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. 2024 Apr 10;72(16):9259–9267. doi: 10.1021/acs.jafc.3c08067

Fermenting Distiller’s Grains by the Domesticated Microbial Consortium To Release Ferulic Acid

Yao Zhang †, Qiang Ye †, Bo Liu †, Zhiping Feng †, Xian Zhang †, Mingyou Luo §, Lijuan Yang †,‡,*
PMCID: PMC11046480  PMID: 38598779

Abstract

graphic file with name jf3c08067_0009.jpg

The microbial consortium FA12 that can release ferulic acid (FA) by fermenting distiller’s grains was screened from Daqu. Taibaiella, Comamonadaceae, and Ochrobacum were highly abundant in FA12 by 16S rRNA gene sequencing. In the process of long-term acclimation with distiller’s grains as a medium, the biomass of FA12 remained stable, and the pH value of fermentation liquid was also relatively stable. Meanwhile, the activities of cellulase, xylanase, and feruloyl esterase secreted by FA12 were stable in the ranges of 0.2350–0.4470, 0.1917–0.3078, and 0.1103–0.1595 U/mL, respectively, and the release of FA could reach 133.77 μg/g. It is proven that the microbial consortium has good genetic stability. In addition, the structural changes of lignocellulose in distiller’s grains before and after fermentation were analyzed by scanning electron microscopy (SEM), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR), and the changes of distiller’s grains weight and lignocellulose content before and after fermentation were also detected. These results all confirmed that FA12 had the function of degrading distiller’s grains. In this study, we explored a method to use microbial communities to release FA from distiller’s grains and degrade lignocellulose in the waste, which opened up a new way for the application of the high value of lost waste.

Keywords: ferulic acid, microbial consortium, lignocellulose, feruloyl esterase, distiller’s grains

Introduction

Liquor distiller’s grains are a large amount of by-waste in the brewing industry. Because of its large quantity and lack of reasonable and effective utilization, it can easily cause environmental pollution. For example, about 39 million tons of distiller’s grains in China are in urgent need of harmless treatment and resource utilization.1−3 Lignocellulose is mainly composed of cellulose, hemicellulose, and lignin forming a stubborn structure. In this structure, enzymes such as cellulases, hemicellulases, and other enzymes (e.g., laccase, catalase) are required for the complete degradation of lignocellulose and hemicellulose.4 However, at present, the enzyme degradation efficiency of a single strain is low, and the combined cultivation of different strains can effectively improve substrate degradation and product yield.5,6

Ferulic acid (FA) is a phenolic acid that exists widely in nature. It has physiological functions such as antioxidant, antithrombotic, hypolipidemic, prevention and treatment of coronary heart disease, antibacterial, anti-inflammatory, antimutation, and anticancer. It is widely used in food additives, health products, and pharmaceutical industries.7−11 For example, FA has been found to have significant anti-inflammatory effects by reducing proinflammatory cytokines such as IL-6, IL-1, and tumor necrosis factor (TNF), or by increasing the expression of anti-inflammatory cytokines and certain stress-response genes, as well as some antioxidant molecules that regulate cell signaling pathways.12 Pang et al. demonstrated the therapeutic effect of FA on COVID-19 and osteosarcoma using bioinformatics analysis and effective identification of pharmacological molecules by targeting autophagy.13 Unfortunately, FA is mainly bound in cereal bran as an ester bond, preventing people from using FA. The FA content in many lignocellulosic wastes is 0.5–3.0% (w/w), which is a good raw material for FA production. Current studies have found that chemical and enzymatic hydrolysis methods can be used to produce FA. Compared with chemical methods, the enzymatic hydrolysis method has the advantages of higher catalytic efficiency, lower energy consumption, safety, and environmental protection, and strong specificity.14

Feruloyl esterase (EC 3.1.1.73, FAE), also known as cinnamate esterase, is a key enzyme in the release of FA, which can hydrolyze the ester bond between FA and hemicellulose or lignin in plant cell walls to release free FA or FA dimer.15,16 Xu et al. expressed FAE from Lactobacillus crispatus in Escherichia coli BL21 (DE3), added 2 g of purified FAE enzyme directly to destarch wheat bran (DSWB), and the yield of FA reached 1.86 mg. Studies have shown that FAE and xylanase have synergistic effects on each other, and they also produce good results in the degradation of lignocellulose on a variety of substrates from different sources.17 Dilokpimol et al. expressed FAE derived from Asperginus niger heterognomically in Pichia pastoris X-33, and the FA yield could reach 10 mg/L. By combining this enzyme with commercial xylanase, the FA yield could be increased by 6 times.18

