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/lp/springer-journals/bioconversion-of-ferulic-acid-and-vanillin-to-vanillic-acid-by-cold-FJ00qXgD7J
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- Springer Journals
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- Copyright © The Author(s) 2023
- ISSN
- 1590-4261
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- 1869-2044
- DOI
- 10.1186/s13213-023-01714-x
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Abstract
Purpose Biovalorization of lignin-derived aromatic monomers such as ferulic acid (FA) has attracted considerable interest. The cold-adapted strain Paraburkholderia aromaticivorans AR20-38 converts FA to the value-added product vanillic acid ( VA), without further VA degradation. The efficiency of the bioconversion of FA to VA was optimized by studying culture conditions. Methods Various cultivation parameters (agitation, temperature, FA concentration, nutrient supplementation) were assessed to increase biomass production and shorten the cultivation time, while obtaining high VA production yields. The fate of the intermediate vanillin was also studied. Lignin monomers and degradation products (FA, vanillin, VA) were quantified via UV/Vis-HPLC. Result Full bioconversion of 5 mM FA occurred over a broad temperature range of 5–30 °C. Concentrations up 30 mM FA were utilized as the sole carbon source at 20 °C. Molar VA yields (> 90%) produced from 5 to 12.5 mM FA and from 15 to 17.5 mM FA (82–87%) were not significantly different at 10 °C and 20 °C. The supplementation of the mineral medium with monosaccharides (glucose, fructose, mannose) and/or N-rich complex compounds (yeast extract, casamino acids) resulted in high biomass production, accelerated FA bioconversion, and high molar yields (96–100%). The presence of the N-rich compounds alone or in combination with glucose reduced the incubation time necessary to convert FA to VA. Vanillin, formed as an intermediate during FA degradation, was consumed and converted to VA before FA metabolization, when added in combination with FA. Vanillin bioconversion was signifi- cantly accelerated in the presence of glucose. Conclusion The variation of culture conditions improved the efficiency of the studied strain to convert FA via vanillin to VA and demonstrated remarkable FA bioconversion under varying environmental conditions, especially tempera- ture, substrate concentration, and nutrient availability, which is of importance for potential future application. Keywords Lignin-derived aromatic monomers, Ferulic acid, Vanillin, Vanillic acid, Bioconversion, Cold-adapted Paraburkholderia Background Lignin, one of the most abundant polymers on earth *Correspondence: (Ganewatta et al. 2019), is the largest renewable reservoir Rosa Margesin rosa.margesin@uibk.ac.at of organic material and could be used for the substitu- Department of Microbiology, University of Innsbruck, Technikerstrasse 25, tion of petroleum-based fuels and aromatic chemicals 6020 Innsbruck, Austria © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Ludwikowski et al. Annals of Microbiology (2023) 73:11 Page 2 of 17 (Chauhan 2020; Wang et al. 2019; Xu et al. 2019). The val - further degradation to metabolites that enter the TCA orization of lignin is a great challenge due to its complex cycle, is requested. Their further metabolization can and heterogeneous structure. Several chemical and phys- be prevented by genetic engineering (dos Santos et al. ical methods, such as pyrolysis, hydrogenation, selective 2022; Luziatelli et al. 2019). However, the accumula- oxidation, and treatment under hydrothermal and super- tion of VA from FA degradation by native bacterial (and critical conditions, have been developed for lignin depo- fungal) strains has been described in a number of stud- lymerization. However, these technologies use expensive ies (e.g., Ashengroph et al. 2012; Brunati et al. 2004; chemical catalysts and require extensive separation and Ghosh et al. 2007; Lu et al. 2020; Mishra et al. 2016). purification procedures (Chauhan 2020; Xu et al. 2019 In an earlier study, we described the stable accumula- and refs. therein). The biological treatment of the recal - tion of high amounts of VA by the cold-adapted bacte- citrant polymer lignin is an ecologically friendly alterna- rial strain Paraburkholderia aromaticivorans AR20-38 tive for lignin valorization (Chauhan 2020; Sharma et al. isolated from Alpine forest soil (Margesin et al. 2021). 