just not all the lignin breakdown products
[00:02:05.720]presented here but to showcase p-Coumarate
[00:02:07.703]and coniferyl alcohol,
[00:02:09.430]because they grew without having to be
[00:02:11.440]supplemented with acetate into two
[00:02:13.750]or three times that of the biomass.
[00:02:16.970]We also looked at Kraft lignum
[00:02:18.590]because it's 96% of the worldwide lignin that's produced
[00:02:22.160]so we thought that would be kind of a good example of
[00:02:25.400]how our boosters is able to catabolized lignin.
[00:02:31.080]So once we had this foundation of
[00:02:33.330]how it's able to grow, under what conditions
[00:02:35.390]on different lignin breakdown products,
[00:02:37.120]we wanted to do something with it, right?
[00:02:38.530]And we want it to produce bioplastics.
[00:02:41.090]So here's a general overview of how we accomplish this.
[00:02:44.290]We would feed the lignin breakdown products
[00:02:46.130]as a carbon source or substrate for fermentation.
[00:02:49.550]We would induce stress that
[00:02:52.090]kind of fosters the bioplastic granules
[00:02:54.910]that are produced inside of the cells.
[00:02:56.780]In our case, we,
[00:02:58.743]we did a study and selected nitrogen starvation
[00:03:00.790]as the optimal condition.
[00:03:02.440]We would extract it
[00:03:03.350]and then quantify it with gas chromatography.
[00:03:08.450]So first you see that we were able to
[00:03:11.230]decipher that our post chart shows growth
[00:03:12.710]to a higher biomass on these lignum breakdown products
[00:03:15.210]which led us to think,
[00:03:16.043]okay maybe these might be good candidates
[00:03:18.240]for a bioplastic production.
[00:03:21.320]So we did a starvation condition analysis
[00:03:24.330]because some microbes produce more bioplastics
[00:03:26.730]under certain stress conditions.
[00:03:28.130]And so we did an analysis and decipher
[00:03:30.190]that nitrogen versus phosphorous,
[00:03:33.110]nitrogen is a better starvation condition
[00:03:36.200]to produce more bioplastics.
[00:03:38.970]And then we did Polyhydroxybutyrate,
[00:03:41.380]which is the most common type of bioplastic.
[00:03:44.820]We analyze production on p-Coumarate,
[00:03:46.790]conifer alcohol, butyrate and acetate.
[00:03:49.400]And what we found is there was no production on acetate
[00:03:52.030]despite it having the same carbon content
[00:03:54.300]as butyrate and p-Coumarate.
[00:03:56.300]So that was pretty baffling to us as why,
[00:03:58.410]why would we feed the micro the same amount of carbon
[00:04:00.960]but yet p-Coumarate and butyrate
[00:04:03.220]would yield more bioplastics.
[00:04:06.240]Also, you note here that p-Coumarate
[00:04:08.244]and coniferyl alcohol,
[00:04:09.080]coniferyl alcohol has a higher carbon content,
[00:04:11.600]yet they peaked at the same
[00:04:14.580]maximum, tighter or production of PHB.
[00:04:19.210]Why is this the case?
[00:04:20.610]So that was also a baffling to us.
[00:04:25.140]We were able to conduct
[00:04:26.600]a hydrogen production analysis
[00:04:28.070]because there's trade-offs in reducing potential
[00:04:30.790]where that may funnel to bioplastic
[00:04:33.220]versus hydrogen production.
[00:04:34.370]And also, so what we found is that,
[00:04:36.520]when at day five, when bioplastic production peaked,
[00:04:40.570]hydrogen production kicked off.
[00:04:42.340]And so this list I think there was still
[00:04:45.360]reducing potential to be analyzed.
[00:04:49.320]So thence, to help answer these questions that we had
[00:04:52.310]we conducted transmission electron microscopy
[00:04:54.480]to image the granules inside of the cell.
[00:04:56.910]And so the white granules you see here
[00:04:58.490]in panel B and panel C
[00:05:00.370]are the bioplastic granules inside of the cell.
[00:05:03.410]And you can see a clear discrepancy
[00:05:04.890]between that and acetate
[00:05:06.540]which virtually had no bioplastic production.
[00:05:09.940]Or what's notable is there to one large granule
[00:05:11.880]inside of the cell which led us to believe
[00:05:13.840]that cytoplasmic space
[00:05:15.240]maybe a limiting factor for bioplastic production.
[00:05:20.320]So now that we have all these questions
[00:05:22.360]we had really robust experimental analysis.
[00:05:24.330]We wanted to conduct metabolic modeling to try
[00:05:26.420]and answer these questions about why this lignum
[00:05:28.910]breakdown products were producing more bioplastics.
[00:05:32.570]And this was conducted by my colleague Adil Al-siyabi,
[00:05:36.270]and he utilized a specific type of fluorometry
[00:05:39.440]and a thermo-kinetic analysis that also fed
[00:05:41.590]into the metabolic model.
[00:05:44.490]And ultimately this yielded three different
[00:05:46.520]design strategies that could be useful
[00:05:49.730]to any bacteria or expanded to all PHB producing bacteria.
[00:05:54.540]So it's not just specific to our polluted stress.
[00:05:56.980]And this was to utilize a carbon source
[00:05:59.110]or substrate that bypasses this ketothiolase reaction
[00:06:03.150]which is a thermodynamically expensive reaction.
[00:06:07.180]And so if you can utilize a substrate that bypasses that,
[00:06:09.760]you can produce more bioplastics.
[00:06:12.600]As well as utilizing reduced substrates,
[00:06:14.470]as I spoke about before the trade off
[00:06:16.160]between reducing potential and
[00:06:19.030]trade offs between producing potential,
[00:06:20.980]as well as using high molecular weight substrates.
[00:06:24.550]So again, these can be expanded to all
[00:06:26.230]bioplastic producing bacteria
[00:06:28.290]and provide a good design strategies
[00:06:31.050]for overproducing bioplastics
[00:06:32.760]on lignin breakdown products.
[00:06:36.800]So in summary, we wanted to use lignin
[00:06:38.730]as a cheaper renewable carbon source,
[00:06:40.380]and R. palustris is an ideal candidate for this.
[00:06:42.920]We conducted a robust growth analysis on Kraft lignin
[00:06:46.630]and many of these lignin breakdown products
[00:06:48.420]which gave us a good foundation
[00:06:50.400]to analyze bioplastic productions on
[00:06:53.270]lignin breakdown products.
[00:06:55.020]And through some of these unique experimental findings,
[00:06:57.770]we were able to apply the metabolic model
[00:07:00.660]and come up with some design strategies
[00:07:02.450]for the overproduction of bioplastics from lignin.
[00:07:07.470]So we would like to thank you guys
[00:07:08.940]for the invite for a student research days
[00:07:11.070]and for attending our presentation
[00:07:12.540]and to thank our funding resources,
[00:07:14.710]the Nebraska Center for Energy Sciences Research
[00:07:17.080]in particular has been very supportive
[00:07:19.480]for us on this project.
[00:07:21.610]Our collaboration with
[00:07:22.460]Industrial Agricultural Products Center
[00:07:24.270]and Dr. Mark Wilkins,
[00:07:25.500]and of course my lab.
[00:07:27.180]Thank you and we look forward
[00:07:28.160]to hearing all of the presentations.
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