Although the commercial FAE enzyme production of FA is efficient and specific, the production cost is prohibitive. The microbial community culture plays a direct role in the degradation of lignocellulose and shortens the purification process of the enzymes. At the same time, the multibacterial collaboration can achieve substrate degradation and product acquisition more effectively.5,6 Collaborative treatment of lignocellulose by microbial communities such as bacteria, actinomyces, and fungi shows many advantages over single species or enzyme treatments.19 The microbial consortium is well-adapted in complex environments, flexible enough to a range of contaminants, easy to control pH during degradation, and cost-effective operation under nonsterile conditions.5,20 In this study, the microbial community FA12, which can release FA through fermented distiller’s grains, was screened and domesticated from Daqu. The composition diversity and abundance of the microbial community were obtained from 16S rRNA sequencing. Furthermore, in this work, the genetic stability of the microbial community, enzyme production performance (FAE, xylanase, and cellulase), and production of FA release were evaluated. At the same time, the degradation capacity of the lignocellulosic microbial community was confirmed by the physical characterization of the distiller’s grains after degradation. In this study, a short-term and effective microbial flora screening strategy was developed, and a new microbial source was obtained for FA production and lignocellulosic degradation, thus promoting the research progress of FA bioproduction and agricultural waste reuse.

Materials and Methods

Experimental Materials

The by-product of liquor brewing is known as distiller’s grains. Distiller’s grains consist primarily of cellulose, hemicellulose, lignin, crude starch, nitrogen, phosphorus, potassium, and other inorganic components. (Table 1).21 The Distiller’s grains are dried and crushed to a size below 60 mesh. Liquor Daqu and distiller’s grains were collected from a winery in Yibin, China; peptone, yeast extract, NaCl, (NH4)2SO4, KH2PO4, MgSO4·7H2O, CaCl2·H2O, and FeCl3 were purchased from Kelong Chemical Co., Ltd. (Chengdu, China); FA and ethyl 4-hydroxy-3-methoxycinnamate were purchased from Yuanye Biotechnology Co., Ltd. (Shanghai, China).

Table 1. Ingredients of Distiller’s Grains.

name of the substrate water content/% organic matter/% crude fiber/% total nitrogen/% total phosphorus/% total potassium/% C/N reference
distiller’s grains 58.42 79.49 28.57 3.26 0.32 1.13 14.17 (21)
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Culture Conditions

The enrichment medium used for screening is an LB medium. The medium used for acclimation of microbial consortium consisted (g/L) of 100 g of distiller’s grains and pH = 7. The screening medium used to detect FAE consisted (g/L) of ethyl-4-hydroxy-3-methoxycinnamate 5, sodium chloride 5, and beef extract 1.5. The fermentation medium used for degrading lignocellulose to release FA is composed of (g/L): (NH4)2SO4 1.3, KH2PO4 0.37, MgSO4·7H2O 0.25, CaCl2·H2O 0.07, FeCl3 0.02, yeast extract 5.0, and distiller’s grains 10, pH 6.5. All flasks were incubated at 37 °C under a continuous rotary shaker at 180 rpm/min.

Screening and Domestication of Microbial Consortium

Baijiu Daqu (1 g) was loaded into the LB medium (50 mL) for enrichment and culture for 24 h. Distiller’s grains (10 g) and 100 mL H2O were put into the culture medium (100 mL), and 10 mL of enriched LB culture medium were inoculated into the domesticated culture medium. This screening scheme was repeated three times. After 3 days of culture, 10% of the inoculum volume (vol/vol) in each fully mixed medium was transferred to a fresh medium. In the process of continuous subculture, the pH changes and biomass of each generation of the medium were detected. The biomass was measured by using a nonmicrobial medium as blank control, and the absorbance value of the sample was measured by a UV spectrophotometer at 600 nm wavelength after filtering the distiller’s grains. After more than 10 consecutive generations of transfer culture, the pH and biomass remained stable for 10 consecutive generations. It could be considered that this colony can adapt to growth and reproduction with distiller’s grains as a single nutrient. After 10 generations of continuous transfer culture, an ideal microbial consortium, named “FA12”, has been obtained from Baijiu Daqu. The microbial consortium is preserved at −80 °C with glycerol for standby.

Evaluation of Microbial Consortium’s Ability To Produce Enzymes, Release FA, and Degrade Lignocellulose

In the evaluation process, three parallel samples were set for each sample detection. The experiment was carried out in 50 mL of medium containing 1% distiller’s grains. A seed volume (v/v) of 2% was inoculated. In the process of biotransformation, the pH value and biomass of the fermentation broth were measured every day. The CMCase activity, xylanase activity, and FAE activity of FA12 were measured on the third day of five consecutive passages. The kinds of phenolic acids released by the microbial consortium and the amount of FA release were studied by LC–MS qualitative and quantitative analysis.22 At the same time, the weight loss rate and changes of four components (cellulose, hemicellulose, acid-insoluble lignin, and acid-soluble lignin) during the microbial treatment were determined. Scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), and X-ray diffraction (XRD) were used to study the change in the structure characteristics of the distiller’s grains before and after microbial treatment.5,6

