Supplemental Information. Temperature-Phased Conversion of Acid. Whey Waste Into Medium-Chain Carboxylic. Acids via Lactic Acid: No External e-donor

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1 JOUL, Volume 2 Supplemental Information Temperature-Phased Conversion of Acid Whey Waste Into Medium-Chain Carboxylic Acids via Lactic Acid: No External e-donor Jiajie Xu, Jiuxiao Hao, Juan J.L. Guzman, Catherine M. Spirito, Lauren A. Harroff, and Largus T. Angenent

2 Equations Volumetric LFLA loading rate (mmol C L -1 d -1 ): (!!!"!!!!!!!"!)!! Where: C L = concentration of lactose in influent, mm C F = concentration of fructose in influent, mm C LA = concentration of lactic acid in influent, mm f = effluent flow rate, L d -1 V = the volume of reactor, L (Eq. S1) Volumetric SCOD loading rate (g COD L -1 d -1 ):!"#$!! Where: (Eq. S2) SCOD = the concentration of soluble chemical oxygen demand in influent, g COD L -1 f = effluent flow rate, L d -1 V = the volume of reactor, L Volumetric production rate on day n (g COD L -1 d -1 ): Where:!!,!!!"# + (!!,!!!!,!!! )!!!!!!!!!!!"""# (Eq. S3) C!,! = concentration of carboxylic acid in effluent on the day n, mm V = volume of reactor, L HRT = hydraulic retention time on the day n, d C!,!, C!,!!! = concentrations of carboxylic acid in the stripping solution on the day n and n-1, mm V! = volume of the stripping solution on the day n, L T! = the day n, d M = conversion factor from mmol to g COD, g O 2 mmol -1 ; for example, acetic acid was g O 2 mmol -1 (Table S1) Product-to-CA production ratio (% mmol C): γ s!!!! γ i Where: γ! = production rate of specific product, mmol C L -1 d -1 γ! =production rate of all carboxylic acids, mmol C L -1 d -1 (Eq. S4)

3 Substrate-into-product conversion efficiency (% mmol C): γ s!!!!!!!!! /! Where: γ! = production rate of specific product, mmol C L -1 d -1 C i = concentration of specific substrate, mm f = effluent flow rate, L d -1 N i = the number of carbons in a specific substrate V = the volume of reactor, L (Eq. S5) SCOD conversion efficiency (% g COD) Where: γ s!"#$!" (Eq. S6) γ! = production rate of specific product, g COD L -1 d -1 SCOD LR = volumetric SCOD loading rate, g COD L -1 d -1

4 Figures Figure S1. Same as Fig. 2 except that the preliminary experiment with the same thermophilic bioreactor is included. The operating and performance parameters of the lactic acid-producing bioreactor are shown with the volumetric lactic acid production rate (A) and the effluent concentrations (B). The period of Day -150 to Day 0 (Day 0 is the same as in Fig. 2-3) represent the time period that acid whey from the Fage yogurt plant from 2/17/2016 was used as the substrate. Period A-I and Period A-II represent the time period when acid whey from Byrne (10/4/2016) was fed. On Day -12 this bioreactor became the Phase A of the temperaturephased system. The data represents a 6-day moving average (each average contains three sampling points).

5 Figure S2. Same as Fig. 3 except that the preliminary experiment for the same mesophilic bioreactor is included. On Day -12 the single bioreactor became the Phase B bioreactor of the temperature-phased system. The operating and performance parameters of chain-elongating bioreactor are shown with the volumetric carboxylic acid production rates (A) and the effluent carboxylic acid concentrations (B). The period of Day -150 to Day 0 (Day 0 is the same as in Fig. 2-3) represent the time period that acid whey from the Fage yogurt plant from 2/17/2016 was used as the substrate. Period B-I, Period B-II, and Period A-III represent the time period when acid whey from Byrne (10/4/2016) was fed into the temperature-phased system with effluent from the Phase A bioreactor being used as the influent for Phase B. The data represents a 6-day moving average (each average contains three sampling points).

6 Figure S3. Same as Fig. 2, but without the 6-day moving average. Sampling was performed each other day. The operating and performance parameters of the lactic acid-producing bioreactor (Phase A bioreactor) are shown with the volumetric lactic acid production rate (A) and the effluent concentrations (B).

7 Figure S4. Same as Figure 3, but without the 6-day moving average. Sampling was performed each other day. The operating and performance parameters of chain-elongating bioreactor (Phase B bioreactor) are shown with the volumetric carboxylic acid production rates (A) and the effluent carboxylic acid concentrations (B).

