Environmental Science: Processes & Impacts

Mandelic Acid and Phenyllactic Acid “Reaction Sets” for Exploring the Kinetics and Mechanism of Oxidations by Hydrous Manganese Oxide (HMO)

Journal: Environmental Science: Processes & Impacts

Manuscript ID EM-ART-03-2019-000128.R1

Article Type: Paper

Date Submitted by the 30-Apr-2019 Author:

Complete List of Authors: Xia, Xiaomeng; Johns Hopkins University - Homewood Campus, Environmental Health and Engineering Stone, Alan; Johns Hopkins University - Homewood Campus, Environmental Health and Engineering

Page 1 of 41 Environmental Science: Processes & Impacts

1 2 3 4 1 Mandelic Acid and Phenyllactic Acid “Reaction Sets” for Exploring 5 6 7 2 the Kinetics and Mechanism of Oxidations by Hydrous Manganese 8 9 10 3 Oxide (HMO) 11 12 13 4 Xiaomeng Xia, Alan T. Stone 14 15 16 17 5 Department of Environmental Health and Engineering 18 6 19 7 Whiting School of Engineering 20 8 21 9 Johns Hopkins University 22 10 23 11 Baltimore, MD 21218 24 25 12 26 13 Submitted to: Environmental Science: Processes & Impacts, February 11, 2019 27 14 28 29 30 15 31 32 33 34 16 35 36 37 17 Graphical Abstract 38 39 40 41 Two hydroxy acids reaction sets are used to explore the kinetics, the 42 role of physicochemical properties, and medium effects of the 43 44 heterogenerious reactions on hydrous manganese oxide (HMO) 45 surface. O 46 O C 47 + - - * + - - 2H - 2e C O + 2H2O - H2CO3 -2H -2e 48 O 49 R - k O O 50 HO H C k1 3 C C O- 51 k k2 O 4 R 52 R CH 53 + - + H O - H CO * - H+ - 2e- R + H2O - 3H - 2e 54 18 2 2 3 55 56 57 58 1 59 60 Environmental Science: Processes & Impacts Page 2 of 41

1 2 3 20 Abstract 4 5 6 7 21 At pH 4.0, hydrous manganese oxide (HMO) oxidizes mandelic acid by two mole- 8 9 22 equivalents of electrons, yielding phenylglyoxylic acid and benzaldehyde. These intermediates, 10 11 23 in turn, are oxidized by two mole-equivalents of electrons to the same ultimate oxidation 12 13 14 24 product, benzoic acid. The four compounds of the "reaction set" just described are conveniently 15 16 25 monitored using capillary electrophoresis (CE) and HPLC. Extents of adsorption are negligible 17 18 26 and their sum exhibits mass balance. Concentrations of phenylglyoxylic acid, benzaldehyde, and 19 20 27 benzoic acid can therefore be used to calculate mole-equivalents delivered to HMO for 21 22 II 23 28 comparison with experimentally-determined dissolved Mn concentrations. Semi-log plots 24 25 29 (ln[substrate] versus time) and numerical analysis can also be used to explore rates of oxidation 26 27 30 of the functional groups represented, i.e. an -hydroxycarboxylic acid, an -ketocarboxylic acid, 28 29 30 31 and an aldehyde. Inserting a -CH2- group between the benzene ring and the functional groups 31 32 32 just described yields a new reaction set comprised of phenyllactic acid, phenylpyruvic acid, and 33 34 33 phenylacetaldehyde, plus the C-1 ultimate oxidation product, phenylacetic acid. At pH 4, mass 35 36 37 34 balance for phenyllactic acid oxidation fell short by ~ 9 %. Phenyllactic acid was oxidized 2.7- 38 39 35 times more slowly than mandelic acid, while phenylpyruvic acid was oxidized 12.7-times faster 40 41 36 than phenylglyoxylic acid. Unlike benzaldehyde, oxidation rates for phenylacetaldehyde were 42 43 44 37 too fast to measure. Under pH 4.0 conditions, this reaction set approach was used to explore the 45 46 38 acceleratory effects of increases in HMO loading and inhibitory effects of 500 M phosphate 47 48 39 and pyrophosphate additions. Mandelic acid and phenyllactic acid were oxidized by HMO far 49 50 51 40 more slowly at pH 7.0 than at pH 4.0. At pH 7.0, 2 mM MOPS and phosphate buffers did not 52 53 41 yield appreciable dissolved MnII, despite oxidation of organic substrate. 2 mM Pyrophosphate, in 54 55 42 contrast, solubilized HMO-bound MnII and MnIII. 56 57 58 2 59 60 Page 3 of 41 Environmental Science: Processes & Impacts

1 2 3 43 Environmental significance 4 5 6 44 Within surface waters, soils, and sediments, manganese oxyhydroxides likely serve as 7 8 45 reactive sinks for siderophores and other natural products that contain -hydroxycarboxylic acid, 9 10 11 46 -ketocarboxylic acid, and aldehyde functional groups. These aliphatic functional groups also 12 13 47 likely contribute to the reductant capacity and reactivity of natural organic matter (NOM). 14 15 Within water supply plants, HMO generated through permanganate reduction or through MnII 16 48 17 18 49 oxidation by chlorine, chloramines, ozone, and other disinfectants may convert - 19 20 50 hydroxycarboxylic acids into -ketocarboxylic acids and aldehydes, and -ketocarboxylic acids 21 22 23 51 and aldehydes into carboxylic acids. By altering the suite of organic compounds present, 24 25 52 identities and amounts of disinfection byproducts generated by downstream processes may be 26 27 53 altered. 28 29 30 54 31 32 55 1. Introduction 33 34 35 56 Manganese oxyhydroxides are important transitional redox zone oxidants, found within 36 37 38 57 the water column of stratified lakes, inlets, and seas, as nodules, dendrites, and crusts within 39 40 58 soils, and as nodules and crusts at sediment-water interfaces.1-3 In water supply plants, 41 42 59 manganese oxyhydroxides are formed when O2, permanganate, and commonly-employed 43 44 60 disinfectants (e.g., chlorine, chloramines, ozone) oxidize Mn2+.4-6 Many organic compounds are 45 46 47 61 oxidized rapidly by manganese oxyhydroxides. Although a great deal is known about oxidations 48 49 62 of phenol- and aniline-bearing contaminants, relatively little is known about oxidations of 50 51 63 compounds bearing aliphatic reductant groups.7 52 53 54 55 56 57 58 3 59 60 Environmental Science: Processes & Impacts Page 4 of 41

1 2 3 64 To learn more about the reactivities of important aliphatic reductant groups with 4 5 6 65 manganese oxyhydroxides, we have selected two "reaction sets" depicted in Figure 1. Each 7 8 66 contributing compound possesses a benzene ring that facilitates UV detection, but is not itself 9 10 67 oxidized or reduced by manganese oxyhydroxides. Compounds in the phenyllactic acid reaction 11 12 set possess a -CH - group located between the benzene ring and the oxidizable moieties, while 13 68 2 14 15 69 those in the mandelic acid reaction set do not. With each set we will quantify hydroxy acid, keto 16 17 70 acid, and C-1 acid by capillary electrophoresis, and aldehyde by HPLC. 18 19 20 71 The eight organic compounds comprising the two reaction sets are representative of a 21 22 72 great many other organic compounds with widespread occurrence and considerable 23 24 25 73 environmental significance, briefly outlined in Electronic Supplementary Information (S1). The 26 27 74 electrophilicity of keto acids and aldehydes is particularly noteworthy. It has been established 28 29 75 that pyruvic acid, oxaloacetic acid, and -ketoglutaric acid protect cell cultures from in situ 30 31 32 76 generated hydrogen peroxide by forming adducts that irreversibly break down into the 33 34 77 corresponding C-1 acid and bicarbonate ion.8-13 Comparable reactions have been confirmed for 35 36 78 phenylglyoxylic acid14 and phenylpyruvic acid.15 Keto acid groups generated through oxidation 37 38 39 79 of hydroxy acids within natural organic matter (NOM) may similarly serve as sinks for hydrogen 40 41 80 peroxide. Aldehydes like benzaldehyde and phenylacetaldehyde, do not serve in a protective 42 43 81 role, but instead may be toxic.16 44 45 46 82 Electronic effects arising from the phenyl or benzyl groups of the compounds shown in 47 48 Figure 1 will be addressed in this work. Many cured-in-place pipe (CIPP) formulations are 49 83 50 51 84 styrene-based. The two styrene oxidation products included in our study, benzaldehyde and 52 53 85 benzoic acid, have been associated with CIPP installation.17, 18 4-Hydroxy-3-methoxymandelic 54 55 86 acid has been detected during lignin degradation by white rot fungus.19, 20 (4-Hydroxy-3- 56 57 58 4 59 60 Page 5 of 41 Environmental Science: Processes & Impacts

1 2 3 87 methoxyphenyl)-glyoxylic acid is generated during the polysulfide treatment of lignin.21 These 4 5 6 88 two examples from the wood, pulp, and paper literature should not come as a surprise, given that 7 8 89 lignin is the product of biologically controlled radical coupling of the hydroxy-/methoxy- 9 10 90 substituted styrenes coniferyl alcohol, sinapyl alcohol, and coumaryl alcohol.22, 23 11 12 13 91 Our objective is to show how reaction set organic substrates can be used to explore the 14 15 92 kinetics of heterogeneous redox reactions. The hydrous manganese oxide (HMO) that we use as 16 17 2+ - 18 93 oxidant, formed from the oxidation of Mn by MnO4 , is typical of the low crystallinity -MnO2 19 20 94 found in soils, sediments, and water supply plants. Simultaneous monitoring of hydroxy acid, 21 22 23 95 keto acid, aldehyde, and C-1 acid concentrations as a function of time is a central feature of this 24 25 96 work. As noted in Figure 1, electron mole-equivalents transferred in each of the four reaction 26 27 97 steps are known. If we can demonstrate that these compounds do not adsorb significantly to 28 29 98 manganese oxyhydroxides and that mass balance involving the four compounds in each set is 30 31 32 99 achieved, then we have the means of determining electron mole-equivalents delivered to 33 34 100 manganese oxyhydroxides. We will also employ semi-log plots (ln[substrate] versus time) and 35 36 101 numerical analysis to obtain rates and rate constants for the four reaction steps. This provides a 37 38 39 102 basis for exploring medium effects, e.g. effects of added phosphate and pyrophosphate and 40 41 103 changes to suspension pH. Comparing results of the mandelic acid reaction set with those of the 42 43 104 phenyllactic acid reaction set will shed light on electronic effects arising from the aromatic ring. 44 45 46 105 47 48 49 106 2. Materials and Methods 50 51 52 107 All solutions and suspensions were prepared using 18 MΩ•cm resistivity distilled, 53 54 108 deionized water (DDW, Millipore Corp., Milford, MA). All bottles and glassware were rinsed 55 56 57 58 5 59 60 Environmental Science: Processes & Impacts Page 6 of 41