Enzyme Activity Determination

For enzyme activity analysis, 10 mL of fermentation broth was centrifuged at 7000 rpm (10 min), and the supernatant was collected as the crude enzyme. CMCase activity was determined by spectrophotometry with CMC-Na as a substrate. Incubating the crude enzyme solution (1 mL) in 4 mL of 50 mM sodium citrate buffer containing 1% (w/v) CMC-Na (pH 5.0) at 50 °C for 30 min, add 1.5 mL DNS to terminate the reaction, and measure the amount of reducing sugar with a spectrophotometer at 540 nm.23 One unit (U) of CMCase activity was defined as the required enzyme amount for releasing 1 μmol of reducing sugar in 1 min under the standard conditions above. Xylanase activity was determined by spectrophotometry with beech xylan as the substrate. The crude enzyme solution (1 mL) was added to 2 mL of 50 mM sodium citrate buffer containing 1.0% (w/v) beech xylan (pH 5.0), incubated at 50 °C for 10 min, and 2 mL DNS was added to terminate the reaction. The production of xylose was measured with a spectrophotometer at 540 nm.24 One unit (U) of xylanase activity was defined as the required enzyme amount for releasing 1 μmol of xylose in 1 min under the standard conditions above. The enzyme activity of FAE was determined by spectrophotometry with ethyl 4-hydroxy-3-methoxycinnamate as a substrate. The crude enzyme solution (1 mL) was added to 2 mL of 100 mM Tris HCl (pH 9.0) buffer solution containing 1% (w/v) ethyl 4-hydroxy-3-methoxycinnamate. After incubation at 37 °C for 2 h, the reaction was terminated in an ice bath for 10 min. The resulting FA was measured with a spectrophotometer at 320 nm. The above enzyme activity determination methods take the substrate without an enzyme as the control.

FA12 Diversity Analysis

16S rRNA gene sequencing technology was used to analyze the microbial diversity of FA12. The bacteria in the fermentation broth were collected in glycerol tubes to extract their total DNA. Primers 515F and 907R were used to amplify the V4–V5 hypervariable region of bacteria for 16S rRNA gene sequencing. PCR amplification products were sequenced through the Illumina Miseq platform to produce paired readings of about 500 bp. Sequencing data was applied to the picking of operational taxonomic units (OTUs) at a critical value of 97% and UCLUST was applied to allocate OTUs at a 70% confidence level in the Silva database (SRA data: PRJNA971861).25

Methods for the Detection of Cellulose, Hemicellulose, and Lignin

About 1 g of the fermented and unfermented waste substrate was weighed and added to a flat-bottomed flask with 25 mL of a nitric acid-ethanol mixture. A reflux condenser is installed in a flat-bottomed flask and added to the boiling water bath for 1 h. After 1 h, the condensing tube is removed and left to stand, waiting for the bottom to be drained. The filtrate was centrifuged at 7000 r/min for 10 min. Twenty-five milliliters of the nitric acid-ethanol mixture was measured and transferred to the flat-bottomed flask a fraction of times, and then the residue was continued in the water bath for 1 h, followed by filtration and centrifugation. The above operation is repeated four times until the fiber color is white. The above fibers were filtered, cleaned with 10 mL nitric acid-ethanol solution, washed with hot water, and tested with methyl orange reagent until the outflow liquid showed an acidic reaction.26

graphic file with name jf3c08067_m001.jpg

where m1 = residue weight of filter and sample after drying (g); m2 = filter weight (g); m0 = initial mass of sample (g); w = sample moisture content (%).

Accurately 0.5 g of waste was weighed and added to a round-bottom flask; 10 g of sodium chloride, several small glass beads, and 100 mL of 12% hydrochloric acid solution were added. 30 mL of 12% hydrochloric acid was poured into a condenser and the funnel was dripped. The furnace temperature was adjusted to allow the round-bottom flask contents to boil and thereafter 30 mL of 12% hydrochloric acid was added to the flask for every 30 mL of liquid distilled. While 300 mL of liquid was waiting to be distilled, furfural was tested for complete distillation with an aniline acetate solution. After completion of furfural distillation, the distillate was transferred from the receiving flask to a 500 mL volumetric flask, and 12% hydrochloric acid was added until the scale was shaken well. The measured 200 mL of distillate was placed in a 1000 mL conical flask, crushed ice prepared by 250 g of distilled water was added, 25 mL of sodium bromate-sodium bromide solution was added, and the bottle was plugged tightly and placed in the dark for 5 min. At this time, the solution temperature should be kept at 0 °C. After reaching the time, 100 g/L potassium iodide solution was added to 10 mL, the bottle was firmly plugged, shaken well, placed in the dark for 5 min, titrated with 0.1 mol/L sodium thiosulfate solution, and when the solution turned light yellow, 5 g/L starch solution was added to 2–3 mL, and continued to titrate until the blue disappears. The blank experiment was carried out with 200 mL of 12% hydrochloric acid solution.26