2018; Xu et al. 2019). Microbial biotransformations are The potential of representatives of the genus to degrade important tools for the renewal of natural resources by aromatic compounds has been reported recently (Lee conversion into commercially valuable products (Brunati et al. 2019; Vanwijnsberghe et al. 2021). To benefit et al. 2004; Mishra et al. 2016). In nature, lignin depolym- from bioconversion, high yields from high substrate erization is initiated by fungi, especially wood-rotting amounts, in particular under varying environmental basidiomycetes, the produced products (lignin mono- conditions (such as fluctuating temperature), are desir - mers, dimers, and other low molecular mass aromatic able for biotechnological applications. Therefore, it was compounds) can be utilized efficiently by bacteria (Lub - the aim of this study to optimize the bioconversion of bers et al. 2019; Xu et al. 2019). Cellular constituents, FA to VA by strain P. aromaticivorans AR20-38. We such as bacterial enzymes, are a promising source for assessed various cultivation parameters (agitation, tem- complete lignin depolymerization (Chauhan 2020). perature, FA concentration, nutrient supplementation) Since lignin is a complex matrix of aromatic (phenolic) to increase biomass production and shorten the culti- and aliphatic compounds, it is a rich resource of renew- vation time, while obtaining high VA production yields. able aromatic compounds that could be of biotechnologi- We also studied the fate of the intermediate vanillin, cal interest (Brink et al. 2019; Lubbers et al. 2019). In the which could not yet be demonstrated by cultivation. context of the bio-valorization of lignin-derived aromatic compounds, ferulic acid (FA) has attracted considerable interest. FA is a very abundant and (almost) ubiquitous Materials and methods plant constituent that can be found in the cell wall of a Strain wide range of vegetation (Rosazza et al. 1995) and thus in The bacterial strain P. aromaticivorans AR20-38 used in common agricultural residues, such as cereal brans and this study was isolated from soil from an Alpine conif- sugar-beet pulp (Oddou et al. 1999). FA bioconversion erous forest site (Franca et al. 2016) and identified as represents a promising way for the production of com- described (Berger et al. 2021; GenBank accession no. mercially valuable fine chemicals such as vanillin and MT281269). The whole genome was sequenced and vanillic acid (VA) (Kaur and Chakraborty 2013; Rosazza the resulting draft genome sequence of the strain has et al. 1995; Xu et al. 2019). Both compounds are inter- been described (Poyntner et al. 2020; GenBank BioPro- mediates of microbial FA degradation (Brink et al. 2019; ject number PRJNA624061). The strain was deposited Bugg et al. 2011; Lubbers et al. 2019; Xu et al. 2019). Van- in the China General Microbiological Culture Collec- illin is one of the most important components of natural tion Center (CGMCC 1.18749). The strain was stored flavors and widely used in the food, pharmaceutical, and at − 80 °C using ROTI©Store cryovials. medical industry (Kaur and Chakraborty 2013; Rosazza et al. 1995; Xu et al. 2019). VA has a wide range of appli- cations and is used as a flavoring agent and food pre - Chemicals servative, in the synthesis of polyester and oxygenated Trans-ferulic acid (FA; Sigma-Aldrich 128,708), vanillin aromatic chemicals, such as vanillin, and in biomedical (Merck 818,718), and vanillic acid (VA; Merck 841,025) treatments due to its analeptic and antibacterial activity were of chromatographic pure grade. Stock solutions (Baniahmad et al. 2020; Rosazza et al. 1995; Upadhyay (0.5–1 M) were prepared in DMSO and stored at 4 °C. et al. 2020). Preliminary studies showed that the final amount of For the biotechnological application of FA biocon- DMSO in the cultivation flasks did not affect bacterial version to vanillin and VA by robust cell factories, the growth. stable production of these compounds, without their L udwikowski et al. Annals of Microbiology (2023) 73:11 Page 3 of 17 Eec ff t of cultivation parameters (temperature, agitation, To test the effect of various nutrient supplements on and FA concentration) on bioconversion of FA to VA bioconversion of 5 mM FA, the strain was grown at 20 °C Biodegradation assays were carried out as previously and 150 rpm in MM containing 5 mM FA and 