8 Figure S5. Biogas production (A) and biomass concentration (OD 600 ) (B) for the Phase A bioreactor during Periods A-I and A-II. During Period A-I, a gravity-settler unit retained biomass in the bioreactor. During Period A-II, a hollow-fibre membrane module was installed instead of the gravity settler. The data represents a 6-day moving average (each average contains three sampling points).

9 Figure S6. Biogas production for the Phase B bioreactor during the three experimental periods. The data represents a 6-day moving average (each average contains three sampling points).

10 Figure S7. Relative abundance during the operating period of the three OTUs that reached a higher than 1% relative abundance in the Phase A bioreactor. The lowest level taxonomic classification of the OTU is provided as well as the OTU ID.

11 Figure S8. Heat map of relative OTU abundances of four microbiome samples from Phase B (Days 0, 12, 68, and 92). The 26 OTUs listed comprised at least 1% of the relative abundance for one or more of the microbiome samples collected. Phylogenetic similarity is indicated. The OTUs are named based on the lowest taxonomic level to which they could be classified. In addition, the two Lactobacillus spp. OTUs, which appeared at a relatively high abundance in the Phase A bioreactor, are indicated by their ID numbers.

12 Figure S9. Comparison of the microbial community between the nine bioreator samples via the β-diversity analysis program UniFrac. First two axes (PCo1 and PCo2) of principal coordinate analysis (PCoA) based on unweighted (A) and weighted (B) Unifrac distances. Phase A bioreactor samples (Days 0, 12, 48, 62, and 92) are represented by diamonds and Phase B bioreactor samples (Days 0, 12, 68, and 92) are represented by circles. The color scale represents the sampling day (white=day 0 to dark blue=day 92). The weighted UniFrac shows that the composition of the microbiome for the Phase A bioreactor did not change much during the operating period. Microbiomes in Phase A and B were different from each other during the operating period.

13 Figure S10. Setup for the Phase A bioreactor during Period A-I (lactic acid producing bioreactor). (A) Process flow diagram showing the feed container, bioreactor, gravity settler, and effluent container. (B) Picture of the actual bioreactor and gravity-settler setup.

14 Tables

15 Table S1. Carboxylic acid chemical and physical information. Name Lipid number Molecular Solubility in water (g L -1 ) Mass (g mol -1 ) Diagram 1 Exchangeable electrons per C-atom 2 SCOD conversion (g COD mmol -1 ) Acetic acid C2:0 C 2 H 4 O 2 Miscible Lactic acid - C 3 H 6 O 3 Miscible Propionic acid C3:0 C 3 H 6 O 2 Miscible n-butyric acid C4:0 C 4 H 8 O 2 Miscible n-valeric acid C5:0 C 5 H 10 O n-hexanoic acid (n-caproic acid) C6:0 C 6 H 12 O n-heptanoic acid C7:0 C 7 H 14 O n-octanoic acid (n-caprylic acid) C8:0 C 8 H 16 O n-nonanoic acid C9:0 C 9 H 18 O FAVRE, H. A. & POWELL, W. H Nomenclature of organic chemistry : IUPAC recommendations and preferred names 2013, Royal Society of Chemistry. 2 HANSELMANN, K. W Microbial energetics applied to waste repositories. Cellular and Molecular Life Sciences, 47, SEIDELL, A Solubilities of inorganic and organic substances: a compilation of quantitative solubility data from the periodical literature. 2 ed. New York: D. van Nostrand Company The Merck Index: An Encyclopedia of Chemicals, Drugs, and Biologicals. 15 ed.: Royal Society of Chemistry Encyclopedia of Molecular Cell Biology and Molecular Medicine. In: MEYERS, R. A. (ed.) 2 ed.: Wiley-Blackwell.

