Mixed-culture fermentation (MCF) is a key central process to enable next generation biofuels and biocommodity production due to economic and process advantages over application of pure cultures. However, a crucial limitation to the application of MCF is predicting culture product response to environmental stimuli. Certain effects, such as product formation response to pH1,2 and microbiome response to substrate type3, have been well characterized. Other stimuli, such as substrate loading rate, have not been characterized despite some intriguing reports in the literature. Additionally, mixed culture metabolic response is not well understood under any of these conditions, limiting the accuracy of MCF models which currently assume static metabolic arrays4,5.
It is has been shown that volatile fatty acid (VFA) production increases during sudden increases in the organic loading rate in anaerobic reactors6,7. In particular, mixed cultures under these conditions are known to produce elevated levels of lactate and propionate, each valuable chemicals for bioplastics production and in the food industry. These â??shock loadsâ? have previously been maintained for short periods of time, but have not yet been sustained for production at steady state. It is notable, however, that lactate and propionate production remained elevated throughout the duration of the shock loads. Cells use production of lactate and propionate to sink excess electrons and maintain a metabolic redox balance. By contrast, pathways for acetate and butyrate production are used for ATP generation to support cell growth and maintenance. Extended elevated production of lactate and propionate is therefore counterintuitive, especially under conditions where more substrate is available for biomass generation. The ultimate goal of this experiment is to determine the product formation effects of long-term elevated organic loading rates, and whether lactate and propionate production remain elevated beyond a particular loading rate threshold. Product formation trends will then be correlated to enzyme expression to assess metabolic function and signalling dynamics.
A 1.3L chemostat, inoculated with biomass from an anaerobic digester in Brisbane, Australia, has been operated at pH 5.50, 30.0°C, and a dilution rate of 1.0 d-1 for over 90 days. The substrate is glucose in basal anaerobic (BA) media8, acting as a synthetic waste water feed. Initially, the glucose concentration in the feed was 5g/L, and has been stepped up to 10g/L
and then 20g/L, each after a period of steady state was established. The glucose feed concentration will also be increased to 50g/L and then 80g/L. These correspond to loading rates of 5, 10, 20, 50, and 80g/L*d.
Temperature, pH, and gas flow production rate were recorded continuously using LabVIEW. Liquid samples were taken regularly for chemical, microbiome, and metatranscriptome analysis. VFA concentration was measured via HPLC. All cell samples were stored in RNAlater® solution (Life Technologies, NY, USA) at -20?C prior to analysis. The microbiome was analysed via fluorescent in situ hybridisation (FISH) supplemented with 16s rRNA pyrotag sequencing. DNA was extracted for sequencing with the PowerSoil® DNA Isolation Kit (MO BIO, CA, USA). RNA was extracted with the RiboPure� RNA Purification Kit, and then mRNA was purified from total RNA for metatranscriptome sequencing with the MICROBExpress� Bacterial mRNA Enrichment Kit (both by Life Technologies, NY, USA).

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Figure 1: a) Primary VFA production during 5g/L*d loading rate phase. b) Primary and secondary VFA production during 10g/L*d loading rate phase. c) Primary and secondary VFA production during 20g/L*d loading rate phase.

Initial analysis of the steady state product response has been consistent with the literature at the given operational conditions1,2. The initial phase of operation, at a loading rate of 5g/L*d (Figure 1a), butyrate (averaging 0.19g/L), acetate (0.33g/L), lactate (0.66g/L), and propionate (0.21g/L) are all primary products. Butyrate and acetate production are relatively stable, whereas propionate and lactate production are more variable.
During the second phase, with a loading rate of 10g/L*d (Figure 1b), butyrate (0.61g/L) and acetate (0.63g/L) become the primary products after reaching steady state, whereas lactate (0.16g/L) and propionate (0.16g/L) become secondary products. Interestingly, lactate and propionate each spike initially in response to the increased loading rate before giving way to butyrate and acetate production. This is consistent with the literature reports of lactate and propionate spiking during shock loads6. It is not clear at this point why lactate and propionate production again increase toward the end of this phase.
During the third phase, with a loading rate of 20g/L*d (Figure 1c), lactate and propionate again spike in response to the loading rate increase, though the lactate response is notably more pronounced than in the second phase. Following this spike, the culture takes considerably longer to reach steady state production in the third phase compared to the
a)

b) c)

Figure 2: a) Enterobacteriaceae, identified with EBAC1790 probe and shown in magenta, interspersed in bacteria cluster, identified with EUB338 probe and shown in blue. Extracellular polymeric substance (EPS) shown in green. b) Propionicimonas, identified with Pro60 and shown in orange, among bacteria cluster, identified with EUB338 and shown in green. EPS is shown in blue. c) Pseudomonas, identified with PSE227

and shown in yellow/orange, among bacteria cluster, identified with EUB338 and shown in green. EPS is shown in blue.

second phase (roughly 10 days compared to 5 days). However, upon reaching steady state butyrate (6.03g/L) becomes the primary product, acetate (1.14g/L) the secondary product, and lactate (0.13g/L) and propionate (0.37g/L) the tertiary products. It is anticipated that the lactate and propionate spikes will become more pronounced and longer lasting with continuing research into substrate loading rates of 50 and 80g/L*d.
FISH has so far revealed preliminary information about the microbiome. Roughly 25% of the bacteria present are of the class Gammaproteobacteria, and of these greater than 90% are of the family Enterobacteriaceae (Figure 2a). Bacteria of this family have previously been shown to be a dominant species under these operating conditions2, particularly species of
Klebsiella. Species of Clostridia have also been shown to be dominant in similar operating conditions2, however our probes for Clostridia have so far been inconclusive. Propionicimonas (Figure 2b) and Psuedomonas (Figure 2c) were also observed with FISH analysis, each making up 1% or less of the total microbiome. Klebsiella and Psuedomonas are both known for 2,3-butanediol production following exponential growth phase9. Clostridia is generally associated with butyrate production, but certain species are also associated with butanol and acetone production10. Further chemical analysis will reveal whether these products are present, and 16s rRNA pyrotag results will guide further FISH analysis. These results will then be used to construct a metagenome for metatranscriptome analysis. It is anticipated that fermentation enzyme expression levels will be associated with activity of redox repressor enzymes11. This set of enzymes is known to regulate transcription of enzymes associated with electron sinking in response to changes in NAD redox state.
While further experimentation is needed to fully characterize the effect of substrate loading rate on the MCF product spectrum, the results to this point are consistent with continuous MCF systems run at similar conditions. Additionally, the spikes in lactate and propionate production in the transition stages between steady states are consistent with known shock load effects. These results, along with shock load studies in the literature, suggest the possibility of elevated and sustained lactate and propionate production at high loading rates. It is anticipated that metatranscriptomic analysis will reveal correlation of product spectrum to activity of redox repressor enzymes. Full characterization of the effects of substrate
loading rate will add valuable knowledge to the understanding of mixed-culture fermentation dynamics.
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