1 2 3 109 with distilled water, soaked in 5 M HNO3 (J. T. Baker, Phillipsburg, NJ) overnight, rinsed with 4 5 6 110 distilled water, and then rinsed with DDW and air dried prior to use. Glassware, bottles, and 7 8 111 filter holders having prior contact with HMO solutions were first soaked in 1 M ascorbic acid 9 10 112 (Aldrich, Milwaukee, WI) and were rinsed several times with DDW prior to soaking in 5 M 11 12 HNO . 13 113 3 14 15 114 2.1. Chemicals 16 17 18 115 All chemical reagents were of the highest purity available. Mandelic acid was obtained 19 20 21 116 from Acros Organics (NJ) with the purity of 99%. Phenylglyoxylic acid with the purity of 95%, 22 23 117 benzoic acid with the purity of 99.5%, benzaldehyde with the purity of 99+%, phenyllactic acid 24 25 118 with the purity of 98+%, phenylpyruvic acid with the purity of 98%, and phenylacetic acid with 26 27 28 119 the purity of 99% were purchased from Sigma-Aldrich (St. Louis, MO). Phenylacetaldehyde was 29 30 120 purchased from Alfa Aesar with the putiry of 95%. Ionic strength was adjusted using NaCl 31 32 121 (Acros Organics). Sodium hydroxide, acetic acid, hydrochloric acid, and sodium acetate were 33 34 35 122 purchased from J. T. Baker and used to buffer pH. These buffers were selected because they are 36 37 123 poorly oxidized by HMO and complex metals weakly, and therefore do not consume HMO or 38 39 124 influence Mn speciation during the reactions. 3-[N-morpholino] propanesulfonic acid (MOPS, 40 41 125 Sigma-Aldrich), Na HPO (J. T. Baker), and Na H P O (Sigma-Aldrich) were used as buffers at 42 2 4 2 2 2 7 43 44 126 pH 7.0. 45 46 47 127 2.2. HMO Synthesis and Characterization 48 49 -3 50 128 HMO (hydrous manganese oxide) synthesis began by heating a 4.0 × 10 M NaMnO4 51 52 129 (Sigma-Aldrich) and 1.0 × 10-2 M NaOH (J. T. Baker) solution (0.5 L) to 90 oC in a 1 L glass 53 54 -3 130 Erlenmeyer flask. A 6.5 × 10 M MnCl2.4H2O (Sigma-Aldrich) solution (0.5 L) was then added 55 56 57 58 6 59 60 Page 7 of 41 Environmental Science: Processes & Impacts

1 2 3 131 dropwise by manually adjusting the stopcock of a separatory funnel. After addition was 4 5 6 132 complete, the suspension was maintained at 90 °C for an additional two hours, and then allowed 7 8 133 to cool to room temperature. Eight different preparations supplied the HMO for our experiments 9 10 134 (Table S1). To minimize the effect of particle aging, each preparation was used within two 11 12 weeks of synthesis. We used the same reagents as Murray24, and similar concentrations. We 13 135 14 15 136 elected, however, to add a slight stoichiometric excess of MnII, and did not use 16 17 137 dilution/resuspension to lower the ionic strength of the suspensions produced. Most importantly, 18 19 138 synthesis and early aging were performed at 90 °C, while Murray employed room temperature. 20 21 22 139 To determine the total concentration of HMO in the suspension, 100 mL of HMO 23 24 25 140 suspension was transferred to a polypropylene bottle and reduced by an excess amount of 26 27 141 ascorbic acid, then the total dissolved Mn was measured using flame atomic absorption 28 29 142 spectrophotometry (AAS, AAnalyst 100, Perkin-Elmer, Norwalk, CT). Since dissolved Mn of 30 31 32 143 filtered HMO suspension was consistently below the detection limit of 1 µM, the total dissolved 33 34 144 Mn concentration measured after ascorbic acid reduction is equal to the concentration of HMO. 35 36 145 The average oxidation state of manganese was determined by iodometric titration for two of the 37 38 146 HMO preparations.25 10 mL of HMO suspension was transferred to a 50 mL Erlenmeyer flask, 39 40 41 147 and reduced by an excess amount of KI (Fisher Chemical, Pittsburgh, PA) in 0.01 M HCl 42 - 43 148 solution. When the solution became a brown I3 solution, the flask was wrapped with aluminum 44 45 149 foil and incubated in the dark for 2 minutes. The brown I - solution was back-titrated using 0.01 46 3 47 48 150 M Na2S2O3 solution (Fisher Chemical). Right before the end point was reached, several drops of 49 50 151 soluble starch indicator (Fisher Chemical) was added, yielding a blue color. The titration was 51 52 152 continued, with the endpoint corresponding to the point where the suspension became colorless. 53 54 55 153 The titration was repeated twice for each HMO suspension. For both HMO suspensions tested, 56 57 58 7 59 60 Environmental Science: Processes & Impacts Page 8 of 41

1 2 3 154 the average oxidation state was found to be +3.82. If we assume that MnII is not present, then this 4 5 III IV 6 155 average oxidation state corresponds to 18 % Mn and 82 % Mn . 7 8 9 156 2.3. Experimental Design 10 11 12 157 All experiments were conducted in 100 mL polypropylene bottles to prevent adsorption 13 14 158 of metal ions to reactor surfaces. All reactions were conducted in a constant temperature water 15 16 159 bath at 25 ºC ± 0.1 ºC and stirred with Teflon-coated stir bars. Constant pH was maintained using 17 18 160 5 mM acetate (pH 4.0), MOPS (pH 7.0), phosphate (pH 7.0), or pyrophosphate (pH 7.0) buffer, 19 20 21 161 and constant ionic strength was maintained using 10 mM NaCl. Stability of solution pH was 22 23 162 verified by periodic measurement (Fisher Accumet 825MP meter with Orion Combination semi- 24 25 163 micro probe; NIST-traceable standards). 26 27 28 164 The reaction solutions were prepared by first mixing together pH buffer, electrolyte, and 29 30 31 165 organic substrate from aqueous stock solutions. No efforts were taken to exclude molecular 32 33 166 oxygen from the reaction suspension. The reactions were initiated by adding HMO. The initial 34 35 167 concentration of organic substrate in most kinetic experiments was 50 μM, and HMO was 36 37 present in considerable excess (0.5 mM). 4mL reaction suspension aliquots were collected at 38 168 39 40 169 periodic intervals as the reactions proceeded. Filtration using 0.05 m pore diameter track-etched 41 42 170 polycarbonate membranes (Whatman) was used to quantitatively remove HMO particles, 43 44 45 171 quenching the reaction. Filtered solutions were analyzed by AAS for total Mn and by capillary 46 47 172 electrophoresis (CE) for organic species (see later section). As noted before, when filtered, 48 49 173 dissolved Mn in HMO suspensions lacking organic substrates was consistently below this 50 51 174 detection limit. Organic substrates and intermediates reduce HMO to MnII, but are not strong 52 53 III IV 54 175 enough chelating agents to solubilize Mn or Mn . We can therefore assume that dissolved 55 56 57 58 8 59 60 Page 9 of 41 Environmental Science: Processes & Impacts

1 2 3 176 manganese consists entirely of MnII. Medium containing pyrophosphate is an important 4 5 III 26-29 6 177 exception, owing to the ability of pyrophosphate to chelate and solubilize Mn . 7 8 9 178 2.4. Organic Analysis 10 11 12 179 A CE system (Sciex, P/ACE MDQ) with diode-array UV-visible detector was used for 13 14 180 analyzing ionized organic compounds. Bare fused silica capillaries (Polymicro Technologies, 15 16 181 Phoenix, AZ) with 75 μm ID and 60 cm total length were used for all separations. The effective 17 18 182 length defined as the length from the inlet to the detector was 52 cm. NaH PO .H O (J. T. 19 2 4 2 20 21 183 Baker), Na2HPO4 (J. T. Baker), and “electroosmotic flow modifier” 22 23 184 tetradecyltrimethylammonium chloride (TTAC) (Sigma-Aldrich) were used in the back ground 24 25 185 electrolyte (BGE) buffer. BGE buffer contained 25 mM phosphate (pH 7.0), and 0.5 mM TTAC. 26 27 28 186 Between separations, the capillary was sequentially rinsed by pressure flushing DDW for 1 29 30 187 minute, 0.1 M NaOH for 1 minute, DDW for 1 minute, and BGE buffer for 2 minutes. Anion 31 32 188 mode with constant applied voltage (-25 kV) was employed for all separations. Sample injection 33 34 35 189 employed 0.5 psi of positive pressure for 15 seconds. All analyses were conducted at 25 ºC in the 36 37 190 constant temperature chamber within the instrument. Data were collected at a detection 38 39 191 wavelength of 194 nm. Since peak area can be affected by the velocity of the sample, peak areas 40 41 192 were calibrated by dividing them by the migration time.30 Typical detection limits for the organic 42 43 44 193 anions reported here was approximate 1 µM. Representative electropherograms are shown in 45 46 194 Figure S8 and S9. Based on the comparison of electropherograms between filtered reaction 47 48 195 solution and authentic standards, the three peaks are assigned to carboxylic acid, keto acid and 49 50 51 196 hydroxy acid in each reaction set, respectively. 52 53 54 197 Concentrations of the neutral organic species benzaldehyde and phenylacetaldehyde were 55 56 198 determined using reverse-phase HPLC with UV detection at 238 nm. An HPLC system (Waters 57 58 9 59 60 Environmental Science: Processes & Impacts Page 10 of 41

