Lactate-mediated medium-chain fatty acid production from expired dairy and beverage waste

Fruits, vegetables, and dairy products are typically the primary sources of household food waste. Currently, anaerobic digestion is the most used bioprocess for the treatment of food waste with concomitant generation of biogas. However, to achieve a circular carbon economy, the organics in food waste should be converted to new chemicals with higher value than energy. Here we demonstrate the feasibility of medium-chain carboxylic acid (MCCA) production from expired dairy and beverage waste via a chain elongation platform mediated by lactate. In a two-stage fermentation process, the first stage with optimized operational conditions, including varying temperatures and organic loading rates, transformed expired dairy and beverage waste into lactate at a concentration higher than 900 mM C at 43 °C. This lactate was then used to produce >500 mM C caproate and >300 mM C butyrate via microbial chain elongation. Predominantly, lactate-producing microbes such as Lactobacillus and Lacticaseibacillus were regulated by temperature and could be highly enriched under mesophilic conditions in the first-stage reactor. In the second-stage chain elongation reactor, the dominating microbes were primarily from the genera Megasphaera and Caproiciproducens, shaped by varying feed and inoculum sources. Co-occurrence network analysis revealed positive correlations among species from the genera Caproiciproducens, Ruminococcus, and CAG-352, as well as Megasphaera, Bacteroides, and Solobacterium, indicating strong microbial interactions that enhance caproate production. These findings suggest that producing MCCAs from expired dairy and beverage waste via lactate-mediated chain elongation is a viable method for sustainable waste management and could serve as a chemical production platform in the context of building a circular bioeconomy.


Continuous operation of fermentation reactor
For the continuous operation of the fermentation reactor, first, three different temperatures were tested for their impact on lactate concentration and production rate using a heating jacket: Period F-I (30 °C), Period F-II (35 °C), and Period F-III (43 °C).The CM medium stored at 4 °C was diluted with tap water (TW) at a ratio of 1:2 before being used as the influent to the fermentation reactor in Period F-I to F-III, and the hydraulic retention time (HRT) was 4 days.Next, we examined the effect of increasing the organic loading rate on the performance of the fermentation reactor.The HRT was adjusted to 2 days in Period F-IV and F-V, and to further increase the organic loading rate CM with no dilution was used as a feedstock in Period F-V.

Feed substrates during Periods CE-I to CE-VI in chain elongation reactor
Synthetic medium containing 800 mM-C ethanol (EtOH, Sigma-Aldrich) and 400 mM-C sodium acetate (Ac, Sigma-Aldrich) was initially fed into the reactor in Period CE-I to enrich for chain elongation microbes.Given that the main product in the fermentation reactor was lactate, the influent for the chain elongation reactor was adjusted in Periods CE-II to CE-V by gradually adding DL-lactic acid (LA, Sigma-Aldrich) to 1050 mM-C and reducing the concentration of ethanol and acetate to 0 mM-C in Period CE-V.In Period CE-VI, unfiltered and unsterilized fermentation reactor effluent, containing around 810-840 mM-C lactate, was used as the feed for the chain elongation reactor, to test the feasibility of chain elongation platform for the production of MCCAs through a two-stage fermentation process using expired dairy and beverage waste as raw materials.

MCCA extraction from chain elongation reactor
One external extraction system made of hollow-fiber membranes was constructed for the continuous forward and reverse extraction of MCCAs from the chain elongation bioreactor, similar to our previous report [1].The chain elongation broth was continuously circulated through the exterior space of the forward membrane module at a flow rate of 50 ml min −1 .Mineral oil solvent (VWR) with 3% tri-n-octylphosphine oxide (TOPO) (Alfa Aesar) was used as the hydrophobic forward extraction solvent at an upflow rate of 30 ml min -1 , while the alkaline solution buffered with 0.2 M boric acid was utilized for the reverse extraction, which was maintained at a pH of 9-11 with manual addition of 2 M sodium hydroxide solution every 4-7 days.

DNA extraction
DNA extraction of the samples was done using a slightly modified version of the standard protocol for FastDNA Spin kit for Soil (MP Biomedicals, USA) with the following exceptions: 500 μL of sample, 480 μL Sodium Phosphate Buffer and 120 μL MT Buffer were added to a Lysing Matrix E tube.Bead beating was performed at 6 m/s for 4x40s [2].Gel electrophoresis using Tapestation 2200 and Genomic DNA screentapes (Agilent, USA) was used to validate product size and purity of a subset of DNA extracts.DNA concentration was measured using Qubit dsDNA HS/BR Assay kit (Thermo Fisher Scientific, USA).

Sequencing library preparation
Amplicon libraries for the archaea/bacteria/eukarya 16S/18S rRNA gene variable regions 4-8 (abeV48-A) were prepared using a custom protocol.Up to 25 ng of extracted DNA was used as template for PCR amplification of the archaea/bacteria/eukarya 16S/18S rRNA gene variable regions 48 (abeV48A).Each PCR reaction (50 μL) contained 0.5 mM dNTP mix, 0.01 units of Platinum SuperFi DNA Polymerase (Thermo Fisher Scientific, USA), and 500 nM of each forward and reverse primer in the supplied SuperFI Buffer.PCR was done with the following program: Initial denaturation at 98 ∘ C for 3 min, 25 cycles of amplification (98 ∘ C for 30 s, 62 ∘ C for 20 s, 72 ∘ C for 2 min) and a final elongation at 72 ∘ C for 5 min.The forward and reverse primers used include custom 24 nt barcode sequences followed by the sequences targeting the archaea/bacteria/eukarya 16S/18S rRNA gene variable regions 4-8 (abeV48-A): [515FB] GTGYCAGCMGCCGCGGTAA and [1391R] GACGGGCGGTGWGTRCA [3,4].The resulting amplicon libraries were purified using the standard protocol for CleanNGS SPRI beads (CleanNA, NL) with a bead to sample ratio of 3:5.DNA was eluted in 25 μL of nuclease free water (Qiagen, Germany).Sequencing libraries were prepared from the purified amplicon libraries using the SQK-LSK110 kit (Oxford Nanopore Technologies, UK) according to manufacturer protocol with the following modifications: 500 ng total DNA was used as input, and CleanNGS SPRI beads for library clean-up steps.DNA concentration was measured using Qubit dsDNA HS Assay kit (Thermo Fisher Scientific, USA).Gel electrophoresis using Tapestation 2200 and D1000/High sensitivity D1000 screentapes (Agilent, USA) was used to validate product size and purity of a subset of amplicon libraries.

Figure S1
Figure S1 Stacked area charts for the production rate of lactate and volatile fatty acids during

Figure S2
Figure S2 COD conversion efficiency and lactate selectivity calculated based on mM-C among

Figure S4
Figure S4 Heatmap of the relative abundance of the top 20 operational taxonomic units in the

Figure S5
Figure S5 Heatmap of the relative abundance of the top 20 operational taxonomic units in the

Figure S6
Figure S6 Heatmap of the relative abundance of the top 20 operational taxonomic units in the

Table S1
Comparison of caproate production via lactate-driven chain elongation a CSTR, continuously stirred tank reactor.b UASB, upflow anaerobic sludge blanket.c N/A, data not available.d RA, relative abundance of the dominant caproate-producing microbial species.