Develop Rhodococcus opacus and Pseudomonas putida into platforms for polyhdroxyalkanoates and lipids production
Abstract
Many microorganisms can accumulate carbon storage molecules when the carbon source is in excess and nitrogen is limited. Specifically, Rhodococcus opacus can accumulate triacylglycerols (TAGs) as the storage carbon, whereas Pseudomonas putida can accumulate polyhydroxyalkanoates (PHAs) as the storage carbon under the C/N imbalance conditions. TAGs can be an alternative for the fossil fuel, while the PHAs have potential to substitute the petroleum-derived plastics in the future. The aim of my research is to lower the cost of lipids and PHAs production, via reducing the harvesting cost, enhancing the conversion efficiency of waste streams to lipids and PHAs in these two different strains. In Chapter 2, I focused on developing R. opacus into the platform for TAGs production and lowering the capital and operational cost by advancing pelletized cultivation of the R. opacus on lignin stream, simplifying the harvest and increasing lipid yield. In Chapter 3 and Chapter 4, I focused on developing P.putida into the platform for PHAs production and lower bioplastics manufacturing cost by using the inexpensive carbon source and increasing the PHAs content by the rational metabolic engineering of PHA biosynthesis pathway.
In Chapter 2, I discovered that a non-filamentous bacterium R. opacus PD630 could form the pellets during the fermentation utilizing the alkaline pretreatment liquor (APL) containing lignin as the carbon source. This discovery renewed our understanding of the bacterium pelletization, considering that only filamentous fungi and filamentous bacteria were reported to form the pellets without addition of external agents such as flocculants or polymers in the previous research. The study also opened new avenues to decrease harvesting and cultivation cost and energy consumption for microbial fermentation. Several factors were investigated to understand how they affect the process of pelletization. Although nitrogen concentration was not found to affect the pelletization, other factors such as shaking speed and initial OD and carbon source had an impact on the pelletization. The lipid content in the pellets was much higher than in the scattered bacteria at low nitrogen concentration(<0.5g/L) under which condition (high C/N ratio) the industrial microbial production for lipids was carried out. Overall, the highest pellet percentage (⁓60% of the total biomass) was observed at 30 g/L soluble solid content (SSC), 180 rpm, 1.4 g/L (NH4)2SO4, 10 initial OD600 (optical density) and 6000 rpm of centrifugation speed.
In Chapter 3 and Chapter 4, I developed P.putida KT2440 into the platform for PHAs production from industrial waste, including the carboxylic acid derived from municipal waste and waste cooking oil. The strategies were expected to lower the price of PHAs and realize the ultimate goal to substitute the traditional plastic with biodegradable ones. The objectivity of this study is to overcome the current plastics challenge with waste utilization to enable our society more sustainable and environmentally-friendly. In Chapter 3, a group of low-cost carboxylic acids known as volatile fatty acids (VFAs) produced in the anerobic fermentation were utilized as the carbon source for P.putida. VFAs are major products of carboxylic platform established to convert a broad range of waste, including food waste and sludge to value-added products. I found that P.putida could utilize VFAs (acetate, propionate, and butyrate) well for cell growth. However, the PHA content varies depending on the carbon source, where the PHA accumulated in P.putida on the acetate was low as 22.5% and the PHA content could be up to 25.9% and 39.0% on propionate and butyrate respectively. I successfully increased the PHA content to 29.1% and sustained the PHA content of P.putida during fermentation by deleting phaZ, which encodes PHA depolymerase and fadA, fadB which involved in the fatty acid β-oxidation. The triple mutant strain ∆Z∆fadA∆fadB could also show similar performance on the waste organic acid. Ultimately, the engineered strain improved the conversion of organic acids derived from carboxylic anaerobic digestion (AD) conversion of waste. Likewise, in Chapter 4, I tried to convert another low-cost carbon source, waste cooking oil (WCO), into value-added PHA, overcoming WCO’s environmental impact on landfills. The long lag phase of P.putida in waste oil hydrolysates was eliminated by deleting glycerol metabolism repressor and the PHA content increased by 39.6% at 24 hours through engineering PHA synthesis.
In summary, my research provided several promising strategies including novel cell harvest method and metabolic engineering to lower the cost of the lipids and PHAs manufacturing, synergizing the cost-effective waste management with addressing plastics and biofuel challenges.
Citation
Xu, Bing (2021). Develop Rhodococcus opacus and Pseudomonas putida into platforms for polyhdroxyalkanoates and lipids production. Doctoral dissertation, Texas A&M University. Available electronically from https : / /hdl .handle .net /1969 .1 /195345.