1 2 3 199 Corp., Milford, MA) was used with a μ-Bondapac column (3.9 × 300 mm column containing 5 4 5 6 200 µm C18 packing material). The flow rate was set at 1 mL/min and an isocratic eluent (5 mM 7 8 201 acetic acid (J. T. Baker) in 15% acetonitrile (J. T. Baker) /85% H2O v/v) was used. The injection 9 10 202 volume was 200 µL. A representative chromatogram is shown in Figure S10. 11 12 13 203 2.5. Data Analysis 14 15 16 204 We applied the following kinetic model to our time course results: 17 18 19 푑퐴푇 20 = ― 푘1퐴푇 ― 푘2퐴푇 (1) 21 푑푡 22 푑푊푇 23 = 푘1퐴푇 ― 푘3푊푇 (2) 24 푑푡 25 푑푌푇 26 = 푘2퐴푇 ― 푘4푌푇 (3) 27 푑푡 28 푑퐵푇 29 = 푘 푊 + 푘 푌 (4) 30 푑푡 3 푇 4 푇 31 32 33 205 AT, WT, YT, and BT refer to total dissolved concentrations of hydroxy acid, keto acid, aldehyde, 34 35 206 and final carboxylic acid product, respectively. Consequently, under each set of reaction 36 37 207 conditions, it will be necessary to evaluate whether the reactions shown in Figure 1 actually 38 39 40 208 follow pseudo-first-order behavior. When the hydroxy acid serves as substrate, the sum (k1 + k2) 41 42 209 can be obtained as the slope of log(AT) versus time plots. When the keto acid serves as substrate, 43 44 210 k3 can be obtained from plots of log(WT) versus time. When the aldehyde serves as substrate, k4 45 46 47 211 can be obtained from plots of log(YT) versus time. 48 49 50 212 Distinguishing k1 from k2 is desirable, but in order to do this, we need information about 51 52 213 keto acid and aldehyde time course behavior. Concentrations of keto acid and aldehyde as a 53 54 214 function of time aren't determined by source reactions alone; sink reactions, i.e. oxidation of keto 55 56 57 58 10 59 60 Page 11 of 41 Environmental Science: Processes & Impacts

1 2 3 215 acid and aldehyde to C-1 acid, must also be accounted for. Fortunately, the software package 4 5 6 216 SCIENTIST for Windows (v. 3.0, Micromath, Salt Lake City, UT) is available for numerically 7 8 217 fitting entire AT, WT, YT, and BT versus time data sets. The apportionment and optimization 9 10 218 approach implemented by SCIENTIST yields (k1 + k2), k3 and k4 values that can then be 11 12 compared with values obtained from plots of log(A ), log(W ), and log(Y ) versus time. 13 219 T T T 14 15 220 16 17 18 19 221 3. Results and Discussion 20 21 222 3.1. Mandelic Acid Reaction Set: Time Course and Stoichiometries 22 23 24 223 The mandelic acid reaction set corresponds to -R in Figure 1 being a phenyl group (- 25 26 224 C H ). Reactions at mineral/water interfaces evolve differently from reactions in homogeneous 27 6 5 28 29 225 solution owing to possible site-site reactivity variability and chemical changes to the surface that 30 31 226 accompany reaction. Effects of these phenomena should become more acute as the HMO loading 32 33 227 is decreased. 34 35 36 228 As shown in Figure 2, hydroxy acid loss rates decreased substantially as the HMO 37 38 39 229 loading was decreased from 500 to 200, and finally to 100 M. Keto acid concentrations reached 40 41 230 a maximum (20 M) at 7.5 hours when the loading was 500 M. Lowering the HMO loading to 42 43 44 231 200 M did not appreciably affect the concentration achieved at the maximum, but the maximum 45 46 232 occurred later, at 15 hours. Lowering the HMO loading still further to 100 M yielded a plateau, 47 48 233 also at 20 M, during late stages of the experiment. Presumably the keto acid concentration 49 50 51 234 would have reached a maximum and then declined if the duration of the experiment had been 52 53 235 longer. The aldehyde concentrations reached a plateau at all three HMO loadings. There was a 54 55 236 modest decrease in this plateau as the HMO loading was decreased, and the plateau was reached 56 57 58 11 59 60 Environmental Science: Processes & Impacts Page 12 of 41

1 2 3 237 later in the time course when lower HMO loadings were used. C-1 acid concentrations followed 4 5 6 238 S-shaped curves. Inflection points in these curves occurred at later times as the HMO loading 7 8 239 was decreased, and the C-1 concentration achieved by the end of each experiment decreased as 9 10 240 well. This S-shaped profile confirmed that C-1 acid is derived from oxidation of keto acid and 11 12 aldehyde intermediates and not from direct oxidation of hydroxy acid parent; keto acid and 13 241 14 15 242 aldehyde concentrations must build up before appreciable rates of C-1 acid production can occur. 16 17 243 As shown in the figure, summing the concentrations of the four reaction set compounds yielded 18 19 244 good mass balance at all three HMO loadings, indicating that adsorption of hydroxy acid, keto 20 21 22 245 acid, aldehyde, and C-1 acid was negligible. 23 24 25 246 Reaction stoichiometry is important for understanding HMO loading effects. Based upon 26 27 247 the experimentally measured average Mn oxidation state of +3.82, we can represent the 28 29 248 manganese half-reaction as: 30 31 32 + - II MnO1.91(s) + 3.82H + 1.82e = Mn + 1.91H2O (5) 33 34 35 249 Turning our attention to the mandelic acid reaction set, each oxidation step depicted in Figure 1 36 37 250 corresponds to an organic half reaction: one mole of hydroxy acid oxidized to either keto acid or 38 39 251 aldehyde yields two moles of electrons; one mole hydroxy acid oxidized to C-1 acid yields four 40 41 252 moles of electrons. Adding organic half-reactions to the manganese half reaction yields three full 42 43 44 253 reactions: 45 46 - + - 2+ 47 RCH(OH)COO + 1.10MnO1.91 + 2.20H = RC(O)COO + 1.10Mn + 2.10H2O (6) 48 - + 2+ * 49 RCH(OH)COO + 1.10MnO1.91 + 3.20H = RC(O)H + 1.10Mn + H2CO3 + 1.10H2O (7) 50 51 - + - 2+ * RCH(OH)COO + 2.20MnO1.91 + 4.40H = RCOO + 2.20Mn + H2CO3 + 2.20H2O (8) 52 53 54 254 (Derivations are shown in Supporting Information S2.) The fact that mandelic acid reaction set 55 56 255 compounds do not adsorb to any appreciable extent means that experimentally-determined keto 57 58 12 59 60 Page 13 of 41 Environmental Science: Processes & Impacts

1 2 3 256 acid, aldehyde, and C-1 acid concentrations provide a means of predicting the amount of MnII 4 5 6 257 generated, denoted using RS for "reaction set": 7 8 9 [Mn]RS = 1.10×[keto acid] + 1.10×[aldehyde] + 2.20×[C-1 acid] (9) 10 11 258 [Mn]RS can then be compared with [Mn]diss, the quantity obtained by filtering reaction aliquots 12 13 259 followed by AAS analysis. Strong chelating agents were not employed in the experiments 14 15 260 presented in this section. Hence, dissolved MnIII and MnIV are negligible, and [Mn] should 16 diss 17 II 29 18 261 consist entirely of Mn . 19 20 21 262 As shown in Figure 3, [Mn]diss/[Mn]RS was low at the onset of the reaction employing 22 23 263 500 M HMO, but gradually approached 1.0 during 35 hours of reaction. When 200 M HMO 24 25 was employed, the early discrepancy between [Mn] and [Mn] was lower, and the ratio 26 264 diss RS 27 28 265 approached 1.0 sooner, within 20 hours of reaction. When 100 M HMO was employed, only 29 30 266 the first reaction aliquot showed significant discrepancy between [Mn]diss and [Mn]RS. 31 32 33 267 Descrepancies between [Mn]diss and [Mn]RS seen in Figure 3 indicate that particle-bound 34 35 MnIII or MnII is present. MnII adsorption largely explains the discrepancies seen in Figure 3 36 268 37 II 38 269 between [Mn]diss and [Mn]RS. Mn adsorption onto HMO can be significant even at pH values as 39 40 270 low as 4.0 and at HMO loadings comparable to those employed here.31 Under neutral and 41 42 271 alkaline conditions and at strongly acidic pH, adsorbed MnII is known to engage in 43 44 IV 45 272 conproportionation reactions with oxyhydroxide-bound Mn , generating oxyhydroxide-bound 46 47 273 MnIII 32-34 and thereby trapping reducing equivalents within HMO particles. Conproportionation 48 49 274 reactions are not, however, believed to be important near pH 4.33, 35 50 51 52 275 At the end of the reaction employing 500 M HMO, [Mn]RS was found equal to 80 M, 53 54 55 276 i.e. 16 % of total added manganese. The corresponding value for our 200 M HMO experiments 56 57 58 13 59 60 Environmental Science: Processes & Impacts Page 14 of 41

