1 Phosphorus Depletion as a Green Alternative to Biocides for

2 Controlling Biodegradation of Metalworking Fluids

3 Yaldah Azimi, Ian P. Thompson*

4 Department of Engineering Science, University of Oxford, Parks Road, Oxford, United Kingdom OX1

5 3PJ

6 * ian.thompson@eng.ox.ac.uk

8 This supporting information (SI) document contains 4 pages (including cover page),

9 including no figures or tables, and includes the following information:

10 • Description of method for obtaining growth curves

11 • Description of micro-respiration experimental method

12 • Method for comparing toxicity of different chemicals using biosensor Acinetobacter

13 baylyi ADP1_recA_lux.

14 • Method for comparing toxicity of different chemicals using biosensor Escherichia

15 coli HB101_pUCD607_lux.

16 • Preparation procedure of Agrobacterium radiobacter culture for Raman

17 Spectroscopy. Sample preparation method for SEM.

18

19

20

21

22

23

24

S1
25 Optical density measures for determining microbial consortia growth curves

26 Growth of the microbial consortia upon exposure to various concentrations of

27 phosphate was measured. In each well of a 96-well plate 200 µL of solution consisting of 20

28 µL of resuscitated bacterial consortia (with an average concentration of 2.8 x 109 CFU/mL),

29 50 µL test compound solution, 130 µL feed (5g/L glucose, and 815 mg/L NH4Cl) were

30 added. Synergy HT Multi-detection Microplate Reader (Bio-Tek, UK) was used to take

31 absorbance measurements from each well at 630 nm at 10 min intervals over 18 h.

32 Microbial consortia respiration and post-exposure recovery

33 A 96-well block (Corning-High Wycombe, UK) was prepared with each well containing 450

34 µL of solution containing 100 µL milliQ water, 50 µL resuscitated bacterial consortia (with

35 an average concentration of 2.8 x 109 CFU/mL), 50 µL of test compounds at various

36 concentrations, and 250 µL of feed (5g/L glucose, and 815 mg/L NH4Cl). A 96-well plate

37 containing the indicator dye in gel form was placed on top of the block, sealed by silicon

38 rubber and left in a shaking incubator at 20 °C and 120 rpm for 20 h. Note that the results are

39 presented as % control and control wells contained 50 µL distilled water (as test compound).

40 Toxicity measurements using biosensor Acinetobacter baylyi ADP1_recA_lux

41 One colony was inoculated into 5 mL of sterile lysogeny broth-Miller with 10 µg/L

42 kanamycin (Sigma Aldrich) (from here on referred to as LBK10) and incubated at 26 °C for

43 14-16 h at 120 rpm until cells reached mid-exponential growth phase. The cells of the

44 overnight culture were harvested by centrifugation at 3000 rpm for 10 min and resuspended

45 in 5 mL of fresh LBK10. Fifty µL of test compounds (La2O3, La(NO3)3, and formaldehyde of

46 various concentrations) and 150 µL of the cell suspension was then transferred into each well

47 of a 96-well microplate (black, optically clear bottom, Corning). Bioluminescence and optical

48 density (at 630nm) were measured immediately after sample preparation using a Synergy HT

S2
49 Multi-Detection Microplate Reader (Bio-Tek, UK) at 10-min intervals for 6 h. All sample

50 assessments were carried out with 3-12 replicates. Relative bioluminescence (absolute

51 intensity divided by optical density) was calculated for each sample and used as an estimate

52 of the severity of DNA damage of the whole cell bioreporter. Ethidium bromide and

53 Mitomycin C were used as positive control for this experiment.

54 Toxicity measurements using biosensor Escherichia coli HB101_pUCD607_lux

55 One colony was inoculated in 5 mL of sterile lysogeny broth-Miller with 50 µg/mL

56 ampicillin, and incubated at 30 °C and 150 rpm agitation for 8 h until the cells reached mid-

57 exponential growth phase. The culture was then centrifuged at 3000 rpm at 4 °C. The cell

58 pellet was then re-suspended in 5 mL of the prepared broth (i.e. lysogeny broth-Miller with

59 50 µg/mL ampicillin). In each well of 96-well microplate (black, optically clear bottom,

60 Corning), 150 µL of biosensor and LBAMP and 50 µL of test compound was placed.

61 Bioluminescence was measured using Synergy HT Multi-Detection Microplate Reader at

62 time 0 and after 30 minutes of being placed in a shaking incubator at 30 °C at 120 rpm. With

63 this biosensor, lower bioluminescence is associated with toxicity related to metabolic activity.

64 Preparation of Agrobacterium radiobacter for Raman Spectroscopy

65 One colony was inoculated into 5 mL of Luria-Bertani (LB) broth with 5 g/L D-glucose

66 anhydrous (Fisher Scientific), and incubated at 27 °C for 14-16 h on a rotary shaker of 150

67 rpm. The cells were harvested by centrifugation at 3000 rpm for 5 min and resuspended in 5

68 mL of distilled water.

69 Scanning electron microscopy (SEM) detailed procedure

70 The glass coupons coated with microorganisms were treated with 1% osmium tetroxide for

71 30 minutes, and then dehydrated using ethanol in the following concertations: 30%, 50%,

72 70%, and 90% allowing for 10 minutes between each step. Samples were then dehydrated
S3
73 three times with absolute ethanol, with 20 minute intervals between each wash. After

74 dehydration, samples were immersed in hexamethyldisilazane (Sigma-Aldrich), for three

75 minutes and then left to dry for 2 h. Gold coating of the surfaces was conducted using a

76 Q150R ES sputter coater under 20mA for 120 s.

S4