graphic file with name jf3c08067_m002.jpg

where V1 = volume of sodium thiosulfate standard solution consumed 0.1 mol/L in blank experiment; V2 = volume of 0.1 mol/L sodium thiosulfate standard solution consumed by the sample (mL); c = concentration of sodium thiosulfate standard solution (mol/L); 0.048 = 1 mL of sodium thiosulfate standard solution equivalent to furfural mass, 1.38 = conversion coefficient; and m = the mass of the sample (g).

The sample was accurately weighed, and 0.5 g was discarded, wrapped with qualitative filter paper, and tied with cotton thread. The sample was wrapped with benzene alcohol in a cable-type extractor, and finally, the sample was air-dried. The sample extracted by benzene alcohol was transferred to a conical flask with a capacity of 100 mL, and 72% sulfuric acid cooled to 12–15 °C was added to make the sample fully saturated. Then, the conical flask was placed in an 18–20 °C water bath and kept warm at this temperature for 2 h, and the conical flask was shaken from time to time to make the reaction in the flask uniform. After the arrival time, the contents of the conical flask were transferred to a 1000 mL conical flask and rinsed with 575 mL of distilled water. Boil the conical flask on a hot plate for 4 h, during which time water should be added continuously to keep the total volume constant. The acid-insoluble lignin is then allowed to precipitate. The filtrate and acid-insoluble lignin were collected by filtration with constant weight filter paper in a weighing bottle. Then, the filtrate is washed with hot distilled water until the addition of 10% barium chloride solution to the lotion is no longer cloudy, and the edge of the filter paper is no longer acidic when checked by pH test paper. Then, the filter paper was transferred to the original constant weight weighing bottle, dried at 105 °C to constant weight, and the acid-insoluble lignin content (%) in distillers lees was calculated = m1/m0 (where m1 is the mass of acid-insoluble lignin residue after drying, and m0 is the mass of the absolute dry sample). After diluting the filtrate by a certain multiple, the content of acid-soluble lignin was detected by UV external spectrophotometry at a 320 nm wavelength.26

graphic file with name jf3c08067_m003.jpg

where ODAL = the absorbance value detected at a 320 nm wavelength; Vf = the reaction volume (mL); D = the dilution ratio of filtrate; ε = the light absorption property of the material at a specific wavelength; m0 = mass of the sample (g); p = thickness of the cuvette (cm).

Experimental Analysis

After 3 days of fermentation, 70% absolute ethanol (50 mL) in the fermentation broth (50 mL) was extracted in the rotary shaker at 37 °C, 180 r/min for 30 min, and then the extract was taken into a centrifuge tube. After centrifugation at 7000 r/min for 10 min, the supernatant was taken out, and 0.22 μ M filter membrane filtration was carried out for LC–MS analysis of phenolic acid types and FA yield. LC–MS conditions: Waters Acquity UPLC Beh C18 1.7 μm 2.1 × 50 mm, Eluent: 10%, 0.1% acetic acid water, 90% methanol, column temperature 25 °C, flow rate: 0.2 mL/min; mass spectrometry conditions: TEM 500 °C, IS −4500v, CUR 25 psi, GS1 50 psi, GS2 50 psi, detection mode MRM, FA 192.9 > 134, DP-43v, EP-11 V, CE-16 V, CXP −5 V.

For FTIR analysis, the dried samples were mixed with KBr and pressed into a layered form. The FTIR spectrum recording range was 400–4000 cm–1, and the resolution was 4 cm–1. The changes in the chemical structure of the distiller’s grains were characterized by spectroscopy. The physical structure changes of rice straw under 15 kV voltage were observed by SEM. Dry samples were prepared by gold plating to ensure the conductivity of the surface and then observed at different magnifications. X-ray diffractometer is used for crystallinity analysis, and the sample is placed on the sample rack. The radiation source is Cu-Ka, and the working voltage is 40 kV and 40 mA. The scanning condition (2θ) is 5–40°, the scanning speed is 2°/min, and step size is 0.01. The crystallinity of lignocellulose before and after the degradation of waste grains is analyzed.

Data Processing

The experiments in this study had 3 replicates. Data processing used software such as Excel 2016 (Microsoft Corporation) and SPSS Statistics 25.0 (IBM Corporation, New York, NY, USA).