1 2 3 277 was 60 M, 30 % of total added manganese. For the 100 M HMO experiments, the value was 4 5 6 278 34 M, 34 % of total added manganese. Comparable decreases in the net volume of HMO 7 8 279 particles in suspension likely took place. We can conclude that alteration of the remaining HMO 9 10 11 280 surface was least important when the HMO loading was 500 M, and more important in 12 13 281 experiments employing lower HMO loadings. 14 15 16 282 We can also initiate reactions mid-way in the mandelic acid reaction set by using 17 18 283 phenylglyoxylic acid (Figure 4A) or benzaldehyde (Figure 4B) as substrate. Good mass balance 19 20 21 284 ([substrate] + [C-1 acid]) was obtained in both cases. During each time course, dissolved Mn 22 23 285 concentrations matched or slightly exceeded concentrations of C-1 acid product. Carefully taking 24 25 286 into account the average oxidation state of HMO and calculating [Mn]diss/[Mn]RS (Figures S12 D 26 27 287 and E) reveals that the ratio is again low at the onset of reaction, but gradually approaches 1.0 as 28 29 30 288 each reaction progresses. 31 32 33 289 3.2. Mandelic Acid Reaction Set: Rates and Rate Constants 34 35 36 290 As noted in Materials and Methods, (k1 + k2) can be obtained from slopes of ln[hydroxy 37 38 291 acid] versus time plots, k3 from slopes of ln[keto acid] versus time plots, and k4 from slopes of 39 40 292 ln[aldehyde] versus time plots (employing hydroxy acid, keto acid, and aldehyde, respectively, 41 42 43 293 as added substrate). When 500 M HMO loadings were employed, semi-log plots were linear 44 45 294 throughout each time course (Figures 2D, 4D, 4E, and S11), and hence under these conditions it 46 47 295 is reasonable to assume that hydroxy acid, keto acid, and aldehyde consumption were pseudo- 48 49 50 296 first-order. 51 52 53 297 Lowering HMO loadings to 200 and 100 M resulted in semi-log plots that, while 54 55 298 initially linear, exhibited increasing curvature (more pronounced decreases in rates) during later 56 57 58 14 59 60 Page 15 of 41 Environmental Science: Processes & Impacts

1 2 3 299 stages of reaction. As noted in the preceding section, percentages of HMO consumed between 30 4 5 6 300 and 34 % were observed during 35 hours of reaction when these HMO loadings were employed. 7 8 301 Significant loss of HMO surface sites or diminishment of the reactivity of remaining sites are 9 10 302 entirely reasonable explanations. Although strictly not obeying pseudo-first-order behavior, we 11 12 elected to apply this assumption in a limited number of cases for the sake of comparison. 13 303 14 15 304 Assuming that the four contributing reactions are pseudo-first-order simplifies numerical 16 17 305 modeling employing SCIENTIST 3.0. 18 19 20 306 Recall that concentrations of all four reaction set organic compounds as a function of 21 22 307 time were included in the SCIENTIST fitting exercise. In Figure 2C, k , k , k , and k values 23 1 2 3 4 24 25 308 obtained from 500 M HMO data were used to generate time course simulations. The simulated 26 27 309 concentrations and the experimental data agree quite nicely. Simulations can be used to show 28 29 310 how rates of the four contributing reactions changed as reaction progressed. As shown in Figure 30 31 32 311 2F, the time when r1 and r3 crossed corresponded to the maximum in keto acid concentration. r4 33 34 312 was so low that it never crossed r2. For this reason, a plateau in aldehyde concentration was 35 36 313 observed, not a maximum. 37 38 39 314 Fitting parameters obtained from the 500 M HMO experiment yielded excellent fits for 40 41 42 315 the first 10 hours of the 100 M HMO and 200 M HMO time course data (Figure 2A-B). At 43 44 316 later portions of these two runs, however, fitting parameters from the 500 M HMO experiment 45 46 47 317 yielded hydroxy acid loss rates that were too high, keto acid maxima that were too high, and 48 49 318 aldehyde plateau values that were too high. We can conclude that at these two lower HMO 50 51 319 loadings significant consumption of surface sites took place, or that the reactivity of remaining 52 53 54 320 sites diminished as reaction progressed. 55 56 57 58 15 59 60 Environmental Science: Processes & Impacts Page 16 of 41

1 2 3 321 Quite interestingly, using SCIENTIST to extract fitting parameters from 100 M HMO 4 5 6 322 experiments and applying fits to 100 M HMO data yielded initial rates of hydroxy acid 7 8 323 consumption (and production of keto acid and aldehyde) during the first ten hours that were too 9 10 11 324 low (Figure S13A). Fitting parameters obtained from 200 M HMO experiments applied to 200 12 13 325 M HMO data similarly yielded initial rates that were too low (Figure S13B). Explaining these 14 15 326 results is straightforward. Fitting parameters from the 500 M HMO experiment nicely capture 16 17 18 327 the reactivity of pristine surface sites. SCIENTIST fitting to 100 and 200 M HMO 19 20 328 experimental data includes data points collected late in the experiments where the surface sites 21 22 23 329 are no longer pristine and surface site loadings have diminished. These late data points spoil the 24 25 330 fit to early portions of data. 26 27 28 331 When 500 M HMO was employed, (k1 + k2) values obtained by semi-log lot (0.256 29 30 332 (±0.007) hr-1) and by SCIENTIST fitting (0.269 (±0.023) hr-1) are in reasonable agreement. 31 32 33 333 SCIENTIST additionally indicates that hydroxy acid oxidation to keto acid (with k1 = 0.19 34 -1 35 334 (±0.01) hr ) was 2.4-times faster than hydroxy acid oxidation to aldehyde (k2 = 7.91 (±0.95) 36 37 -2 -1 335 ×10 hr ). The two intermediates were ultimately oxidized to C-1 acid. The keto acid (k3 = 7.03 38 39 -2 -1 -3 -1 40 336 (±0.40) ×10 hr ) was 34-times more reactive than the aldehyde (k4 = 2.06 (±0.22) ×10 hr ). 41 42 337 When keto acid and aldehyde were used as substrates, values obtained from semi-log plots (7.35 43 -2 -1 -3 -1 44 338 (±0.70) ×10 hr for k3 and 2.22 (±0.21) ×10 hr for k4) and values obtained from SCIENTIST 45 46 339 fitting are in good agreement. 47 48 49 340 Dividing rate constants by HMO loading (in mole.L-1) yields normalized rate constants 50 51 52 341 (k1', k2', k3', k4') compiled in Table 1 that facilitate comparisons of results obtained using 53 54 342 different HMO loadings. Despite normalization, values of all four normalized rate constants 55 56 57 58 16 59 60 Page 17 of 41 Environmental Science: Processes & Impacts

1 2 3 343 consistently increased as the HMO loading was increased. Again, significant loss of HMO 4 5 6 344 surface sites or diminishment of the reactivity of remaining sites at the lower loadings are likely 7 8 345 explanations. 9 10 11 346 The balance between two competitive reactions in parallel can be evaluated using 12 13 347 normalized rate constants. As shown below, the ratio of hydroxy acid oxidation to keto acid 14 15 348 versus hydroxy acid oxidation to aldehyde increases as the HMO loading is decreased: 16 17 18 19 HMO Loading k1'/k2' 20 21 2.9 22 100 M 23 24 200 M 2.6 25 26 500 M 2.2 27 28 29 349 Keto acid oxidation to C-1 acid versus aldehyde oxidation to C-1 acid also makes for interesting 30 31 350 comparison: 32 33 34 35 HMO Loading k3'/k4' 36 37 100 M - 38 39 40 200 M 40 41 42 500 M 34 43 44 45 351 (Aldehyde oxidation in the 50 M mandelic acid plus 100 M HMO experiment was too slow 46 47 352 for SCIENTIST to obtain a value for k '.) By altering rates of reactions that both produce 48 4 49 50 353 intermediates (keto acid and aldehyde) and consume them (yielding C-1 acid), changes in HMO 51 52 354 loadings bring about significant changes to organic species time course behavior, as shown in 53 54 355 Figure 2. 55 56 57 58 17 59 60 Environmental Science: Processes & Impacts Page 18 of 41

1 2 3 356 3.3. Phenyllactic Acid Reaction Set 4 5 6 357 The phenyllactic acid reaction set corresponds to -R in Figure 1 being a benzyl group (- 7 8 9 358 CH2C6H5), i.e. with an added methylene group between the aromatic ring and the reactive 10 11 359 portion of each molecule. As shown in Figure 5A, hydroxy acid loss rates were lower than 12 13 360 observed in the mandelic acid experiment performed under comparable conditions (Figure 2C). 14 15 361 Experimental keto acid concentrations were barely above the detection limit. The maximum keto 16 17 18 362 acid concentration was 13-times lower than observed in the corresponding mandelic acid 19 20 363 experiment, and occurred at 2.5 hours, not at 8 hours. Aldehyde concentrations were below the 21 22 364 detection limit throughout the experiment. Although concentrations of phenylacetic acid, the C-1 23 24 25 365 acid product, increased as concentrations of phenyllactic acid decreased, mass balance fell short, 26 27 366 by approximately 9 %. As far as reaction stoichiometry is concerned, relationships analogous to 28 29 367 Equations 6-9 apply. Early in the reaction, discrepancies between [Mn]diss and [Mn]RS (Figure 30 31 32 368 5B) are slightly smaller than those observed in the mandelic acid experiment under comparable 33 34 369 conditions (Figure 3C). The ratio [Mn]diss/[Mn]RS (Figure 5D) nears unity a little sooner than 35 36 370 observed in the mandelic acid experiment (Figure 3F). 37 38 39 371 The value of (k1 + k2) obtained using semi-log plots was 7-times lower when phenyllactic 40 41 372 acid served as substrate than when mandelic acid served as substrate. Phenylpyruvic acid is the 42 43 44 373 keto acid for this reaction set. When used as substrate (Figure 4C), phenylpyruvic acid was 12.7- 45 46 374 times more reactive towards HMO than phenylglyoxylic acid, the keto acid of the mandelic acid 47 48 375 reaction set. During the 2.5 hour duration of our experiments employing phenylpyruvic acid as a 49 50 51 376 substrate, the sum of [keto acid] and [C-1 acid] decreased 10 %. This missing mass balance is of 52 53 377 the same magnitude as the missing mass balance seen when phenyllactic acid was used as 54 55 378 substrate. [Mn]RS, calculated from measurements of phenylpyruvic acid and C-1 acid 56 57 58 18 59 60 Page 19 of 41 Environmental Science: Processes & Impacts

