Does glucose affect the de‐esterification of methyl ferulate by Lactobacillus buchneri?

Abstract Silage, the fermented product from anaerobic storage of forage crops with high water contents (50%–70%), is normally used as animal feed but also for the production of biofuels and value‐added products. To improve the utilization of plant fibers during ensiling, previous attempts have aimed at breaking linkages between lignin and hemicellulose by use of Lactobacillus buchneri LN 4017 (ATCC PTA‐6138), a feruloyl esterase (FAE)‐producing strain, but results have been inconsistent. Normally, there are sufficient amounts of readily available substrates for bacterial growth in silage. We thus hypothesized that the inconsistent effect of L. buchneri LN 4017 on the digestibility of silage fibers is due to the catabolic repression of FAE activity by substrates present in silage (e.g., glucose). To test this hypothesis, we analyzed the effect of glucose on the de‐esterification of methyl ferulate (MF), a model substrate used for FAE activity assays. At three glucose:MF ratios (0:1, 1:1, and 13:1), the bacteria continued hydrolyzing MF with increasing glucose:MF ratios, indicating that the de‐esterification reaction was not repressed by glucose. We therefore conclude that the de‐esterification activity of L. buchneri LN 4017 is not repressed by silage substrates during ensiling.

ester bonds with arabinose residues of xylan chains on one side and ether bonds with lignin on the other side (Ralph, 2010;Wong, 2006). The lignin-hemicellulose matrix encrusts the cellulose, and this overall configuration results in recalcitrance of plant fibers (Pu, Hu, Huang, Davison, & Ragauskas, 2013;Rubin, 2008). The ester link between FA and hemicelluloses can be cleaved by feruloyl esterases (FAEs) (EC3.1.1.73), thereby opening the fiber structure and increasing the bioavailability of fiber constituents for fermentation processes.
L. buchneri LN 4017 (ATCC PTA-6138), a FAE-producing strain (Nsereko et al., 2008), was used in several studies as the silage inoculant. While fiber digestibility was improved in some cases (Jin et al., 2015;Kang, Adesogan, Kim, & Lee, 2009), no improvement was found in other studies (Kang et al., 2009;Lynch, Baah, & Beauchemin, 2015). One possible explanation for these inconsistent results could be a catabolic repression of FAE activity of the inoculant, caused by the readily available substrates in the silage (e.g., glucose). Such a hypothesis would parallel previous findings with Aspergillus niger, where FA induces expression of FAE genes (faeA and faeB) but fails to induce the expression of these genes in the presence of glucose (de Vries, vanKuyk, Kester, & Visser, 2002). In the present study, we aimed at investigating the effects of varying glucose concentrations on the hydrolytic conversion of methyl ferulate (MF) to FA, a reaction indicative for FAE activity, by L. buchneri LN 4017. The ability of L. buchneri to grow on the aforementioned compounds was concomitantly assessed.

| MATERIAL S AND ME THODS
Two experiments were performed. In a primary experiment, deesterification of MF by L. buchneri LN 4017 (ATCC PTA-6138) was tested at varying glucose concentrations. To clarify the results obtained, a secondary experiment was conducted to study FA metabolism of this bacterium in the presence of glucose.
De Man, Rogosa and Sharpe (MRS) broth (DSMZ medium 11) without glucose was used as a basal medium. To prepare the inoculum, the bacterium was cultivated anaerobically in MRS broth for 48 hr at 37°C without agitation. Subsequently, 1 ml of bacterial culture was centrifuged at 4,000 g for 5 min (21°C). Bacterial cells were thereafter resuspended in 1 ml basal medium and used as inoculum.

MF (abcr GmbH) and FA (Merck
preparation. The final concentration of DMF in the growth medium was always 0.5% (v/v).

| Secondary experiment
The setup included: (a) basal medium with 0.5% DMF, (b) basal medium with 177 µg/ml FA, and (c) basal medium with 2,515 µg/ ml glucose and 161 µg/ml FA (Glc:FA). Sterile controls were made for incubations with FA and Glc:FA. All treatments were done in triplicate.
Bacterial growth was estimated by measuring optical density at 600 nm. Samples were centrifuged at 20,817 g for 10 min (4°C), and supernatants were stored at −20°C until chemical analyses.
Upon thawing at room temperature, samples were centrifuged at 20,817 g for 20 min (4°C) before analyses for glucose, MF, and FA.
Glucose was measured by HPLC as described by Porsch, Wirth, Toth, Schattenberg, and Nikolausz (2015) with the following modifications: operation temperature was 55°C and flow rate was 0.7 ml/ min. MF and FA were measured by UPLC according to Hofmann and Schlosser (2016) with the modification of using formic acid for acidification of the mobile phase.

| RE SULTS AND D ISCUSS I ON
In all treatments, bacterial growth reached a stationary phase after 12 hr, except in the Glc:FA treatment, in which the stationary phase was reached after 24 hr (Figure 1). Growth curves were similar for cultures incubated in the basal medium, with MF or with FA.
MF disappearance, FA accumulation, and the sum of MF and FA concentrations followed similar trends and magnitudes in incubations with MF and with Glc:MF (1:1) (Figure 2a,b). In the Glc:MF (13:1) treatment, the sum of MF and FA concentrations decreased sharply between 4 and 12 hr before slowing down during the remaining incubation period (Figure 2c).
MF is used as a model substrate to study FAE activity, with FA as a product of MF hydrolysis (Donaghy et al., 1998;Wang et al., 2016). our experimental setup. It should be noted that such ratio varies extensively from case to case as the sugar contents of forages and cell wall-associated ester linkages vary by forage type, forage maturity, climate, etc. Following assumptions were made. (a) Concentration of water-soluble carbohydrates (WSC) in silage crops is on average 15% of dry matter (DM), with glucose comprising 21% of WSC (Müller, Rosen, & Udén, 2008). (b) Neutral detergent fiber (Van Soest, Robertson, & Lewis, 1991), with an average concentration of 49% of DM (Müller et al., 2008), represents plant cell walls. (c) Trans-FA of plant cell walls, with an average concentration of 0.54% of cell walls (Hartley & Jones, 1977), represents cell wall-associated ester linkages. Under these conditions, the ratio of glucose:FA becomes 12:1 on mass basis, in agreement with our experimental setup.
The sterile controls were included to ensure that there was no abiotic degradation of MF and FA. There was an increase in the concentration of FA in the sterile control of Glc:FA treatment between 0 and 8 hr (Figure 3b), likely due to sampling/pipetting errors. As this increase did not interfere with data interpretation, it was ignored.

| CON CLUS IONS
FA released from hydrolysis of MF was further metabolized by L. buchneri LN 4017 but did not support bacterial growth. MF hydrolysis was almost similar at all concentrations of glucose, indicating that the de-esterification activity of L. buchneri LN 4017 was not repressed by glucose. We therefore suggest that de-esterification activity of L. buchneri LN 4017, mediated by the action of FAE, is not repressed by substrates present in silage. Our results, however, should be complemented with transcriptomic/proteomic studies to provide firm conclusions.

This study was partially supported by the Swedish Research Council
Formas under project registration number: 2016-01449.

CO N FLI C T O F I NTE R E S T S
None declared.

AUTH O R S CO NTR I B UTI O N
All authors contributed to conceptualization of the experiment. The raw data supporting the conclusions of this manuscript will be made available on request by the authors, without undue reservation, to any qualified researcher.