Siseon Lee

Environmentally friendly pretreatment of plant biomass by planetary and attrition milling.

This study evaluated the use of planetary and attrition milling as pretreatment processes for lignocellulosic biomass using rice straw. Planetary milling reduced the rice straw crystallinity from 0.48 to 0.11. Since the samples could be milled and enzymatically treated using the same media, loss of the biomass due to washing was effectively eliminated. In contrast, conventional sodium hydroxide and soaking in aqueous ammonia (SAA) processes showed a loss of 34.2% and 14.8%, respectively. Furthermore, milling produced significantly lower concentrations of soluble phenolics than the alkali treatments. Using a bioluminescent bioreporter strain that is sensitive to these phenolics, neither of the milled samples elicited a response while the sodium hydroxide and SAA samples led to a 25.8 and 4.7 -fold induction, respectively. Although planetary milling produced more reducing sugars than attrition milling before saccharification, both had similar monosaccharide yields, i.e., 0.38 and 0.34 g/g-biomass, respectively, when 40 g/l rice straw was treated.

Aqueous two-phase system-derived biofilms for bacterial interaction studies.

We describe patterning of bacterial biofilms using polymer-based aqueous two-phase system (ATPS) microprinting protocols. The fully aqueous but selectively bacteria-partitioning nature of the ATPS allows spatially distinct localization of suspensions of bacteria such as Pseudomonas aeruginosa and Escherichia coli with high precision. The ATPS patterned bacterial suspensions form spatially distinct biofilms over time. Due to the fully aqueous and gentle noncontact printing procedures employed, coculture biofilms composed of multiple types of bacteria could be printed not only adjacent to each other but also directly over another layer of existing biofilm. In addition, the ATPS environment also allows free diffusion of small molecules between spatially distinct and localized bacterial suspensions and biofilms. This enables biofilms to chemically affect or be affected by neighboring biofilms or planktonic cells, even if they consist of different strains or species. We show that a β-lactamase producing biofilm confers ampicillin resistance to neighboring nonresistant planktonic cells, as seen by a 3,600-fold increase in survival of the ampicillin-sensitive strain. These examples demonstrate the ability of ATPS-based biofilm patterning methods to enable unique studies on commensalistic effects between bacterial species.

Identification of Escherichia coli biomarkers responsive to various lignin-hydrolysate compounds.

Aberrations in the growth and transcriptome of Escherichia coli str. BL21(DE3) were determined when exposed to varying concentrations of ferulic acid (0.25-1 g/L), an aromatic carboxylic acid identified within lignin-cellulose hydrolysate samples. The expression of several individual genes (aaeA, aaeB, inaA and marA) was significantly induced, i.e., more than 4-fold, and thus these genes and the heat shock response gene htpG were selected as biomarkers to monitor E. colis responses to five additional hydrolysate-related compounds, including vanillic acid, coumaric acid, 4-hydroxybenzoic acid, ferulaldehyde and furfural. While all of the biomarkers showed dose-dependent responses to most of the compounds, expression of aaeA and aaeB showed the greatest induction (5-30-fold) for all compounds tested except furfural. Lastly, the marA, inaA and htpG genes all showed higher expression levels when the culture was exposed to spruce hydrolysate samples, demonstrating the potential use of these genes as biomarkers.

Detection of toxic lignin hydrolysate-related compounds using an inaA::luxCDABE fusion strain

Real-time quantitative PCR analyses of Escherichia coli str. BL21(DE3) exposed to 0.5 g/L ferulic and coumaric acid showed that the inaA gene was significantly induced (7.7- and 3.6-fold higher, respectively). Consequently, a transcriptional fusion of the inaA promoter with the luxCDABE operon was constructed and characterized with several compounds identified within hydrolysates. Tests demonstrated that the phenolics were major inducers, while acetic acid and furfural had only a minor or no effect on the inaA expression respectively. Additional tests with mutant E. coli strains found that a marA partially abolished the response while a marB knock-out led to a 2-3-fold higher basal level expression as evidenced by the bioluminescent levels of the cultures. However, a significant induction was seen even in the marA mutant, suggesting some other control mechanism is involved in regulating inaA expression during an exposure to the hydrolysate compounds. Finally, E. coli str. BL21(DE3)/pSP4 was used to analyze a spruce hydrolysate sample. Real-time quantitative PCR showed a 2.8-fold induction of the inaA expression level while the bioluminescence from the exposed culture was 22-fold higher than the control, demonstrating the possible application of this reporter strain to analyze hydrolysates for the presence of fermentation-inhibiting phenolics.

Sensing of plant hydrolysate-related phenolics with an aaeXAB::luxCDABE bioreporter strain of Escherichia coli.

A bioluminescent Escherichia coli bioreporter strain to detect hydrolysate related phenolics was developed by cloning the aaeXAB promoter from E. coli upstream of the luxCDABE genes. E. coli str. DH5α carrying this plasmid (pDMA3) was responsive to sub-inhibitory concentrations of plant hydrolysate-related phenolics, such as ferulic and vanillic acids, responding to these compounds at concentrations as low as 9.8 and 4.9 mg/L, respectively. Experiments with a mixture of the compounds showed similar responses as with single compound tests, with a minimum detectable concentration of 19.5mg/L. Finally, tests using rice straw hydrolysates were conducted, with E. coli str. DH5α/pDMA3 showing a maximum induction of 33-fold and a minimum detectable phenolic concentration of 9.3mg/L, based upon Folin-Ciocalteus reagent. These results demonstrate that this bioreporter maintains its sensitivity even with hydrolysate samples and that it can be potentially applied within biofuel industries to detect phenolics present within plant hydrolysates.

Serum complement enhances the responses of genotoxin- and oxidative stress-sensitive Escherichia coli bioreporters

Bacterial bioreporters are limited in their abilities to detect large polar molecules due to their membrane selectivity. In this study, the activity of serum complement was used to bypass this undesired selectivity. Initially, the serum complement activity was assessed using the responses of a bacterial bioreporter harboring a recA::luxCDABE transcriptional fusion when exposed to the chemotherapy drug, mitomycin C (MMC). Using 50 °C-treated serum, the limit of detection for this bacterial sensor was lowered by nearly 450-fold, from 31 μg/L to 0.07 μg/L MMC. Real-time quantitative PCR demonstrated that serum-treated cultures responded more strongly to 100 μg/L MMC, with 3.1-fold higher recA expression levels. Subsequent experiments with other bioreporter strains also found enhanced sensitivities and responses. Finally, combining each of the above findings, tests were performed to demonstrate the potential application of the recA::luxCDABE bioreporter within a lab-on-a-CD platform as a point-of-care diagnostic to measure chemotherapeutic drug concentrations within blood.

Improved Sugar Production by Optimizing Planetary Mill Pretreatment and Enzyme Hydrolysis Process

This paper describes an optimization of planetary mill pretreatment and saccharification processes for improving biosugar production. Pitch pine (Pinus rigida) wood sawdust waste was used as biomass feedstock and the process parameters optimized in this study were the buffering media, the milling time, the enzyme quantity, and the incubation time. Glucose yields were improved when acetate buffer was used rather than citrate buffer. Initially, with each process variable tests, the optimal values were 100 minutes of milling, an enzyme concentration of 16 FPU/g-biomass, and a 12-hour enzymatic hydrolysis. Typically, interactions between these experimental conditions and their effects on glucose production were next investigated using RSM. Glucose yields from the Pinus rigida waste exceeded 80% with several of the conditions tested, demonstrating that milling can be used to obtain high levels of glucose bioconversion from woody biomass for biorefinery purposes.