Results

Screening and Domestication Process of the Microbial Consortium

The microbial consortia FA11, FA12, and FA21 can grow stably in the medium containing only discarded grains. After a long-term process of domestication, microorganisms retained by their natural succession can use distiller’s grains for their own growth and metabolism. In the process of continuous passage, the biomass OD of the three microbial communities can be stabilized between 0.2 and 0.35 after every 3 days of culture, and the pH of the culture medium is stabilized in the range of 5.0–6.5 (Figure 1A). The microbial communities FA11, FA12, and FA21 had good subgeneration stability. Using ethyl 4-hydroxy-3-methoxycinnamate as the substrate, The FAE activities of these three microbial consortia were detected. The FAE activity of FA12 reached 0.1264 ± 0.016 U/mL, which was higher than that of FA11 and FA21 (Figure 1B, P < 0.001). Therefore, FA12 was selected for subsequent enzyme production evaluation, lignocellulose degradation, and FA release.

Figure 1.

Figure 1

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Biomass (OD) and pH of the microbial consortia FA11, FA12, and FA21 were detected on the third day of each passage cycle (A); comparison of FAE enzyme activities among three microbial consortia FA11, FA12, and FA21 on the third day (B). The data are presented as the means ± standard deviations of three independent experiments. Different lowercase letters indicate significant differences between treatments (P < 0.001).

Analysis of Key Enzymes for FA Release by FA12

First, the biomass of FA12 and pH changes in the fermentation medium were measured to predict the time of enzyme secretion. The pH of the fermentation broth during FA12 fermentation was detected (Figure 2A). In the first 3 days, the OD value of FA12 increased rapidly from 0 to about 1.921 within 2 days, and the pH value of the medium increased from the initial 6.99 to about 8.75. Early in culture, FA12 is likely to preferentially consume the favorable substrates (i.e., crude protein and yeast extract) in the culture medium to produce alkaline compounds, the accumulation of which leads to an increase in pH. After that, the microbial consortium entered a stable period (2–7 d), and the OD was stable at about 1.55–1.90. It can be concluded from the growth cycle of FA12 and the change in pH of the fermentation medium that FA12 entered a stable period on the third day. Therefore, the changes in enzyme activities of FA12 key enzymes (FAE, xylanase, and CMCase) in the fermentation microbial community were detected for 5 consecutive times on every third day (Figure 2B, P < 0.05). During the 5 passages, the FAE activity of FA12 was up to 0.4470 U/mL, and the activities of xylanase and CMCase were in the range of 0.1917–0.3078 and 0.1103–0.1595 U/mL, respectively. It was observed that the activities of FAE, xylanase, and CMCase produced by FA12 were maintained above 0.2, 0.19, and 0.11 U/mL, respectively, indicating that the enzyme production capacity of FA12 was relatively stable during each passage.

Figure 2.

Figure 2

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Growth curve of FA12 and the pH change of fermentation broth within 7 days (A); evaluation of FAE, xylanase, and CMCase characteristics of microbial consortium FA12 during five consecutive fermentation processes (B). The data are presented as the means ± standard deviations of three independent experiments. Different lowercase letters indicate significant differences between treatments (P < 0.05).

FA12 Diversity Analysis

FA12 species was investigated by 16S rRNA gene sequencing. The composition of FA12 mainly includes 6 bacterial phyla, which are Proteobacteria (55.5%), Bacteroidota (25.9%), Firmicutes (7.9%), Bdellovibrionot (6.7%), Actinobacteriota (2.5%), and Sumerlaeota (1.4%) (Figure 3A, P < 0.01). Proteobacteria (55.52%) was the most abundant phylum in all samples. The composition of FA12 at the genus level is shown in Figure 3B. Taibaiella (18.9%), Comamonadaceae (17.3%), Ochrobactrum (16.8%), and Escherrichia-Shigella (10.7%) were the dominant bacteria detected in this study, and the sum of their abundances accounted for more than 60%. Notably, the relative abundance of unclassified genera was particularly high, reaching over 10%.

Figure 3.

Figure 3

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16S rRNA gene sequencing technology was used to analyze the microbial diversity of FA12. The bacteria in the fermentation broth were collected into glycerol tubes to extract their total DNA. Primers 515F and 907R were used to amplify the V4–V5 hypervariable region of bacteria for 16S rRNA gene sequencing. Species composition abundance analysis of phylum (A) and genus (B) level of microbial consortium FA12 (P < 0.01).

Analysis of the Types of Phenolic Acids Released by FA12

LC-MS qualitatively detected the types of phenolic acids released by fermentation distiller’s grains of microbial community. As shown in Figure 4A-B, it was found that there was no release of FA in distiller’s grains without microbial treatment. FA, caffeic acid, p-Hdroxyphenylacetic acid, and maltol were detected in distiller’s grains fermentation broth samples after microbial treatment. It is clear from the figure that the peak area of FA is smaller than that of caffice acid. Compared with the control sample, the peak areas of p-hydroxybenzoic acid, syringic acid, phloretic acid, and vanillic acid were reduced.