0.3% (w/v) described (Margesin et al. 2021) in 100-mL Erlenmeyer of D-glucose, D-fructose, D-mannose, YE, or CAS. Each flasks with screw caps containing 20 mL of mineral of the monosaccharides and the N sources was tested salts medium (MM) supplemented with FA as the sole alone or in combination (Table 1). The control contained carbon source. To ensure sufficient aeration, the culture 5 mM FA without nutrient supplementation. Sterile flasks were opened regularly under sterile conditions. controls were prepared additionally. The preculture was The pH-neutral MM contained (compositions indi - obtained as described above. Growth (OD600), pH of cated per liter) Na HPO × 2H O (3.5 g), KH PO (2 g), the cultures, and the concentrations of FA and VA were 2 4 2 2 4 (NH ) SO (1 g), MgSO × 7 H O (0.2 g), Ca(NO ) × 4 monitored in samples collected at regular time intervals. 4 2 4 4 2 3 2 H O (0.05 g), ammonium iron(III) citrate (10 mg), a trace element and a vitamin solution (Margesin and Eec ff t of vanillin on bacterial growth Schinner 1997; Margesin et al. 2021). The growth-inhibiting effect of vanillin was studied in For inoculation, a preculture of P. aromaticivorans triplicate at 10 °C and 20 °C at 150 rpm in MM containing AR20-38 grown in MM containing glucose (2 g/L) as a carbon source was prepared. The bacterial cells were sep - Table 1 Eec ff t of supplementation of MM with nutrients (0.3% arated by centrifugation (10,000 × g for 10 min), washed (w/v)), alone or in combination, on growth and VA production twice with sterile MM and suspended in MM. The initial from 5 mM FA by P. aromaticivorans AR20-38 at 20 °C (t0) optical density at 600 nm (OD600) in the inoculated flasks was adjusted to 0.05 by inoculation with an ade - Nutrient supplement Growth (OD600) Molar VA yield (%) from 5 mM FA quate aliquot of the pre-culture. Two negative controls contained (1) sterile medium supplemented with FA and Without (control) 0.23 a 98.5 a (2) inoculated medium without FA. Growth (OD600), pH Glucose 2.75 c 101.9 a of the cultures, and the concentrations of FA and VA were Fructose 2.90 c 101.4 a monitored in samples collected at regular time intervals. Mannose 2.76 c 100.8 a All experiments were performed in triplicate. Yeast extract 1.59 b 98.9 a To evaluate the effect of agitation on bioconversion of Casamino acids 1.30 b 96.2 a 5 mM FA, cultivation was done at 20 °C without shaking Glucose + yeast extract 3.92 d 101.9 a as well as on a rotary shaker at 150, 200, and 250 rpm. Glucose + casamino acids 4.39 e 95.0 a The effect of temperature on bioconversion of 5 mM FA Fructose + yeast extract 4.34 e 100.6 a to VA was studied at 150 rpm at temperatures ranging Fructose + casamino acids 4.44 e 98.1 a from 1 to 30 °C (5 °C intervals). To determine the effect Mannose + yeast extract 3.89 d 100.3 a of FA concentration on FA bioconversions, the strain was Mannose + casamino acids 3.90 d 96.4 a cultivated at 10 °C and 20 °C at 150 rpm in MM supple- Glucose 2.75 a 101.9 a mented with 5, 10, 12.5, 15, 17.5, 20, 25, and 30 mM FA as Glucose + yeast extract 3.92 b 101.9 a sole carbon source. The pH of the medium was adjusted Glucose + casamino acids 4.39 c 95.0 a to 7.0 after the addition of the various FA concentrations. Fructose 2.90 a 101.4 a Fructose + yeast extract 4.34 b 100.6 a Fructose + casamino acids 4.44 b 98.1 a Eec ff t of medium composition (nutrient supplements) Mannose 2.76 a 100.8 a on bioconversion of FA to VA Mannose + yeast extract 3.89 b 100.3 a The ability of strain P. aromaticivorans AR20-38 to utilize Mannose + casamino acids 3.90 b 96.4 a various sugars (monosaccharides: L-arabinose, D-fruc- Yeast extract 1.59 a 98.9 a tose, galactose, D-glucose, D-mannose, D-xylose; disac- Glucose + yeast extract 3.92 b 101.9 ab charides: cellobiose, lactose, maltose, D-sucrose, starch; Fructose + yeast extract 4.34 c 100.6 ab sugar alcohol: glycerol) and N-rich complex compounds Mannose + yeast extract 3.89 b 100.3 ab (yeast extract (YE), casamino acids (CAS); thereafter Casamino acids 1.30 a 96.2 a named as N sources) for growth was tested by cultivation Glucose + casamino acids 4.39 c 95.0 a in MM supplemented with these compounds (0.2% w/v) Fructose + casamino acids 4.44 c 98.1 a at 20 °C and 150 rpm. Next, the strain was grown with Mannose + casamino acids 3.90 b 96.4 a various concentrations (0.05%, 0.1%, 0.2%, and 0.3% (w/v)) Data represent mean values of three replicates. Different letters (a-f ) indicate of D-glucose, D-fructose, D-mannose, YE, and CAS. statistically significant differences (p ≤ 0.05) between nutrients Ludwikowski et al. Annals of Microbiology (2023) 73:11 Page 4 of 17 Eec ff t of agitation on FA bioconversion 0.2% (w/v) glucose and vanillin at concentrations of 0 Shaking of the culture flasks had a significantly positive (= control), 1, 2, 3, 4, and 5 mM. effect on growth, FA biodegradation and VA production by strain P. aromaticivorans AR20-38 compared to the Eec ff t of medium composition (nutrient supplements) performance under unshaken conditions (Fig. 1). There on bioconversion of vanillin to VA were no significant differences (p ≤ 0.05) between the dif- To study vanillin bioconversion, P. aromaticivorans ferent shaking speeds tested (150, 200, 250 rpm) (Table 2). AR20-38 was grown in triplicate at 10 °C and 20 °C and The highest FA bioconversion was noted after 4 days at 150 rpm in MM containing 5 mM vanillin and supple- 20 °C with shaking, regardless of the shaking speed, while mented with/without 5 mM FA. Additionally, medium 7 days were needed for the same performance without variations were prepared by adding glucose or YE (0.3% shaking. However, the shaking speed had no significant w/v). The pH of the medium was adjusted to 7.0 after the effect (p ≤ 0.05) on the molar VA yield (97–98%) obtained addition of the various supplements. The preculture was in the stationary growth phase of the strain (Table 2). obtained as described above. Growth (OD600), pH of the cultures, and the concentrations of vanillin, FA, and Eec ff t of temperature on FA bioconversion VA were monitored in samples collected at regular time During an incubation period of 18 days, strain P. aromati- intervals. civorans AR20-38 was able to degrade 5 mM FA and fully convert it into VA over a temperature range of 5–30 °C HPLC analysis (Fig. 2). Almost no growth and biodegradation could be The samples for lignin monomer analysis were prepared observed at 1 °C. An increase in biomass production was as described previously (Margesin et al. 2022, 2021) and accompanied by FA degradation and stable VA accumu- quantified via UV/Vis-HPLC (220 nm) using a RFQ Fast lation due to FA bioconversion. The intermediate vanillin Acid column (50 × 7.8 mm, Phenomenex, Germany) as was not detected at any of the incubation temperatures shown in Wagner et al. (Wagner et al. 2017). As external tested and therefore not excreted from cells. standards, FA, VA, and vanillin were used in concentra- Biodegradation (bioconversion) was fastest at tions of 1, 5, and 10 mM. 20–30 °C, where at least 95% degradation was noted within 3 days, closely followed by 15 °C (4 days), while Statistical data analysis 7 days were needed at 10 °C. At 5 °C, growth, and thus The statistical calculations were done using the soft - also biodegradation, were considerably delayed; none- ware Statistica 13. The normal distribution of data was theless, more than 90% of 5 mM FA were degraded confirmed by the Kolmogorov–Smirnov test. Data were after 16 days. When maximum biomass formation had analyzed by ANOVA (p ≤ 0.05), followed by the post occurred, there was no significant difference (p ≤ 0.05) hoc Fisher LSD test (p ≤ 0.05). Differences between cul - between the molar (98–100%) VA yield produced from tivation conditions, growth (OD600), and VA produc- FA at temperatures ranging from 5 to 30 °C (Table 3). The tion (molar yield) were considered significant when p highest biomass formation was noted at 5 °C, followed was ≤ 0.05. by that produced at 25–30 °C (not significantly differ - ent, p ≤ 0.05) and at 10–20 °C (not significantly different, Results p ≤ 0.05). This was also observed in MM containing glu - In all biodegradation experiments, no abiotic losses could cose as the sole carbon source (data not shown). be detected over the whole incubation periods (data not shown). u Th s, the differences between the initial and the Eec ff t of FA concentration on bioconversion residual FA, vanillin, and VA concentrations had to be Remarkably, strain P. aromaticivorans AR20-38 was attributed to bioconversion by strain P. aromaticivorans able to grow well in the presence of 5–30 mM FA AR20-38. The initial pH of 7.0 in the culture media was at 20 °C (within 20 days); growth was always paral- not affected during growth and biodegradation. leled by full FA degradation and stable VA produc- Molar VA yields were calculated from the