16 Table S2. Composition of three different acid-whey feedstocks. Each of the feedstocks was evaluated by analyzing six samples, and the error represents the standard deviation. The acid whey that was collected on 10/21/2015 was only used for this analysis, while the acid whey from 2/17/2016 was used for the preliminary experiments (Day -150 until Day 0 in Fig. S1-S2). Acid whey collected on 10/4/2016 was used for all periods for the temperature-phased bioconversion system (Day 0 until Day 90 in Fig. 2-3). Collection date 10/21/2015 2/17/ /4/2016 Collection site Fage (Johnstown, NY) Fage (Johnstown, NY) Byrne Hollow Farm (Cortland, NY) ph 4.48 ± ± ± 0.02 TS (g L -1 ) ± ± ± 3.72 VS (g L -1 ) ± ± ± 4.27 TCOD (g COD L -1 ) 52.2 ± ± ± 1.00 SCOD (g COD L -1 ) 50.5 ± ± ± 1.00 Ammonia (mg/l) ± ± ± 1.63 Lactose (mm) ± ± ± 1.28 Fructose (mm) ± ± ± 0.89 Lactic acid (mm) ± ± ± 0.97 Lactose (mm C) 1,240 ± ,031 ± ,429 ± 15.4 Fructose (mm C) ± ± ± 5.34 Lactic acid (mm C) ± ± ± 2.91 Lactose* (g COD L -1 ) ± ± ± 0.49 Fructose* (g COD L -1 ) 6.73 ± ± ± 0.17 Lactic acid* (g COD L -1 ) ± ± ± 0.09 Calculated SCOD* (g COD L -1 ) Calculated SCOD*-to-SCOD ratio * COD conversions are: 0.384, 0.192, and g COD mmol -1 for lactose, fructose, and lactic acid, respectively. The total calculated SCOD includes only lactose, fructose, and lactic acid, which are the predominate sources of SCOD in acid whey.

17 Table S3. The operating and performance parameters for the Phase A bioreactor. The parameters are corrected to the volume of the Phase A bioreactor only. Volumetric loading rates were calculated with mmol C and g COD, volumetric production rates and product-to-ca ratios were calculated with mmol C, while the conversion efficiencies were calculated with both mmol C and g COD for carbon products of the Phase A bioreactor for Period A-I and A-II. LF means lactose and fructose, while LA means lactic acid with LFLA being a combination of lactose, fructose, and lactic acid. CO 2 SCCA without LA (C2, C3, C4, and C5) Lactic acid (LA) Phase A bioreactor Period A-I Period A-II Volumetric LFLA loading rate (mmol C L -1 d -1 ) 1642 ± ± Volumetric SCOD loading rate (g COD L -1 d -1 ) ± ± 0.85 Wet volume (L) Flow rate (L d -1 ) HRT (d) Influent dilution of acid whey (times) 100% (0) 100% (0) Volumetric production rate (mmol C L -1 d -1 ) Volumetric production rate (mmol C L -1 d -1 ) 8.21 ± ± 1.52 Volumetric production rate (mmol C L -1 d -1 ) 815 ± ± 16.4 Volumetric conversion rate (minus LA in influent) (mmol C L -1 d -1 ) 583 ± ± 13.9 LA-to-CA production ratio (% mmol C) ~100 ~100 LA-to-SCOD effluent conc. ratio (% g COD) LF-into-LA conversion efficiency (% mmol C) SCOD conversion efficiency (% g COD) BD: Below detection (<0.24 mm C, 0.36 mm C, 0.48 mm C, and 0.60 mm C for C2, C3, C4, and C5, respectively) BD BD

18 Table S4. The operating and performance parameters for the Phase B bioreactor. The parameters are corrected to the volume of the Phase B bioreactor only. Volumetric loading rates were calculated with mmol C and g COD, volumetric production rates and product-to-ca ratios were calculated with mmol C, while the conversion efficiencies were calculated with both mmol C and g COD for carbon products of the Phase B bioreactor for Period B-I, B-II, and B-III. CA is carboxylic acid, SCCA is short-chain carboxylic acid, MCCA is medium-chain carboxylic acid, and LA is lactic acid.

19 Phase B bioreactor Period B-I Period B-II Period B-III Volumetric LA loading rate (mmol C L -1 d -1 ) 183 ± ± ± 2.76 Volumetric SCOD loading rate (g COD L -1 d -1 ) BD: Below detection (<0.3 mm C for lactic acid) Wet volume (L) Flow rate (L d -1 ) HRT (d) Influent dilution of Phase A effluent (times) 40% (2.5) 24% (4.17) 51% (1.96) Effluent from Phase A to Phase B (times) 22.5% (4.46) 15.6% (6.27) 16.8% (5.94) CO 2 Volumetric production rate (mmol C L -1 d -1 ) 1.61 ± ± ± 0.04 CH 4 Volumetric production rate (mmol C L -1 d -1 ) 2.10 ± ± ± 0.04 Lactic acid (LA) SCCA without LA (C2, C3, C4, and C5) n-caproic acid (C6) n-heptanoic acid (C7) n-caprylic acid (C8) n-nonanoic acid (C9) MCCA (C6, C7, C8, and C9) Volumetric production rate (mmol C L -1 d -1 ) BD BD BD Volumetric production rate (mmol C L -1 d -1 ) ± ± ± 6.77 (SCCA no LA)-to-CA production ratio (% mmol C) LA-into-(SCCA no LA) conversion efficiency (% mmol C) SCOD conversion efficiency (% g COD) Volumetric production rate (mmol C L -1 d -1 ) 72.2 ± ± ± 4.52 C6-to-CA production ratio (% mmol C) LA-into-C6 conversion efficiency (% mmol C) SCOD conversion efficiency (% g COD) Volumetric production rate (mmol C L -1 d -1 ) 11.0 ± ± ± 0.04 C7-to-CA production ratio (% mmol C) LA-into-C7 conversion efficiency (% mmol C) SCOD conversion efficiency (% g COD) Volumetric production rate (mmol C L -1 d -1 ) 19.6 ± ± ± 1.05 C8-to-CA production ratio (% mmol C) LA-into-C8 conversion efficiency (% mmol C) SCOD conversion efficiency (% g COD) Volumetric production rate (mmol C L -1 d -1 ) 1.03 ± ± ± 0.01 C9-to-CA production ratio (% mmol C) LA-into-C9 conversion efficiency (% mmol C) SCOD conversion efficiency (% g COD) Volumetric production rate (mmol C L -1 d -1 ) 104 ± ± ± 5.62 MCCA-to-CA prod. ratio (% mmol C) LA-into-MCCA conversion efficiency (% mmol C) SCOD conversion efficiency (% g COD)