Figure 4.

Figure 4

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LC–MS was used to qualitatively analyze the release types of phenolic acids in the fermentation broth. Analysis of species of phenolic acids (A, B). CK: A sample of fermentation broth without microorganisms; 10 times: a sample of fermentation broth the tenth generation of microbial consortium FA12; 20 times: a sample of fermentation broth the 20th generation of microbial consortium FA12; peak area: signal peak area of substances detected by LC–MS. Analysis of FA production released by microbial consortium FA12 (C); the data are presented as the means ± standard deviations of three independent experiments. Different lowercase letters indicate significant differences between treatments (P < 0.001).

FA12 Degrades Distiller’s Grains and Releases FA

FA was obtained from distiller’s grains, and the FA release of the 10th and 20th generation microbial consortium FA12 could reach 82.78 ± 0.75 and 133.77 ± 0.039 μg/g (Figure 4C, P < 0.001), respectively. Compared with the cohydrolysis of purified FAE and xylanase to release FA, this study completed the release of FA through the enzyme secreted by FA12 itself. Although the yield of FA is lower than that of direct use of enzyme preparation, it is more economical and convenient to release FA by microbial community fermentation.

Weight Loss and Change of Lignocellulose after FA12 Fermentation

FA12 has a good degradation effect on waste grain through the coordination of microorganisms. The weight loss of distiller’s grains from 0 to 7 days is shown in Figure 5A (P < 0.01). The weight loss rate of the degradation of distiller’s grains increased exponentially in the first 3 days, and the weight loss reached 18.63 ± 1.2264% on the third day, and the degradation rate slowed down after 3 days. Then, we detected the changes in the content of four components after FA12 fermentation for 3 days. The cellulose content decreased from 37.88 ± 0.36 to 34.96 ± 0.59%, and the hemicellulose content decreased from 13.10 ± 0.60% from the beginning decreased to 11.38 ± 0.72%, the acid-insoluble lignin increased from 14.5 ± 0.01 to 15.53 ± 0.01%, and the acid-soluble lignin increased from 4.22 ± 0.69 to 8.11 ± 0.06% (Figure 5B, P < 0.01). It can be seen that the enzymes secreted by FA12 can degrade lignocellulose, and cellulose and hemicellulose degrade faster than lignin.

Figure 5.

Figure 5

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Changes in weight loss (A) and four components (B) after microbial consortium FA12 degradation; the data are presented as the means ± standard deviations of three independent experiments. Different lowercase letters indicate significant differences between treatments (P < 0.01).

Electron Microscopic Structure, Functional Group Characteristics, and Crystallinity Changes of FA12 before and after Degrading Distiller’s Grains

The changes in the epidermal morphology of distiller’s grains before and after fermentation by FA12 were observed to reveal the degradation process of lignocellulose by SEM (electron microscope magnification 5000× and 10,000×) (Figure 6A–D). Figure 6A,B shows unfermented distiller’s grains. It can be observed that the surface is groove-shaped and relatively smooth without obvious voids, and a small amount of flake particles are attached to the surface. Figure 6C,D shows the discarded grains after 3 days of fermentation. The surface is honeycomb-shaped, with a large number of holes. The particles are relatively fine and the lamellar structure is obvious. Through the observation of the changes in the epidermal morphology and tissue structure of discarded particles by SEM, it can be inferred that FA12 may secrete related enzymes to decompose cellulose, hemicellulose, and other substances in the distiller’s grains, and destroy the cuticle layer of the distiller’s grains, so that the original massive overall structure is decomposed into broken sheet structure. It seemed that the microorganisms and their enzymes in FA12 have the ability to decompose distiller’s grains.

Figure 6.

Figure 6

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Surface degradation of distiller’s grains before (A, B) and after (C, D) microbial treatment was detected by SEM; A and C are the results observed by SEM at 10,000 times magnification; B, D are the results observed by SEM at 5000 times magnification. The microbial treatment time of distiller’s grains was 3 days.

FTIR was used to study the changes in the molecular structure and functional group characteristics before and after the degradation of the distiller’s grains (Figure 7). The wavelength of 3419 cm–1 is the characteristic absorption peak of hydroxyl group, and the peak value decreases with the degradation of FA12, indicating that the molecules between the lost fibers will change the hydrogen bond structure.27 The peak at 2974 cm–1 is the C–H vibrational band inside the cellulose, and the peak at 1078 cm–1 is the C–O extended vibrational band in the cellulose. The absorption peak is significantly weakened after microbial treatment, indicating that the cellulose is destroyed after treatment.28,29 The peak at 1636 cm–1 belongs to the stretching vibration of the aromatic skeleton, indicating the decomposition of lignin by the FA12.30 This series of phenomena shows that the structure of each component in lignocellulose changes after the treatment of FA12, and the destruction of the stubborn structure of lignocellulose increases the chance for biological enzymes and substrates to increase the yield of products. The degradation of lignocellulose in the distiller’s grains is more beneficial to the application of downstream industries, such as distiller’s grains as feed, fertilizer, etc.