1 2 3 379 concentrations, fell considerably short of [Mn]diss (Figure S12F). We hypothesize that the portion 4 5 6 380 missing from mass balance arises from oxidation of phenylpyruvic acid to a product that is not 7 8 381 detected by CE, and that generation of this product yields more than two electron-equivalents. 9 10 11 382 Phenylacetaldehyde, the aldehyde for the phenyllactic acid reaction set, is at least five 12 13 383 orders-of-magnitude more reactive towards HMO than benzaldehyde, the aldehyde for the 14 15 384 mandelic acid reaction set. Oxidation of phenylacetaldehyde by HMO was so fast that 16 17 18 385 phenylacetaldehyde could not be detected in an aliquot collected at 1 minute, immediately 19 20 386 filtered and injected into HPLC. The C-1 acid concentration in this sample, measured by CE, 21 22 387 equaled the concentration of phenylacetaldehyde reactant used. [Mn] of this sample was 53.2 23 diss 24 25 388 µM measured by AAS, which is agreement with [Mn]RS (55 µM), calculated using the measured 26 27 389 C-1 acid concentration and Reaction S10 of Supporting Information. 28 29 30 390 3.4. Organic Reactivity Comparisons 31 32 33 391 The two mechanisms for mandelic acid oxidation by transition metal-containing species 34 35 392 that have been presented in the literature are shown in Figure S14. In Scheme I, mandelic acid 36 37 oxidation begins with hydrogen atom abstraction, which generates a free radical intermediate.36, 38 393 39 40 394 37 In Scheme II, mandelic acid oxidation begins with hydride ion transfer, which generates a 41 42 395 carbocation intermediate.36, 38 Electronic interactions with the benzene ring would be expected to 43 44 396 stabilize free radical or carbocation intermediates generated from mandelic acid oxidation 45 46 47 397 relative to those generated from phenyllactic acid oxidation. Although we cannot establish which 48 49 398 oxidation mechanism is operative, the higher reactivity of mandelic acid in comparison with 50 51 399 phenyllactic acid is reasonable. 52 53 54 55 56 57 58 19 59 60 Environmental Science: Processes & Impacts Page 20 of 41

1 2 3 400 The keto acid phenylpyruvic acid can form gem-diols via hydration and can form enols 4 5 6 401 (Figure S15). Although thermodynamic information isn't available for phenylpyruvic acid, it is 7 8 402 available for pyruvic acid, and their speciation should be similar. As discussed in Supporting 9 10 403 Information S3, the two gem-diols (HXo and X-) represent 7.7 % of the total pyruvic acid 11 12 concentration at pH 4.0; only one molecule in 73,000 is found in one of the enol forms. The keto 13 404 14 15 405 acid phenylglyoxylic acid, in contrast, can form gem-diols via hydration but cannot form enols. 16 17 406 Gem-diols only comprise 0.1 % of total phenylglyoxylic acid at pH 4.0. Gem-diols have been 18 19 407 reported to be much more readily oxidized than corresponding keto forms. Hence, the 13-fold 20 21 22 408 higher reactivity of phenylpyruvic acid in comparison to phenylglyoxylic acid is quite 23 24 409 reasonable. 25 26 27 410 Phenylacetaldehyde can form a gem-diol via hydration and can also form cis- and trans- 28 29 411 enols. According to Chiang et al.,39 the gem-diol represents 75 % of total phenylacetaldehyde in 30 31 32 412 solution, whereas only one molecule in 2850 exists in one of the enol forms. Only about 0.8 % of 33 34 413 total benzaldehyde is found in the gem-diol form, and formation of an enol is not possible.40, 41 35 36 414 The preponderance of the more oxidizable gem-diol of phenylacetaldehyde, and its high 37 38 415 reactivity towards oxidants, quite readily explains why it doesn't appear when phenyllactic acid 39 40 41 416 serves as substrate, and why it is entirely consumed by the time the first aliquot is collected when 42 43 417 added as substrate. 44 45 46 418 3.5. pH 4.0 Experiments with Added Phosphate and Pyrophosphate 47 48 49 419 In our next set of experiments (Figure 6), we continued to maintain pH 4.0 using 5.0 mM 50 51 420 acetate, but augmented the reaction medium with ten-fold lower concentrations (500 M) of 52 53 54 421 phosphate or pyrophosphate. Linear regression of the logarithm of the mandelic acid 55 56 422 concentration as a function of time indicated that phosphate lowered the rate of mandelic acid 57 58 20 59 60 Page 21 of 41 Environmental Science: Processes & Impacts

1 2 3 423 oxidation by 49 %, and pyrophosphate lowered the rate by 80 %. Rate constants for each step in 4 5 6 424 the reaction set were derived from experimentally measured concentrations and included in 7 8 425 Table 1. As shown in Figure S17, numerical simulations based on these rate constants fit time 9 10 426 course data reasonably well. k1 (conversion of hydroxy acid to keto acid) and k2 (conversion of 11 12 hydroxy acid to aldehyde) are lowered by phosphate and pyrophosphate additions to similar 13 427 14 15 428 extents. k3, the rate constant for conversion of keto acid into C-1 acid, decreased 56 % when 16 17 429 phosphate was added, and 83 % when pyrophosphate was added. k4, the rate constant for 18 19 430 conversion of aldehyde into C-1 acid, was already low in the absence of additive. Phosphate and 20 21 22 431 pyrophosphate diminished k4 to negligible levels. 23 24 25 432 In control experiments free of organic substrate, neither 500 M phosphate nor 500 M 26 27 433 pyrophosphate yielded any appreciable dissolved Mn after 30 hours of reaction. In mandelic 28 29 434 acid-containing suspensions, Equation 9 can be used to convert measured concentrations of keto 30 31 II 32 435 acid, aldehyde, and C-1 acid into [Mn]RS, the expected amount of Mn generated by redox 33 34 436 reaction, which can be compared with direct [Mn]diss measurements. As noted earlier, [Mn]diss 35 36 437 falls significantly short of [MnII] throughout the entire time course when a 500 M HMO 37 RS 38 39 438 loading is used and mandelic acid is added in the absence of phosphate or pyrophosphate. 40 41 439 [Mn]diss/[Mn]RS plotted as a function of time makes this discrepancy easier to discern (Figure 42 43 440 6G). In the presence of 500 M pyrophosphate, a slight discrepancy is observed, but only in the 44 45 46 441 earliest four data points. In the presence of 500 M phosphate, a slightly larger discrepancy is 47 48 442 seen, but it is again restricted to the earliest data points. 49 50 51 443 Phosphate and pyrophosphate effects on reaction pathways and rates likely arise from 52 53 54 444 their coordination properties: (i) adsorption onto HMO surface sites, which lowers site 55 56 445 accessibility to reductants, (ii) ligand-assisted dissolution of surface-bound MnII and MnIII, and 57 58 21 59 60 Environmental Science: Processes & Impacts Page 22 of 41

1 2 3 446 (iii) formation of dissolved MnII and MnIII complexes. Adsorption of phosphate and other 4 5 6 447 oxyanions onto HMO at acid, neutral, and slightly alkaline pH is surprisingly robust, given that 7 42 43 8 448 pHzpc values for these materials are less than 4. Villalobos attributed this adsorption to 9 10 449 oxyanion-reactive sites at sheet edges, and stated that they are relatively few in number in 11 12 comparison to cation-reactive vacancy sites within basal planes. Being relatively few in number 13 450 14 15 451 would be relevant to ligand-assisted dissolution, since MnIII-oxyanion complexes are also 16 17 452 attracted to these sites. Wang and Stone29 concluded that pyrophosphate concentrations in excess 18 19 453 of 1 mM must be added before all oxyanion-reactive sites are occupied, forcing MnIII-oxyanion 20 21 22 454 complexes into solution. Apparently phosphate and pyrophosphate concentrations in the 23 24 455 experiments described in this section were too low for this to occur. 25 26 27 456 The tendency for MnII to adsorb or precipitate is less than that for MnIII. Both phosphate 28 29 457 and pyrophosphate are viable ligands for MnII, and can bring about release of surface-bound 30 31 II 32 458 Mn , as reflected in nearly equal values of [Mn]RS and [Mn]diss observed when 500 M 33 34 459 phosphate and pyrophosphate are present. 35 36 37 460 3.6. pH 7 Experiments: MOPS, Phosphate, and Pyrophosphate Buffers 38 39 40 461 Mandelic acid and phenyllactic acid oxidation by HMO was also investigated at pH 7.0 41 42 462 in suspensions buffered using 2.0 mM MOPS, phosphate, or pyrophosphate. In MOPS and 43 44 45 463 phosphate buffer, concentrations of dissolved Mn obtained by filtering reaction solutions and 46 47 464 measured by AAS stayed below the detection limit throughout the duration of each experiment. 48 49 465 As shown in Figure 7, the time courses for mandelic acid oxidation in MOPS and phosphate 50 51 466 buffer look very similar, with keto acid as the only organic product detected, with good mass 52 53 -3 54 467 balance. Slopes of ln[mandelic acid] versus time yielded rate constants of 4.89 (±0.11) ×10 55 56 468 hour-1 in MOPS buffer and 5.40 (±0.15) ×10-3 hour-1 in phosphate buffer (Figure S18). This 57 58 22 59 60 Page 23 of 41 Environmental Science: Processes & Impacts