initial con- tion (Fig. 3). Generally, increased FA concentrations centrations of FA and vanillin and the produced VA con- resulted in increased biomass formation but delayed FA centration. Bioconversion per cell dry mass could not bioconversion. Full degradation of 5–17.5 mM FA was be calculated because cells of strain P. aromaticivorans obtained within 3 days (5 mM), 5 days (10 mM),7 days AR20-38 do not sediment sufficiently after centrifuga - (12.5 mM), 12 days (15 mM), or 14 days (17.5 mM). tion. However, biomass-related VA production was high- Of 20–25 mM and 30 mM FA, less than 5% of the ini- est, when OD600 values were low (indicated in tables and tial concentration was left after 15–16 and 20 days of figures) and thus followed tendentially the opposite trend incubation, respectively. While molar VA yields > 90% of biomass production. L udwikowski et al. Annals of Microbiology (2023) 73:11 Page 5 of 17 Fig. 1 Eec ff t of agitation on growth (upper panel), degradation of 5 mM FA (middle panel), and VA production (lower pannel) by P. aromaticivorans AR20-38. Data represent mean values of three replicates (SDs < 10%) (91–100%) were obtained from bioconversion of initial lowest molar yield compared to that from lower FA FA concentrations of 5–12.5 mM and 15–17.5 mM FA, concentrations) were produced from 30 mM FA (Fig. 3, respectively, only 62% VA (the significantly (p ≤ 0.05) Table 4). Ludwikowski et al. Annals of Microbiology (2023) 73:11 Page 6 of 17 Table 2 Eec ff t of aeration on growth and VA production from 5 mM FA by P. aromaticivorans AR20-38 at 20 °C rpm Growth (OD600) Molar VA yield (%) from 5 mM FA 0 0.155 a 97.1 a 150 0.208 b 97.7 a 200 0.208 b 97.7 a 250 0.211 b 97.9 a Data represent mean values of three replicates. Different letters (a, b) indicate statistically significant differences (LSD, p ≤ 0.05) between shaking speeds At 10 °C, however, growth and biodegradation were significantly delayed (by 2–3 days) in comparison with the performance at 20 °C: for example, 15 and 28 days were needed for full degradation of 10 and 17.5 mM FA, respectively. Of 20 mM FA, 21% remained untouched after 32 days; however, the stationary growth phase was not yet reached. Molar VA yields > 90% (92–99%) and > 80% (82–87%), respectively, were obtained from bioconversion of initial FA concentrations of 5–12.5 mM and 15–17.5 mM FA, respectively; how- ever, a significantly (p ≤ 0.05) lower yield of 68% was produced from 20 mM FA (Fig. 3, Table 4). Due to the delayed growth with 20 mM FA, higher concentrations (25 mM and 30 mM) were not tested at 10 °C. Nonetheless, molar VA yields produced from 5 to 12.5 mM FA (> 90%) and from 15 to 17.5 mM FA (82–87%) were comparable at 10 °C and 20 °C. In fact, there were no significant (p ≤ 0.05) differences between molar VA yields produced from 5 to 17.5 mM at 10 °C and 20 °C; only yields from 20 mM FA were signifi - cantly higher at 20 °C (78%) than at 10 °C (68%) (Fig. 4). Eec ff t of medium composition (nutrient supplements) on bioconversion of FA to VA Strain P. aromaticivorans AR20-38 was able to utilize all monosaccharides tested (growth was lowest with L-ara- binose and D-xylose), glycerol as well as YE and CAS for growth. The tested disaccharides resulted in absent or very weak growth. The highest biomass formation was obtained with the monosaccharides D-fructose, D-glu- cose, and D-mannose, and with the N sources CAS and YE extract (data not shown). Therefore, these compounds were used to test the effect of nutrient supplementation on FA bioconversion. Next, the strain was grown with various concentra- Fig. 2 Eec ff t of temperature on growth (upper panel), degradation tions (0.05%, 0.1%, 0.2%, and 0.3% (w/v)) of D-glucose, of 5 mM FA (middle panel), and VA production (lower pannel) by D-fructose, D-mannose, YE, and CAS. P. aromaticivorans P. aromaticivorans AR20-38. Data represent mean values of three AR20-38 could utilize all tested compounds in all added replicates (SDs < 10%) concentrations; the higher the concentration, the higher biomass formation was (Fig. 5). Biomass formation from L udwikowski et al. Annals of Microbiology (2023) 73:11 Page 7 of 17 Growth‑inhibiting effect of vanillin the three sugars was always comparable and significantly The effect of vanillin on the growth of strain P. aromati - higher than that produced from CAS and YE. civorans AR20-38 in MM supplemented with glucose Finally, it was evaluated whether