Article Figures & Data
Figures
- FIG 1
Rhodamine B agar screen for lipase activity. Master plates of mixed colonies were generated by soaking soil samples in water and collecting the supernatant to spread on LB plates. Individual plates with growth (A and C) were stamped onto rhodamine B plates (4.0% wt/vol) (B and D) to screen for lipase activity. The presence of orange or yellow halos under 365-nm UV exposure indicates lipase positive colonies (indicated with arrows). After, individual colonies in lipase-positive areas were spotted onto new rhodamine plates to isolate the lipase producers, and positive spots were restreaked onto LB agar for purification.
- FIG 2
Lipase production by strains plated on rhodamine B agar containing olive oil demonstrating lipolytic activity. Lipase negative E coli strain MC4100 (1) was a negative control. Strains 9.1 (2), 9.2 (3), 10 (4), 13.1 (5), and 13.2 (6) were inoculated on LB and olive oil-rhodamine B plates grown for 48 h at 26°C without UV exposure (A and B) and with UV light at 365 nm (C and D). (C) LB plates show natural fluorescence. (D) The rhodamine B plate shows lipolytic activity indicated by white halos for strains 9.2, 10, and 13.2.
- FIG 3
The full consortium grows faster, with higher yields on PET and BHET, than individual consortia or strains. Strains were grown on UV-treated postconsumer 2.0% wt/vol PET (A), 2.0% wt/vol UV-treated granular PET pellets (B), or 0.25% wt/vol BHET (C) as sole carbon sources. All strains were grown overnight in LCFBM supplemented with yeast extract and normalized by OD600 to ensure equal amounts of bacteria were added to all samples. Cultures containing PET were incubated statically, while those with BHET were shaken at 225 rpm at 30°C. Growth was measured every 24 h by an OD600 with a noninoculated sample as a blank. All experiments were performed in triplicate. Error bars indicate standard error.
- FIG 4
Differential growth of isolates on the surface of PET in LCFBM. All isolates were grown in LCFBM supplemented with granular (A) or postconsumer (B) PET for 6 weeks. By standard plate count, the total CFU/g of bacteria in the media were compared by Student's t test to CFU on the surface of substrate (P values < 0.05 were considered significant: *, P < 0.05; **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001). Data are representative of three biological replicates, and means are reported above each bar with standard deviation.
- FIG 5
SEM images show surface modification of amorphous PET plastic. LCFBM containing amorphous PET and the full consortium were grown at 30°C for 40 days. Samples were treated with a mild detergent, saponin (A to C), to remove biofilms or with proteinase K (D to F) to remove remaining, tightly adherent bacteria prior to SEM. (A and D) Samples were uninoculated. Note, Pseudomonas (B) and Bacillus (C) indicated by arrows, and the cavities (E) and groove underlying lysed Bacillus bacteria (F) also indicated by arrows. Bars, 20 μm (A to C) and 5 μM (D to F).
- FIG 6
Secreted enzymes from the Bacillus species efficiently convert BHET to TPA. (A) PET degradation pathway. (B) Enzymes secreted by strains 9.1, 9.2, 10, 13.1, 13.2, and consortium 9, consortium 13, and the full consortium (FC) were added to 0.25% wt/vol BHET. Significant differences between TPA and BHET signals are represented as <0.05 (*), <0.01 (**), and <0.001 (***). The quantity of TPA from BHET cleavage was normalized by the presence of the BHET signal. For the full consortium, BHET was below detectable limits, suggesting there was near-complete conversion to TPA, unlike that observed for the other consortia and strains.
- FIG 7
Infrared spectra of postconsumer PET plastics incubated with lipase positive consortia 9 and 13, strain 10, and E. coli. (A) Averaged ATR-FTIR spectrum acquired of the method blank (Buv) compared with consortium 13 (13uv) to illustrate representative peaks. (B) Comparison of PET difference spectra with UV (dashed line) and without UV (solid line) pretreatment prior to inoculation. Difference spectra (D) are vertically offset for clarity and were produced by spectral subtraction of the method blank (Buv) from the inoculated PET (9uv, 10uv, 13uv, or Euv). Direction of arrows indicates the growth or loss of a peak after incubation, signifying a relative increase or decrease in abundance of that bond, respectively. All spectra were averaged (n = 9) and normalized by peak area to the asymmetric bending mode (δ) at 1,408 cm−1 prior to spectral math.
- FIG 8
Acetaldehyde, methyl, glycol, and carboxylic acid by-products detected from granular PET treated with consortia. Representative 1H NMR spectra from leached granular PET treated with nobacteria (black), full consortium (red), consortium 13 (blue), and consortium 9 (green). Specific regions of interest are highlighted for unique methyl, hydroxyl, aldehyde, and carboxylic acid species. Unique signals are highlighted in red dotted boxes. 1H NMR (DMSO-d6) spectra include 1.04 (t, −CH3), 1.88 (m, −CH2-CH3), 2.12 (d, 3H, acetaldehyde), 2.28 (t, −CH2-OH) 3.26 (m, CH2-OH), 3.43 (m, CH2-OH), 4.28 (m, Ph-CH2-CH2-OH), 9.66 (q, 1H, acetaldehyde), 10.02 (s, −COOH), 10.87 (s, −COOH).
