Physiological changes in Rhodococcus ruber S103 immobilized on biobooms using low-cost media enhance stress tolerance and crude oil-degrading activity

For economic feasibility, sugarcane molasses (0.5%, w/v) containing K2HPO4 (0.26%, w/v) and mature coconut water, low value byproducts, were used in cultivation of Rhodococcus ruber S103 for inoculum production and immobilization, respectively. Physiological changes of S103 grown in low-cost media, including cell hydrophobicity, saturated/unsaturated ratio of cellular fatty acids and biofilm formation activity, enhanced stress tolerance and crude oil biodegradation in freshwater and even under high salinity (5%, w/v). Biobooms comprised of S103 immobilized on polyurethane foam (PUF) was achieved with high biomass content (1010 colony-forming units g−1 PUF) via a scale-up process in a 5-L modified fluidized-bed bioreactor within 3 days. In a 500-L mesocosm, natural freshwater was spiked with crude oil (72 g or 667 mg g−1 dry biobooms), and a simulated wave was applied. Biobooms could remove 100% of crude oil within only 3 days and simultaneously biodegraded 60% of the adsorbed oil after 7 days when compared to boom control with indigenous bacteria. In addition, biobooms had a long shelf-life (at least 100 days) with high biodegradation activity (85.2 ± 2.3%) after storage in 10% (w/v) skimmed milk at room temperature. This study demonstrates that the low-cost production of biobooms has potential for future commercial bioremediation.


S1
Text S1: Arabian light (AL) crude oil source and properties Arabian light (AL) crude oil was kindly provided by the Thai Oil Public Company Limited (Thailand). The viscosity, density and API gravity were 13 cP, 0.86 g cm -3 and 31° API, respectively 1 . The composition was a mixture of 40% saturated hydrocarbons, 26% aromatic hydrocarbons, 24% resins and 10% asphaltenes, which was analyzed by thin-layer chromatography with the flame ionization method (Iatron Labs, Tokyo, Japan) according to Nopcharoenkul,et al. 2 .

Text S2: Selection of low-cost media
The growth characteristics of Rhodococcus ruber S103 were preliminarily determined in different concentrations of molasses and mature coconut water (CW) using 96-well plates at room temperature (30-33 ºC). The inoculum (20 µL) was added to 180 µL of the tested media to obtain an initial cell concentration of 10 7 CFU mL -1 . After incubation, the growth of S103 was measured for optical density at 540 nm using a multimode plate reader (PerkinElmer, Finland). S103 grew well in 100%CW, 0.25xLB and 0.5-1.0%M, respectively (Fig. S2). The lowest growth of S103 was obtained from 0.5-1.0%M since this condition might be an unfavorable pH condition. S103 preferred the optimum pH for growth as neutral to mildly alkaline conditions (Fig. S3). This strain could not grow in 100%CW (pH ~5) without pH adjustment (data not shown). The pH of the sterilized CW could be maintained after adjustment with NaOH to obtain pH 7.0, while the pH of molasses dissolved in distilled water still changed after sterilization (121 ºC, 15 min). Saejung and Puensungnern 3 observed a drastic pH drop from 6.8 (initial pH) to 3.4 in the culture broth when molasses concentrations ranged from 2 to 10 g L -1 , at which buffering action was needed. Thus, low-cost inorganic phosphate salts were added to maintain a pH of 0.5%M. The growth of S103 was promoted in 0.5%M added 0.26% (w/v) K2HPO4, named as 0.5%MK, when compared to other phosphate salts, and the pH was S2 adjusted with 1 M NaOH before and after sterilization (Fig. S4). It is important to note that the use of only raw materials should be considered pH control, especially to scale up biomass production. In this study, 100%CW and 0.5%MK were selected for further study prior to being used as alternative media.

Text S3: Crude oil adsorption capacity
The oil adsorption capacity was determined using a slightly modified method from Songsaeng,et al. 4 . Briefly, 2 g crude oil was added into 30 mL of freshwater. The PUF cubes (1x1x1 cm 3 ) were weighed before their immersion into the oil-freshwater system at room temperature. After 20 min, the PUF was removed from the system. The excess oil on the PUF surface was removed, and the PUF were weighed. All experiments were performed in triplicate.
The adsorption capacity was calculated as follows: adsorption capacity (g g -1 ) = (W1-W0)/W0, where W0 and W1 represent the weight of before and after adsorption of PUF, respectively.

References
Remark: S1-S4, sampling points from surface water, *Sum of remaining crude oil detected from the surface area. 1, 2 and 3: the code of each boom and bioboom, *Average of the remaining crude oil detected from the boom and bioboom in triplicate.

Fig. S1
Actual experimental tanks of the simulated freshwater environments, natural attenuation, control and bioaugmentation under natural conditions used in this study (a).
Schematic representation of crude oil remediation in mesocosm tanks (control and bioaugmentation) (b).

Fig. S2
Growth characteristics of R. ruber S103 in sterilized molasses (pH ~5.8) and mature coconut water (pH ~7). Preculture of S103 was prepared in the same medium. Growth was performed in 96-well plates at room temperature for 24 h and determined by measuring the optical density at 540 nm. The lowercase letters above the vertical bars represent significant differences in the culture media (P < 0.05).

Fig. S3
Growth characteristics of R. ruber S103 determined by the most probable number (MPN) in LB medium (pH 4-12) at room temperature for 7 days. The different letters on the same day represent significant differences in pH (P < 0.05).

Fig. S4
Growth characteristics of R. ruber S103 in sterilized molasses (pH ~5.8) and sterilized molasses with inorganic phosphate salts (0.56% Na2HPO4·12H2O, 0.05% KH2PO4 and 0.26% K2HPO4). Preculture of S103 was prepared in the same medium. Growth was performed in 96well plates at room temperature for 24 h and determined by measuring the optical density at 540 nm. The lowercase letters above the vertical bars represent significant differences in the culture media (P < 0.05).

Fig. S5
A typical time course of growth of R. ruber S103 in 0.25xLB and low-cost media, 0.5%MK and 100%CW. Growth conditions were performed in 250-mL Erlenmeyer flasks containing 120 mL of medium at room temperature and 200 rpm for 120 h. The different letters on the same day represent significant differences at P < 0.05.

Fig. S6
Biodegradation efficiency of crude oil (a) and bacterial numbers (b) of R. ruber S103 in CFMM medium containing various concentrations of AL crude oil at room temperature and 200 rpm for 7 days. A preculture was grown in 0.25xLB. The lowercase letters above the vertical bars represent significant differences in the crude oil concentration (P < 0.05).

Fig. S7
A scanning electron micrograph at different magnifications, 250x (a, e and i), 2,500x (b, f and j), 15,000x (c, g and k) and 20,000x (d, h and l) of S103 immobilized on PUF in 100%CW at room temperature and 200 rpm for 1 (a-d), 3 (e-h) and 5 (i-l) days.

Fig. S8
Removal and biodegradation efficiency of crude oil by biobooms in CFMM with 0-5% (w/v) NaCl and 2,500 mg L -1 AL crude oil at room temperature with shaking at 200 rpm for 3 days. The lowercase letters above the vertical bars represent significant differences in NaCl concentrations (P < 0.05).