the supplementa- (0.2% w/v) as carbon source was determined at 10 °C tion of MM with the C or/and N sources, alone or in and 20 °C, since good growth and FA bioconversion were combination, would stimulate or accelerate 5 mM FA observed at these two temperatures (Fig. 2). biodegradation at 20 °C. Indeed, the MM supplementa- A decrease in temperature resulted in an increased tion with nutrients influenced growth (incubation time growth-inhibiting effect of vanillin (Table 5). At 20 °C, and biomass production) and FA bioconversion signifi - 1–4 mM vanillin had no growth-inhibiting effect and did cantly (Table 1, Fig. 6). There was no FA release or VA not delay growth; the stationary phase in the presence of production from any of the tested nutrient supplements 0–4 mM vanillin was reached after 2 days. Only a concen- as observed in control samples. Biomass production in tration of 5 mM vanillin resulted in significant (p ≤ 0.05) the stationary growth phases with 5 mM FA as the sole growth inhibition of 11% (which was significantly lower carbon source was almost sixfold higher in the presence than growth inhibition at 10 °C) and an increased lag of an additional N source, about 12-fold higher in the phase (stationary growth phase after 4 days). At 10 °C, presence of an additional carbon source (without sig- already a concentration of 1 mM vanillin inhibited nificant differences between glucose, fructose, and man - growth significantly, however only by 8%; 2–5 mM vanil - nose) and even 17–19-fold higher if monosaccharides lin had a significantly (p ≤ 0.05) higher negative effect and and N sources were combined. Among additionally inhibited growth by 30% on average (Table 5). The higher added C sources (sugars), the lag phase was shortest in the vanillin concentration, the longer was the time to the presence of glucose. The stationary growth phases reach the stationary growth phase. were obtained after 2 days with glucose and fructose and after 3 days with mannose, which were also needed Eec ff t of medium composition (nutrient supplements) in case of FA as the sole C source and in case of sup- on bioconversion of vanillin to VA plementation with YE and CAS. In case of YE and CAS, Figure 8 (A-C) shows the time course of growth as well as growth after 1 day was already close to the stationary VA production from 5 mM vanillin and 5 mM FA at 10 phase (Fig. 6). °C and 20 °C. All parameters were delayed at 10 °C com- VA yield produced from 5 mM FA correlated with pared to 20 °C, however, with the same final result. growth. But in contrast to biomass formation, molar VA Independent of the cultivation temperature, the strain yield in the stationary growth phase was not significantly showed almost no growth in the presence of 5 mM van- influenced by MM supplementation with nutrients, nei - illin as the sole carbon source or in combination with ther alone nor in combination, and was always in the 5 mM FA, while considerable growth was obtained when range of 96–100% (Table 1). the medium was supplemented with 0.3% (w/v) YE. Bio- Importantly, nutrient supplements influenced the incu - mass formation even was approx. threefold (and thus bation time required for full FA biodegradation (and significantly) higher in the presence of 0.3% (w/v) glu - thus VA production). With 5 mM FA as the sole carbon cose compared to YE. Biomass formation in the presence source, full FA bioconversion was obtained after 3 days, of FA was higher than in its absence. These results were as it was the case when MM was supplemented with one obtained at both cultivation temperatures. With nutri- of the three monosaccharides tested alone or with combi- ent supplementation, growth at 10 °C (stationary growth nations of mannose with YE or CAS or with fructose and phase after 7–8 days) was delayed compared to growth at YE. The presence of YE, CAS as well as the combination 20 °C (stationary growth phase after 3 days of incubation) of glucose with YE or CAS reduced the incubation time (Fig. 8A, B). from 3 to 2 days. In contrast, the supplementation with The medium supplementation with glucose or YE fructose and CAS delayed the incubation time to 4 days resulted in full vanillin degradation after 2 days at 20 °C (Fig. 7). and after 7 days at 10 °C, independently of the presence When 5 mM FA was the sole carbon source, still 65% of or absence of FA (Fig. 8A, B). The additional presence of the initial concentration was detected