1 2 3 469 amounts to a 50-fold decrease in the rate of mandelic acid loss in comparison to the pH 4.0 4 5 6 470 experiments presented earlier. 7 8 9 471 At pH 7.0, 2.0 mM phosphate yielded a rate of phenyllactic loss that was 1.43-times 10 11 472 higher and produced twice as much keto acid than was observed in 2.0 mM MOPS (Figures 7 12 13 473 and S18). Concentrations of phenylacetic acid produced in the two reaction suspensions were 14 15 474 approximately the same, and no aldehyde production was observed. Good mass balanced was 16 17 18 475 obtained. On average, phenyllactic acid was oxidized 30-fold more slowly at pH 7.0 than at pH 19 20 476 4.0. 21 22 23 477 Unlike results obtained using MOPS and phosphate buffers, pH 7.0 pyrophosphate 24 25 478 experiments produced substantial concentrations of dissolved manganese (Figure 8). In an 26 27 28 479 organic substrate-free control experiment, ~ 5 % of total added HMO became dissolved during 29 30 480 the first 6 hours. Between 6 and 150 hours, further dissolved manganese was released, but only 31 32 481 added an additional 1.5 %. The total amount of manganese released in the control experiment, 33 33 34 III 35 482 M, was a little more than one-third of the HMO Mn content at the loading employed (500 36 37 483 M). UV/visible spectra of filtered solutions exhibited a peak at 260 nm (Figure S19A), 38 39 matching a peak reported by other investigators in the 6.5 pH 8 range,26, 28, 29, 44 attributed to 40 484   41 42 485 dissolved MnIII-pyrophosphate complexes. Linear regression of absorbance versus dissolved 43 44 486 manganese yielded a molar absorptivity of 6.17×103 M-1cm-1 (Figure S19D), which matches the 45 46 pH 6.7 value reported by Cabelli and Bielski44 (6.2×103 M-1cm-1). 47 487 48 49 50 488 In the presence of 50 M mandelic acid or phenyllactic acid (Figure 8A,B) time courses 51 52 489 for dissolved manganese during the first six hours of reaction were nearly the same as observed 53 54 490 in the organic substrate-free control experiment. From six hours to the end of each experiment, 55 56 57 58 23 59 60 Environmental Science: Processes & Impacts Page 24 of 41

1 2 3 491 however, substantially greater increases in dissolved manganese were observed. The peak at 260 4 5 III 6 492 nm attributable to Mn -pyrophosphate complexes was observed and increased substantially 7 8 493 during both experiments. Absorbance versus [Mn]diss was linear within early portions of each 9 10 494 time course, but at later times exhibited downward curvature (Figure S19E,F). We elected to use 11 12 the calibration curve obtained from the organic substrate-free experiment (Figure S19D) to 13 495 14 III 15 496 calculate [Mn ]diss organic substrate-containing experiments (Figure 8D,E). Substracting 16 III II 17 497 [Mn ]diss from [Mn]diss yielded [Mn ]diss. At the end of the mandelic acid experiment, 17 % of 18 19 498 dissolved manganese was MnII and 83 % was MnIII. At the end of the phenyllactic acid 20 21 II III 22 499 experiment, 12 % was Mn and 88 % was Mn . 23 24 25 500 Mandelic acid and phenyllactic acid loss were slightly faster when pyrophosphate 26 27 501 replaced MOPS or phosphate as buffer (Figure 8). Mandelic acid oxidation again yielded only 28 29 502 keto acid. Phenyllactic acid oxidation yielded both keto acid and C-1 acid. Pyrophosphate was 30 31 32 503 similar to phosphate in that keto acid production was twice that obtained when MOPS served as 33 34 504 buffer. In all experiments employing 2 mM pyrophosphate buffer, good mass balanced was 35 36 505 obtained. 37 38 39 506 Pyrophosphate exhibited the ability to "mine" a portion of the MnIII content of the HMO 40 41 507 suspensions employed, and yielded quite substantial dissolved MnII and MnIII concentrations as 42 43 44 508 reactions with mandelic acid and phenyllactic acid progressed. The fact that rates of organic 45 46 509 substrate loss and identities/yields of oxidized products changed so little is rather surprising. We 47 48 510 can speculate that hydroxy acid, keto acid, and aldehyde oxidations by HMO-bound MnIV are far 49 50 III III 51 511 more rapid than oxidations by HMO-bound Mn or by the dissolved Mn -pyrophosphate 52 53 512 complexes that predominate at the pH and pyrophosphate concentration employed here. 54 55 513 Removing HMO-bound MnIII apparently had little effect on the reactivity of the remaining MnIV. 56 57 58 24 59 60 Page 25 of 41 Environmental Science: Processes & Impacts

1 2 3 514 4 5 6 515 4. Conclusions 7 8 9 10 516 At pH 4.0, the reaction set comprised of mandelic acid, phenylglyoxylic acid, 11 12 517 benzaldehyde, and benzoic acid exhibited mass balance during oxidations by HMO, and extents 13 14 518 of adsorption were negligible. We were therefore able to calculate mole-equivalents delivered to 15 16 II 17 519 HMO for comparison with experimentally-determined dissolved Mn concentrations. Because 18 19 520 concentrations of phenylglyoxylic acid and benzaldehyde were appreciable and exhibited 20 21 521 distinctive time course behavior, it was possible using numerical approaches to distinguish and 22 23 522 quantify rates of contributing reactions occurring in series and in parallel. With phenyllactic acid, 24 25 26 523 concentrations of phenylpyruvic acid were near the detection limit and concentrations of 27 28 524 phenylacetaldehyde were not discernible. There was more and more deviation from mass balance 29 30 525 as reactions progressed. Nevertheless, the phenyllactic acid reaction set revealed important 31 32 33 526 aspects of -hydroxycarboxylic acid, -ketocarboxylic acid, and aldehyde reactivity. The 34 35 527 inserted -CH2- group either raised the extent of hydration, yielding gem-diols, or raised the 36 37 528 extent of tautomerization, yielding enols. Gem-diols and enols are far more reactive towards 38 39 40 529 oxidants like HMO than corresponding keto forms. 41 42 43 530 Environmentally-relevant redox reactions are often far from simple. The reaction set 44 45 531 approach entails finding the means of distinguishing and quantifying all reaction intermediates 46 47 532 and products, so that contributing reaction steps can be fully and clearly discerned. We have 48 49 50 533 demonstrated the utility of this approach for documenting HMO loading effects and effects of 51 52 534 500 M phosphate and pyrophosphate on reactions at pH 4.0, and the effects of 2 mM phosphate 53 54 535 and pyrophosphate on reactions at pH 7.0. In our subsequent publications, we will use the 55 56 57 58 25 59 60 Environmental Science: Processes & Impacts Page 26 of 41

1 2 3 536 reaction set approach to explore HMO-driven autocatalysis during permanganate oxidations, and 4 5 6 537 other catalytic effects on permanganate oxidations. 7 8 9 538 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 26 59 60 Page 27 of 41 Environmental Science: Processes & Impacts

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1 2 3 584 chemical air emissions and worker exposure during sanitary sewer and stormwater pipe 4 585 rehabilitation using cured-in-place-pipe (CIPP), Environ. Sci. Technol. Lett., 2017, 4, 5 6 586 325-333. 7 587 18. K. Ra, S. M. T. Sendesi, J. A. Howarter, C. T. Jafvert, B. M. Donaldson and A. J. 8 588 Whelton, Critical review: surface water and stormwater quality impacts of cured-in-place 9 589 pipe repairs, J. - Am. Water Works Assoc., 2018, 110, 15-32. 10 590 19. C. Chen, H. Chang and T. K. Kirk, Aromatic-acids produced during degradation of lignin 11 591 in spruce wood by phanerochaete-chrysosporium, Holzforschung, 1982, 36, 3-9. 12 20. R. F. P. Nogueira, R. A. Pilli and N. Duran, Degradation of β-O-4 lignin model and 13 592 14 593 related-compounds by the ascomycete chrysonilia-sitophila (TFB-27441 strain), Appl. 15 594 Biochem. Biotechnol., 1992, 33, 169-176. 16 595 21. F. Berthold, C. T. Lindgren and M. E. Lindstrom, Formation of (4-hydroxy-3- 17 596 methoxyphenyl)-glyoxylic acid and (4-hydroxy-3,5-dimethoxyphenyl)-glyoxylic acid 18 597 during polysulfide treatment of softwood and hardwood, Holzforschung, 1998, 52, 197- 19 598 199. 20 21 599 22. M. Ragnar, C. T. Lindgren and N. O. Nilvebrant, pKa-values of guaiacyl and syringyl 22 600 phenols related to lignin, J. Wood Chem. Technol., 2000, 20, 277-305. 23 601 23. L. Pollegioni, F. Tonin and E. Rosini, Lignin-degrading enzymes, FEBS J., 2015, 282, 24 602 1190-1213. 25 603 24. J. W. Murray, Surface chemistry of hydrous manganese-dioxide, J. Colloid Interface Sci., 26 604 1974, 46, 357-371. 27 605 25. E. W. Rice, Baird, R.B., Eaton, A. D., Clesceri, L. S., editors, Standard Methods for the 28 29 606 Examination of Water and Wastewater., American Public Health Association, American 30 607 Water Works Association, Water Environment Federation, 22nd ed., 2012. 31 608 26. J. E. Kostka, G. W. Luther and K. H. Nealson, Chemical and biological reduction of 32 609 Mn(III)-pyrophosphate complexes - potential importance of dissolved Mn(III) as an 33 610 environmental oxidant, Geochim. Cosmochim. Acta, 1995, 59, 885-894. 34 611 27. J. K. Klewicki and J. J. Morgan, Kinetic behavior of Mn(III) complexes of 35 pyrophosphate, EDTA, and citrate, Environ. Sci. Technol., 1998, 32, 2916-2922. 36 612 37 613 28. J. K. Klewicki and J. J. Morgan, Dissolution of β-MnOOH particles by ligands: 38 614 Pyrophosphate, ethylenediaminetetraacetate, and citrate, Geochim. Cosmochim. Acta, 39 615 1999, 63, 3017-3024. 40 616 29. Y. Wang and A. T. Stone, Phosphonate- and carhoxylate-based chelating agents that 41 617 solubilize (hydr)oxide-bound Mn(III), Environ. Sci. Technol., 2008, 42, 4397-4403. 42 618 30. D. R. Baker, Capillary Electrophoresis, John Wiley & Sons, Inc., New York, US, 1995. 43 44 619 31. J. W. Murray, Interaction of metal-ions at manganese dioxide solution interface, 45 620 Geochim. Cosmochim. Acta, 1975, 39, 505-519. 46 621 32. E. J. Elzinga, Reductive transformation of birnessite by aqueous Mn(II), Environ. Sci. 47 622 Technol., 2011, 45, 6366-6372. 48 623 33. E. J. Elzinga, 54Mn radiotracers demonstrate continuous dissolution and reprecipitation of 49 624 vernadite (δ-MnO2) during interaction with aqueous Mn(II), Environ. Sci. Technol., 2016, 50 625 50, 8670-8677. 51 52 626 34. H. Y. Zhao, M. Q. Zhu, W. Li, E. J. Elzinga, M. Villalobos, F. Liu, J. Zhang, X. H. Feng 53 627 and D. L. Sparks, Redox reactions between Mn(II) and hexagonal birnessite change its 54 628 layer symmetry, Environ. Sci. Technol., 2016, 50, 1750-1758. 55 56 57 58 28 59 60 Page 29 of 41 Environmental Science: Processes & Impacts