Figure 7.

Figure 7

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FTIR spectra of distiller’s grains before and after the degradation of microbial consortium FA12; 0 d: a sample of distiller’s grains without microbiological treatment; and 3 d: distiller’s grain samples after 3 days of microbiological treatment.

The changes in the crystallinity of lignocellulose in distiller’s grains before and after fermentation by FA12 were analyzed by XRD (Figure 8). After microbial treatment, the diffraction peak at 22.5° sharpens, and a new prominent diffraction peak appears at 18.5°. The sharping of the diffraction peak and the generation of a new peak indicate that the crystallinity is higher, and the cellulose chain in the waste grains is hydrolyzed after microbial treatment or the characteristic transformation of natural cellulose from cellulose I to cellulose II. The crystallinity of cellulose is related to its structural stiffness, and compared to cellulose I, cellulose II has a stronger hydrophobic effect, which contributes to its more stable crystal morphology.30,31

Figure 8.

Figure 8

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XRD spectra of distiller’s grains before and after the degradation of microbial consortium FA12; 0 d: a sample of distiller’s grains without microbiological treatment; and 3 d: distiller’s grains samples after 3 days of microbiological treatment.

Discussion

In this study, the method of releasing FA from distiller’s grains by domesticating the microbial community was investigated. This production mode is economical and convenient, which makes it possible to use distiller’s grains with high value. How to obtain a microbial community with stable genetic and productive performance has become a key issue. Using distiller’s grains as domestication medium, microbial communities that can release FA were screened and domesticated from microbial-rich Daqu, and microbial biomass, pH of domestication medium, and the key enzymes were used as basic evaluation indexes. Many research results have confirmed that pH has a far-reaching impact on the growth and metabolism of microorganisms (Thacker, 2021; Tien et al., 2021). Many of these characteristics (cell membrane potential, nutrient availability, enzyme activity, organic carbon characteristics, and salinity) are usually directly or indirectly related to pH. In addition, the above factors may change the diversity of microbial communities, affect metabolic pathways, and determine the release of products (Zhou Wen, 2021). The results showed that the biomass of FA12 and the pH of the culture medium increased in the first 2 days, and entered a stable period on the third day so 3 days was chosen as the passage period of FA12. The final pH value is stable at around 8.5. The change in pH provides an alkaline environment for microbial degradation of lignocellulosic acid to release FA, which may be more conducive to the release of FA. FAE, CMCase, xylnase, and other enzyme activities of FA12 maintained a relatively stable state during the five consecutive passage processes. The results are similar to previous studies. Previous studies have found that when multiple enzymes are used to DSWB, FAE, and related enzymes of lignocellulosic degradation can play a synergistic role in increasing the release of FA.19,32 However, the difference in this study is that it is more convenient to use the mixed culture method to directly degrade the waste grains and release FA. At the same time, it also provides a new idea for the analysis of the process of degradation of lignocellulose by the microbial consortium. The biomass, key enzyme activity, and pH of the culture medium of each generation of the microbial community demonstrated that the microbial community of FA12 with good genetic stability and FAE release was obtained.

FA accounts for up to 3% of the dry weight of cells in plants.33 Purified enzymes are often used to extract FA from plants. The feruloyl esterase PcFAE1 of Penicillium chrysogenum 31B was overexpressed in P. pastoris KM71H and then purified to release FA from natural substrates. The previous study showed that the yield of FA from DSWB by alkali extraction was 4.04 mg/g, and the FA released by enzymatic reaction accounted for 70% of the total.34 Xiaoli et al. found that when using purified FAE and commercial xylanase alone, the FA released from DSWB was only 5 and 2% respectively, but in the presence of FAE and xylanase, the amount of FA released was approximately increased by 10 times compared to FAE alone.19 Zhenshang et al. used FAE derived from Lactobacillus to release a maximum of 199 μg FA from 0.2 g destarched wheat bran.35 Yin et al. used A. niger for solid fermentation of wheat bran to release free FA.36 The difference between this study and previous studies is that FA in distiller’s grains is obtained by semisolid fermentation of microbial consortium. The domesticated microbial consortium showed excellent performance, releasing FA up to 133.77 μg from 0.5 g of waste grains. This method saves the enzyme purification process, while the mixed fermentation culture process is easier to control, thus, promoting FA production. Through the results of weight loss, SEM, FTIR, and XRD, it was found that FA12 had a good degradation effect on lignocellulose, and its degradation ability on other biomasses can be studied in the future. The compact structure of lignocellulose in distiller’s grains treated by reducible microorganisms is changed, and it is easier to absorb and utilize by animals when it is used as animal feed. The study of waste grains after fermentation as animal feed may be able to improve the utilization rate of feed.