20 Table S5. The operating and performance parameters for the overall temperature-phased system. A carbon balance is shown to estimate the LFLA-into-measured carbon conversion efficiency (% mmol C) by including the loading rates and production rates of all measured carbon substrates (lactose, fructose, and lactic acid [LFLA]) and products (CO 2, CH 4, SCCAs, and MCCAs). Since the volumetric lactic acid production rates for the thermophilic Phase A bioreactor were considerably larger than the mesophilic volumetric MCCA production rates, we pretended for this balance that the Phase B bioreactor volume had been large enough to convert all produced lactic acid from the Phase A bioreactor. To do this, the actual production rates from the Phase B bioreactor were multiplied by the times from Effluent from Phase A to Phase B in Table S4. For example, during Period B-I the multiplication was The mesophilic production rates for Phase B in this table are, therefore, larger than what we actually observed. This is clearly noted by an *. Daily loading rates were calculated with mmol C and g COD, daily production rates were calculated with mmol C, while the conversion efficiencies were calculated with both mmol C and g COD for carbon products of the Phase A and Phase B bioreactors for Period A-I and B-I, A-II and B-II, and A-II and B-III. The overall SCOD conversion efficiencies include both Phase A and B bioreactors, and represent the conversion of soluble substrates from acid whey into SCCAs and MCCAs.

21 CO 2 CH 4 SCCA without LA (C2, C3, C4, and C5) MCCA (C6, C7, C8, and C9) Lactic acid (LA) Overall temperature-phased system Period A-I and B-I Period A-II and B-II Period A-II and B-III LFLA loading rate (mmol C d -1 ) 903 ± ± ± 11.1 SCOD loading rate (g COD d -1 ) 29.1 ± ± ± 0.47 Production rate Phase A (mmol C d -1 ) Production rate Phase B* (mmol C d -1 ) Total production rate* (mmol C d -1 ) LFLA-into-CO 2 conversion efficiency (% mmol C) Production rate Phase B* (mmol C d -1 ) LFLA-into-CH 4 conversion efficiency (% mmol C) Production rate Phase B* (mmol C d -1 ) LFLA-into-(SCCA no LA) conversion efficiency (% mmol C) Overall SCOD conversion efficiency (% g COD) Production rate Phase B* (mmol C d -1 ) LFLA-into-MCCA conversion efficiency (% mmol C) Overall SCOD conversion efficiency (% g COD) Production (left-over) rate Phase B* (mmol C d -1 ) 4.51 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± BD BD BD Sum of measured carbon production rates (mmol C d -1 ) LFLA-into-measured carbon conversion efficiency (% mmol C) * Corrected with Effluent from Phase A to Phase B by multiplying by the volume of Phase B that would have been necessary to convert all substrate in the effluent of Phase A (times in Table S4). BD: Below detection (<0.3 mm C for lactic acid)

22 Table S6. Alpha diversity for the Phase A bioreactor samples (Days 0, 12, 48, 62, and 92) and the Phase B bioreactor samples (Days 0, 12, 68, and 92). Mean values and standard deviations of three different alpha diversity metrics (based on ten rarefactions at a depth of 36,330 sequences per sample for each sample): Shannon diversity, Gini coefficient, and observed OTUs are reported for each set of bioreactor samples. Phase Shannon Diversity Gini Coefficient Observed OTUs Phase A ± ± ± 4 Phase B ± ± ± 35