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1 2 3 654 Figures and Tables 4 5 6 7 655 Table 1. Medium conditions and rate constants for the oxidation of the four organic substrates 8 656 comprising the mandelic acid and phenyllactic acid reaction sets. Rate constants have been 9 10 657 normalized with respect to HMO loading (ki' = ki[HMO]). Values in brackets were obtained 11 12 658 from slopes of ln[substrate] as a function of time. The remaining values were obtained using 13 14 659 SCIENTIST fits to concentration versus time data of all organic compounds comprising each 15 660 reaction set. Uncertainties indicate 95% confidence intervals. 16 17 Reactants 18 -1 -1 19 (HMO Age, Days) Medium Normalized Rate Constants (M hr ) 20 2 21 50 M Mandelic Acid 5 mM Acetate (pH 4.0) k1' = 2.52 (±0.99) ×10 22 1 100 M HMO-A(2) 10 mM NaCl k2' = 8.61 (±1.77) ×10 23 2 24 k3' = 1.14 (±0.61) ×10 25 k4' = ~0 26 2 (k1' + k2') = 3.38 (±1.19) ×10 27 2 28 [4.98 (±0.28) ×10 ] 29 2 30 50 M Mandelic Acid 5 mM Acetate (pH 4.0) k1' = 3.13 (±0.78) ×10 2 31 200 M HMO-A(8) 10 mM NaCl k2' = 1.20 (±0.32) ×10 32 k ' = 1.22 (±0.54) ×102 33 3 0 34 k4' = 3.02 (±0.66) ×10 2 35 (k1' + k2') = 4.33 (±1.83) ×10 36 [5.30 (±0.50) ×102] 37 38 2 39 50 M Mandelic Acid 5 mM Acetate (pH 4.0) k1' = 3.80 (±0.24) ×10 2 40 500 M HMO-B(2) 10 mM NaCl k2' = 1.58 (±0.19) ×10 41 2 k3' = 1.40 (±0.08) ×10 42 0 43 k4' = 4.12 (±0.44) ×10 2 44 (k1' + k2') = 5.38 (±0.43) ×10 45 [5.12 (±0.14) ×102] 46 47 50 M Mandelic Acid 5 mM Acetate (pH 4.0) k ' = 3.78 (±0.25) ×102 48 1 2 49 500 M HMO-F(2) 10 mM NaCl k2' = 1.62 (±0.21) ×10 2 50 k3' = 1.36 (±0.11) ×10 51 k ' = 4.13 (±0.39) ×100 52 4 2 53 (k1' + k2') = 5.40 (±0.46) ×10 54 [5.12 (±0.16) ×102] 55 56 57 58 30 59 60 Page 31 of 41 Environmental Science: Processes & Impacts

1 2 3 2 50 M Mandelic Acid 5 mM Acetate (pH 4.0) k1' = 1.96 (±0.56) ×10 4 3- 1 5 500 M HMO-F(2) 500 M PO4 k2' = 8.03 (±1.09) ×10 1 6 10 mM NaCl k3' = 6.22 (±2.13) ×10 7 0 k4' = 4.12 (±1.88) ×10 8 2 9 (k1' + k2') = 2.76 (±0.67) ×10 10 [2.72 (±0.59) ×102] 11 1 12 50 M Mandelic Acid 5 mM Acetate (pH 4.0) k1' = 7.80 (±2.20) ×10 13 4- k ' = 2.81 (±0.58) ×101 14 500 M HMO-F(2) 500 M P2O7 2 1 15 10 mM NaCl k3' = 2.41 (±1.44) ×10 16 k4' = ~ 0 17 (k ' + k ') = 1.06 (±0.29) ×102 18 1 2 2 19 [1.07 (±0.01) ×10 ] 20 0 21 50 M Mandelic Acid 2 mM MOPS (pH 7.0) (k1' + k2') = [9.78 (±0.21) ×10 ] 22 500 M HMO-D(2) 15 mM I (using NaCl) 23 24 3- 1 25 50 M Mandelic Acid 2 mM PO4 (pH 7.0) (k1' + k2') = [1.08 (±0.03) ×10 ] 26 500 M HMO-D(2) 15 mM I (using NaCl) 27 28 2 mM P O 4- (pH 7.0) (k ' + k ') = [1.54 (±0.07) ×101] 29 50 M Mandelic Acid 2 7 1 2 30 500 M HMO-D(2) 15 mM I (using NaCl) 31 2 32 50 M Phenylglyoxylic 5 mM Acetate (pH 4.0) k3' = 1.49 (±0.17) ×10 33 10 mM NaCl [1.47 (±0.06) ×102] 34 Acid 35 500 M HMO-C(10) 36 37 0 50 M Benzaldehyde 5 mM Acetate (pH 4.0) k4' = 4.50 (±0.58) ×10 38 0 39 500 M HMO-G(3) 10 mM NaCl [4.43 (±0.41) ×10 ] 40 1 41 50 M Phenyllactic Acid 5 mM Acetate (pH 4.0) (k1' + k2') = [7.22 (±0.32) ×10 ] 42 500 M HMO-C(3) 10 mM NaCl 43 44 3 45 50 M Phenylpyruvic 5 mM Acetate (pH 4.0) k3' = [1.86 (±0.79) ×10 ] 46 Acid 10 mM NaCl 47 500 M HMO-H(5) 48 49 5 50 50 M 5 mM Acetate (pH 4.0) k4' > [4.69×10 ] 51 Phenylacetaldehyde 10 mM NaCl 52 500 M HMO-G(3) 53 54 0 55 50 M Phenyllactic Acid 2 mM MOPS (pH 7.0) k1' + k2' = [1.86 (±0.34) ×10 ] 56 57 58 31 59 60 Environmental Science: Processes & Impacts Page 32 of 41

1 2 3 500 M HMO-E(3) 15 mM I (using NaCl) 4 5 3- 0 6 50 M Phenyllactic Acid 2 mM PO4 (pH 7.0) k1' + k2' = [2.66 (±0.29) ×10 ] 7 500 M HMO-E(3) 15 mM I (using NaCl) 8 9 50 M Phenyllactic Acid 2 mM P O 4- (pH 7.0) k ' + k ' = [3.78 (±0.14) ×100] 10 2 7 1 2 11 500 M HMO-E(3) 15 mM I (using NaCl) 12 661 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 32 59 60 Page 33 of 41 Environmental Science: Processes & Impacts

1 2 3 4 Hydroxy Acid Reaction Set 5 6 Keto Acid O 7 8 Hydroxy O C 9 + - - * + - - 2H - 2e C O + 2H2O - H2CO3 -2H -2e 10 AcidO 11 R - k W- k O O 12 HO H C 1 3 C C-1 13 C O- k k 14 2 O 4 R Acid 15 R A- CH B- 16 + - * + - 0 + H O - 3H - 2e 17 + H2O - H2CO3 - H - 2e R Y 2 18 19 Aldehyde 20 21 22 HW0 23 24 HA0 Phenylglyoxylic Acid HB0 25 R = 0 26 Mandelic Acid Y Benzoic Acid 27 28 Benzaldehyde 29 30 HW0 31 32 HA0 Phenylpyruvic Acid HB0 R = 33 CH2 0 34 Phenyllactic Acid Y Phenylacetic Acid 35 Phenylacetaldehyde 36 662 37 38 663 Figure 1. Pathways for hydroxy acid oxidation. Hydration and enolization reactions for the keto 39 664 acid and aldehyde intermediates are also shown. 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 33 59 60 Environmental Science: Processes & Impacts Page 34 of 41

1 2 3 4 100 M HMO 200 M HMO 500 M HMO 5 (A) (B) (C) 6 50 50 50 7 Mandelic acid 8 40 Phenylglyoxylic acid 40 40 9 Benzaldehyde ) 10 Benzoic acid )  30 Mass balance 30  30   (

11 (

i ] i ] [

12 [ 20 13 20 20 14 15 10 10 10 16 17 0 0 0 18 0 10 20 30 0 10 20 30 0 10 20 30 19 Time (Hour) Time (Hour) Time (Hour) 20 0.3 21 0 (D) (E) (F) 22 8 ) 1

23 - ) r ) u )

24 0 1

( r o -1 - 0.2 6 1 T r

25 h u • A /

o r 26 )

M 2 t

h ( y = -0.0498x-0.00780 6 ( T - 27 2

s 4 r

R = 0.997 0 A -2 b 3 (

28 o 1

k (

29 l n 0.1 r

e 4 30 y = 4.79x10-4x+4.91x10-3 t a 2 y = -0.256x-0.0225 2 R = 0.997 R 31 2 y = -0.106x+0.00462 -3 R = 0.998 32 R2 = 0.994 33 0.0 0 34 0 10 20 30 40 0 100 200 300 400 500 600 0 10 20 30 35 Time (Hour) [HMO] () Time (Hour) 36 37 38 Figure 2. Oxidation of 50 µM mandelic acid by (A) 100 M, (B) 200 M, and (C) 500 M HMO. Phenylglyoxylic acid, 39 benzaldehyde, and benzoic acid products are also shown. (D) Semi-log plots for mandelic acid loss for the three HMO loadings. 40 Fitting was applied to the data delineated by continuous lines; dashed lines are extrapolated. (E) First-order rate constants for mandelic 41 42 43 44 45 34 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 35 of 41 Environmental Science: Processes & Impacts