It was found that the peak area of FA was smaller than that of caffeic acid, indicating that the content of caffeic acid in the product was higher than that of FA. It is speculated that FA released by FA12 may be further converted to caffeic and vanillic acid. A study found that in Enterobacter cloacae, the side chain of FA was demethylated into caffeic acid, which was further dehydroxylated into cinnamic acid, and then into phenylpropionic acid.37 In the metabolic path of Pseudomonas putida, FA is catalyzed by the thioester condensation reaction with Coenzyme A under the catalysis of CoA ferulic synthetase to form CoA FA. Then, under the action of CoA hydratase, the hydroxyl group is connected to the 2′-carbon of the side chain, followed by the acetal reaction to form vanilaldehyde. Then, vanillic acid is oxidized by vanillic aldehyde dehydrogenase to vanillic acid, which is further converted to protocatechuic acid by vanillic acid demethylase, and then converted to β-Ketoadipate, which is finally decomposed to form acetylkievase A and enters TCA to supply energy for bacterial growth.38,39 The reduction of the peak area of p-hydroxybenzoic acid, syringic acid, phloretic acid, and vanillic acid shows that for the phenolic acid substances that inhibit the growth of microorganisms, microorganisms in order to maintain their normal growth and metabolism, consume or secrete enzyme liquids to fight the toxic substance, and then remove or transform it. Previous studies have shown that lactic acid bacteria have good degradability to chlorogenic acid (Caroline Fritsch, 2016).

Bacteroides and Pseudomonas were detected in the results of microbial diversity, which further supported previous reports. It has been reported that Bacteroides can degrade complex arabinoxylan in dietary fiber and release FA (Yasuma Taro, 2021). The cellulose-degrading bacterium Pseudomonas poae, belonging to the phylum Proteobacteria, can utilize its carboxymethyl CMCase for bioethanol production (Emad Abada, 2019). At the same time, when brewer’s grains were used as the inducing carbon source, Pseudomonas could produce FAE and hydrolyze brewer’s grains to release FA (Kim Yong Gyun, 2007). Figure 2 shows that the fermentation environment becomes alkaline gradually, which is caused by the growth and metabolism of Alcaligenace in the microbial consortium. These unclassified microbial taxa suggest that they might work synergistically with the host microbes in the microbiota during lignocellulose degradation. The diversity of bacterial communities not only plays an important role in lignocellulose decomposition and phenolic acid release but also helps maintain the stability of biological production and degradation processes.

Conclusions

A novel microbial consortium FA12 with stable biomass and enzyme activity in the process of passage was obtained through short-term screening and domestication, which was used to degrade the distiller’s grains waste and release FA. The FA12 was mainly composed of Taibaiella (18.9%), Comamonadaceae (17.3%), Ochrobactrum (16.8%), and Escherrichia-Shigella (10.7%). After 3 days of treatment, FA release of distiller’s grains, the loss of weight, cellulose degradation rate, and hemicellulose degradation rate can reach 133.77 μg/g, 18.63, 7.70, and 13.12%, respectively. The SEM results showed that degradation was significant. FTIR analysis confirmed that FA12 can significantly break the chemical bonds in the waste, leading to the deconstruction of stubborn lignocellulose. XRD results showed the change in crystallinity in the treated grains. A method was developed to degrade distiller’s grains to produce FA, which promoted the development of FA bioproduction and the high-value utilization of distiller’s grains waste.

Data Availability Statement

The authors declare that the data supporting the findings of this study are available within the paper. The data that support the findings of this study are available from the corresponding author, upon reasonable request. Microbial diversity data entry number is PRJNA971861.

Author Contributions

∥ Y.Z. and Q.Y. contributed equally to this work.

Author Contributions

L.Y. conceived and designed the experiment. Q.Y., B.L. and Y.Z. completed most of the experimental work. M.L. carried out the material pretreatment Y.Z. analyzed the experimental results and wrote and received the manuscript. X.Z. assisted in data analysis. L.Y. and Z.F. supervised the overall work, revised the manuscript, and all authors read and approved the final manuscript.

This work was supported by the project for Liquor Making Bio-Technology & Application of Key Laboratory of Sichuan Province Fund (NJ-2021-04), Natural Science Foundation of Sichuan Province (2022NSFSC0246) and The Innovation Fund of Postgraduate, Sichuan University of Science & Engineering (y2021038).

The authors declare no competing financial interest.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The authors declare that the data supporting the findings of this study are available within the paper. The data that support the findings of this study are available from the corresponding author, upon reasonable request. Microbial diversity data entry number is PRJNA971861.

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