Tables
- TABLE 1
Consortium 9 synergistic lipase activitya
↵a Strain 9.1 B. thuringiensis strain C15 and strain 9.2 Pseudomonas sp. B10 (consortium 9), strains 9.1, 9.2, or strain 10 Pseudomonas sp. SWI36 alone were swabbed from cultures with postconsumer PET as the sole carbon source and then inoculated onto rhodamine B plates with olive oil as a substrate (n = 5). The growth of each strain and the corresponding halo diameters were measured after 48 h. All diameters were quantified in ImageJ with a column average plot across each halo, and the ratio of halo to growth for each strain was compared to the two pseudomonads alone. By standard plate count, approximately equal numbers (∼200 CFU/ml) of strains 9.2 and 9.1, comprising consortium 9, were released from the PET plastic, upon gentle vortexing, after 8 weeks of incubation at room temperature. Strain 9.1 alone was unable to grow using PET as a sole source of carbon.
↵b P values < 0.05 were considered significant.
- TABLE 2
Substrates used for screening for various hydrolytic abilities of consortia strainsa
↵a Short-, medium-, and long-chained substrates were used to assess specificity and promiscuity of esterase and lipase activities among strains. Simple triglyceride tributyrin was used to screen for basic ester cleavage. Tween 20 and Tween 80 were used to screen hydrolytic activities classified between esterases and true lipases. Coconut oil is a medium-chain fatty acid and was used to detect distinctly different lipase and esterase activities than those screened with Tween. Olive oil, a long-chain fatty acid, indicated true lipase activity. Polycaprolactone, a biodegradable polyester, was used to detect polymer hydrolysis/polyurethane esterase activities.
- TABLE 3
Molecular index and confidence intervals calculated by relative intensities of vibrational bands from ATR-FTIR analysis of commercial PET with and without UV pretreatment incubated in carbon-free medium for 6 weeksa
- TABLE 4
1H NMR assignments for unique acetaldehyde, methyl, glycol, and carboxylic acid by-products detected from granular PET treated with consortia
↵a H NMR spectra of leached chemical species from granular PET treated with no bacteria was compared to treatment with the full consortium, consortium 13, and consortium 9. Unique chemical shifts upon bacteria treatment are reported.
↵b For each unique chemical shift, the consortium that generated the signal is reported. C9, consortium 9; C13, consortium, C13; FC, full consortium.
↵c Validated by standard in DMSO-d6.
- TABLE 5
Strain genomes encode hydrolytic enzymes that may be associated with PET degradation
↵a Lipases, esterases, α/β-fold hydrolases, and carboxylesterases were found to be encoded in all isolates. All isolates shared the conserved protein domain EstA; specifically, Bacillus sp. 9.1 and 13.1 encoded a triacylglycerol esterase/lipase, while Pseudomonas strains 9.2, 10, and 13.2 encoded a triacylglycerol lipase-like from the SGNH hydrolases subfamily. Only the Pseudomonas strain 9.2 encoded an acetyl esterase/lipase from the Aes domain. Only Pseudomonas spp. encoded bifunctional outer membrane translocase/extracellular lipase (PlpD) and a predicted carboxylesterase, YpfH, whereas only Bacillus spp. encoded an α/β-fold hydrolase, FrsA. Like EstB in Enterobacter sp. HY1, FrsA has been shown to display esterase activity on pNP-butyrate, which is correlated with BHET cleavage.
↵b Genomes/genes annotated via the IMG suite from the Joint Genome Institute (JGI).
↵c SignalP was used to identify putative signal sequences, indicating secretion.
- TABLE 6
Enzymes associated with ethylene glycol and terephthalic acid metabolism, associated metabolites, and the strains that encode them
↵a Only Pseudomonas 10 and 13.2 encode all genes associated with EG metabolism. Pseudomonas strains 9.2, 10, and 13.2 encode enzymes responsible for TPA metabolism/downstream metabolites protocatechuate (PCA) and catechol (CAT), while neither Bacillus strains encode this capability. EG, ethylene glycol; TPA, terephthalic acid.
TABLE S1
Predicted secretion signals of putative hydrolytic enzymes determined using SignalP 5.0. Download Table S1, PDF file, 0.1 MB.
Copyright © 2020 Roberts et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
TABLE S2
Enzymes encoded in the genomes implicated in ethylene glycol and acetaldehyde metabolism. Download Table S2, PDF file, 0.1 MB.
Copyright © 2020 Roberts et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
FIG S1
BHET is hydrolyzed by secreted enzymes generating ethylene glycol and terephthalic acid. Consortium 9 and the full consortium were grown with BHET substrate as the only carbon source. Native secreted proteins were then collected from filtered culture supernatants. The full collection of secreted proteins were incubated with fresh BHET substrate. Resulting insoluble degradation products were analyzed by 1H NMR in DMSO-d6 for comparison to an untreated control. A glycol methylene triplet of BHET (3.73 ppm) was used as the reference peak for 4 hydrogens. BHET uninoculated (A), BHET treated with secreted proteins from consortium 9 (B), and the full consortium (C). Note peak at 13.28 ppm (1H, s, TPA carboxylic acid) absence in the uninoculated control (A), with increasing intensity when inoculated with consortium 9 (B) and then the full consortium (C). Download FIG S1, PDF file, 1.3 MB.
Copyright © 2020 Roberts et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