1 2 3 acid loss as a function of HMO loading. (F) Rates of contributing reactions calculated for 500 M HMO, based on the numerical 4 5 approach described in the text. Reaction medium: 5.0 mM acetate buffer (pH 4.0), and 10 mM NaCl. 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 35 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Environmental Science: Processes & Impacts Page 36 of 41

1 2 3 4 5 100 M HMO 200 M HMO 500 M HMO 6 80 7 8 [Mn] 60 diss 9 [Mn] 10 ) RS  

11 (

] 40 i 12 [ 13 14 20 15 16 (A) (B) (C) 17 0 18 19 1.0 20 S R 21 ] 22 n M [ /

23 s s i d

24 ] 0.5 25 n M 26 [ 27 28 (D) (E) (F) 0.0 29 0 10 20 30 0 10 20 30 0 10 20 30 30 31 Time (Hour) 32 33 34 Figure 3. Using reaction stoichiometry to evaluate the oxidation of 50 M mandelic acid by (A, D) 100 M, (B, E) 200 M, and (C, 35 F) 500 M HMO. Upper plots: [Mn]diss (×) refers to dissolved Mn measured by filtration and AAS. [Mn]RS (▽) is calculated from 36 measurements of A , W , Y , and B (reaction set organic compounds), and expected reaction stoichiometries as discussed in the text. 37 T T T T 38 Lower plots: [Mn]diss/[Mn]RS as a function of time. Reaction conditions: 5 mM acetate buffer (pH 4.0), and 10 mM NaCl. 39 40 41 42 43 44 45 36 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 37 of 41 Environmental Science: Processes & Impacts

1 2 3 4 5 Phenylglyoxylic acid Benzaldehyde Phenylpyruvic acid 6 7 50 50 50 8 9 40 40 40

10 )

Phenylglyoxylic acid  )

) Benzaldehyde 11 30 30  30 (

 Benzoic acid

Phenylpyruvic acid 

Benzoic acid ]  i 

12 ( [Mn] [

diss ( Phenylacetic acid ]

[Mn] diss i ] 20

13 [ 20 Mass balance i 20 [Mn] [ Mass balance diss 14 Mass balance 15 10 10 10 16 (A) (B) (C) 17 0 0 0 18 0 10 20 30 0 40 80 120 0 1 2 3 4 19 Time (Hour) Time (Hour) Time (Hour) 20 21 22 0 0 0 23 ) 24 y = -0.0737x-0.0277 )

0 y = -0.931x-0.103 ( ) 2 25 ) ) T 2 R = 0.994 ) R = 0.999 0 y = -0.00220x-0.0224 0 ( 26 W ( /

T 2 T -1 -1 R = 0.981 ) -1 27 t Y ( W / / T ) ) t

28 t ( ( W T T 29 ( Y n l ( W (

30 n l n 31 l -2 -2 (E) -2 32 (D) (F) 33 0 10 20 30 0 40 80 120 0 1 2 3 4 34 Time (Hour) Time (Hour) 35 Time (Hour) 36 37 38 Figure 4. Using keto acids and aldehydes as substrates. Phenylglyoxylic acid (A) and benzaldehyde (B) are from the mandelic acid 39 reaction set, while phenylpyruvic acid (C) is from the phenyllactic acid reaction set. Corresponding semi-log plots for loss of substrate 40 41 are shown in D-F. Reaction conditions: 50 M substrate, 500 µM HMO, 5 mM acetate buffer (pH 4.0), and 10 mM NaCl. 42 43 37 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Environmental Science: Processes & Impacts Page 38 of 41

1 2 3 4 5 80 6 50 7 [Mn] diss 8 [Mn] 60 RS 9 40 10 ) 11 ) 30 

 40  

12 ( (

] ] i i 20 13 [ [ 14 Phenyllactic acid 20 15 10 Phenylpyruvic acid 16 Phenylacetic acid (B) Mass balance (A) 17 0 0 18 0 10 20 30 0 10 20 30 19 Time (Hour) 20 Time (Hour) 21 22 0.0 23 1.0

) y = -0.0361x-0.00717 S 24 ) R

0 2 ] 25 ( R = 0.990 T -0.5 n A M

26 / [ ) / t s ( 27 s i T d 0.5 ] 28 A ( n n l 29 -1.0 M [ 30 31 (C) (D) 32 -1.5 0.0 33 0 10 20 30 0 10 20 30 34 Time (Hour) Time (Hour) 35 36 37 38 39 Figure 5. Oxidation of 50 µM phenyllactic acid by 500 M HMO at pH 4.0 (5.0 mM acetate 40 buffer, 10 mM NaCl). (A) and (B) are time course results, while (C) is a semi-log plot for 41 42 phenyllactic acid loss. (D) explores reaction stoichiometry using [Mn]diss/[Mn]RS ratios. 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 38 59 60 Page 39 of 41 Environmental Science: Processes & Impacts

1 2 3 4 5 50 Mandelic acid (A) (B) (C) (D) 6 Phenylglyoxylic acid Benzaldehyde Benzoic acid 7 8 40 No additives Phosphate 9 )

10  30 Pyrophosphate  ( 11 i ] 12 [ 20 13 14 10 15 16 0 17 0 10 20 30 0 10 20 30 0 10 20 30 0 10 20 30 18 Time (Hour) Time (Hour) Time (Hour) Time (Hour) 19 20 21 22 0 [Mn] (F) (G) (E) 80 diss [Mn] 1.0 23 RS S

24 R ] ) -1 ) n

25 ) 60 0 ( M [ T 

26 /  s A ( s / i

) 27 y = -0.0534x-0.00911 d ] t -2 ]

( 2 n 40 n 28 T R = 0.999 0.5 M A M [ ( 29 [

30 l n y = -0.256x-0.0225 -3 2 20 Black: No additives 31 R = 0.998 y = -0.136x-0.0152 Blue: Phosphate 32 R2 = 0.999 Red: Pyrophosphate 33 -4 0 0.0 34 0 10 20 30 0 10 20 30 0 10 20 30 35 Time (Hour) Time (Hour) Time (Hour) 36 37 38 Figure 6. pH 4.0 experiments examining the effects of adding 500 M phosphate or pyrophosphate on the reaction of mandelic acid 39 with HMO. (A) Mandelic acid, (B) phenylglyoxylic acid, (C) benzaldehyde, and (D) benzoic acid product time course plots. (E) Semi- 40 log plots for mandelic acid loss. (F) [Mn]diss and [Mn]RS and (G) [Mn]diss/[Mn]RS as a function of time. 41 42 43 39 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Environmental Science: Processes & Impacts Page 40 of 41

1 2 3 4 Mandelic acid 5 Phenyllactic acid 6 7 50 50 8 MOPS 9 40 40 ) ) MOPS 10 Mandelic acid Phenyllactic acid    11 30  30 Phenylpyruvic acid ( Phenylglyoxylic acid (

] ] i 12 Mn i Phenylacetic acid [ diss [ Mn 13 20 Mass balance 20 diss 14 Mass balance 15 10 10 (C) 16 (A) 17 0 0 18 0 20 40 60 80 100 0 50 100 150 19 20 21 50 50 22 Phosphate 23 40 40 ) 24 ) Phosphate  25   30  30 ( (

]

26 ] i i [ 27 20 [ 20 28 (D) 29 10 10 30 (B) 31 0 0 32 0 20 40 60 80 100 0 50 100 150 33 Time (Hour) Time (Hour) 34 35 36 37 Figure 7. pH 7.0 experiments. Oxidation of 50 µM (A-B) mandelic acid or (C-D) phenyllactic 38 acid by 500 µM HMO medium employing 2.0 mM MOPS or phosphate buffer. An ionic strength 39 of 15 mM was fixed using NaCl addition. 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 40 59 60 Page 41 of 41 Environmental Science: Processes & Impacts

1 2 3 4 Phenyllactic acid 5 Mandelic acid 80 6 7 50 50 8 60 9 ) 40 40  )

10  ) ( 

s Phenyllactic acid  11  30 Mandelic acid 30 s ( i

40 

d ] ( Phenylpyruvic acid ] i 12 Phenylglyoxylic acid [ ] n i Phenylacetic acid 13 [ 20 Mass balance 20 M

[ Mass balance 14 20 Control (E) 15 With MA 10 10 With PLA 16 (A) (C) 17 0 0 0 18 0 50 100 150 0 20 40 60 80 100 0 50 100 150 19 20 80 80 80 21 22 60 60 60

23 ) ) ) 24  M  M  ( 

(

25 ( s [Mn]

s 40 40 diss s 40 i s s i d s III 26 i ] d [Mn] d [Mn ] ] diss ] n diss

27 n III n II

M [Mn ]

[ diss [Mn ] M M 28 diss [ 20 [ 20 II 20 [Mn ] 29 diss 30 (B) (D) 31 0 0 0 (F) 32 0 5 10 15 0 20 40 60 80 100 0 50 100 150 33 Time (Hour) TIme (Hour) TIme (Hour) 34 35 36 37 Figure 8. pH 7.0 experiments employing 2 mM pyrophosphate buffer. Reactions were performed in substrate-free suspension 38 (control) or in suspensions containing 50 µM mandelic acid (MA) or phenyllactic acid (PLA). Suspensions all contained 500 µM 39 HMO. An ionic strength of 15 mM was fixed using NaCl addition. (A-B) Concentrations of dissolved Mn in 150 hours and the first 15 40 hours. (C) and (E) show concentrations of organic substrates. (D) and (F) show concentrations of dissolved MnII and MnIII. 41 42 43 41 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60