after 2 days, while 5 mM FA demonstrated clearly that vanillin was always nutrient supplementation had already resulted in more degraded first. FA consumption was completed after than 90% or even full FA degradation (Fig. 6). After 1 day 3–4 days at 20 °C and after 8 days at 10 °C. Both, 5 mM of incubation, FA degradation was fastest in the presence vanillin and 5 mM FA, were fully converted to VA, inde- of YE. Interestingly, the presence of YE or CAS resulted pendent of the temperature, with the exception of YE in a comparable bioconversion speed like the presence supplementation at 10 °C which resulted in a 90% molar of monosaccharides despite significantly lower biomass VA yield. Notably, full bioconversion of vanillin, both, production. Ludwikowski et al. Annals of Microbiology (2023) 73:11 Page 8 of 17 Table 3 Eec ff t of temperature on growth and VA production Here, we report the optimization of the bioconver- from 5 mM FA by P. aromaticivorans AR20-38 sion of FA and the intermediate product vanillin by increasing biomass production and shortening culti- Temperature (°C) Growth (OD600) Molar VA yield (%) from 5 mM FA vation time, thereby maintaining high VA production yields. For this, various cultivation parameters (agita- 5 0.314 c 97.7 a tion, temperature, FA concentration, nutrient supple- 10 0.218 a 99.1 a mentation) were assessed in detail. 15 0.218 a 98.3 a The evaluation of the effect of agitation demonstrated 20 0.232 a 99.5 a clearly the positive effect of shaking. Better oxygen sup - 25 0.272 b 99.5 a ply under shaking culture conditions resulted in accel- 30 0.275 b 99.2 a erated performance (growth and FA bioconversion) Data represent mean values of three replicates. Different letters (a-c) indicate compared to unshaken conditions. However, there was statistically significant differences (LSD, p ≤ 0.05) between temperatures no significant effect between the shaking speeds tested (150–250 rpm); therefore, all further biodegradation experiments were carried out at a shaking frequency in the absence and in the presence of FA, was already of 150 rpm. The cultivation of aerobic microorganisms obtained after 3 days at 20 °C if the medium was sup- requires sufficient oxygen supply. Due to its low solu - plemented with glucose. In contrast, 8 days were needed bility, dissolved oxygen can become limiting for micro- with YE supplementation (Table 6). bial growth under unfavorable conditions (Schiefelbein VA was even produced from 5 mM vanillin in the et al. 2013). Agitation (shaking) increases the surface absence or presence of FA without nutrient supple- area and subsequently the oxygen concentration in the mentation (Fig. 8C). The degradation of 5 mM vanil - medium. lin as the sole carbon source was accompanied by VA Temperature is an important factor in biotechno- production with molar VA yields of 23% and 31% after logical applications. In this study, we demonstrated 11 days at 10 °C and after 8 days at 20 °C, respectively. the capability of strain P. aromaticivorans AR20-38 In the presence of 5 mM FA, molar VA yields of 15% to degrade and fully convert 5 mM FA into VA over a and 20% were obtained after 11 days at 10 °C and after broad temperature range of 5–30 °C. Despite the signifi - 8 days at 20 °C, respectively. After 32 days of incuba- cantly highest biomass formation at 5 °C, temperature tion at 20 °C, the molar VA yield increased to 68 ± 7.3% (5–30 °C, 5 °C intervals) had no significant effect on the (Table 6). Full biodegradation could not be obtained, molar VA yield (98–100%) obtained from FA bioconver- since the incubation time used in this experiment was sion. Mass transfer rates were dramatically increased too short for the strain to reach the stationary growth with increasing temperatures with no significant dif - phase. ference between incubation temperatures of 20–30 °C. No vanillin or FA degradation occurred in sterile con- Under those conditions, maximum FA degradation and trols, nor was there any release of FA, vanillin, or VA bioconversion were achieved after 3 days. However, at from nutrient supplements, which demonstrates that VA the time of maximum biomass production (which was production in all experiments had to be attributed to the delayed at lower temperatures), molar VA yields were activity of the studied strain. not significantly different over the entire temperature range tested. Unfortunately, we cannot compare our Discussion data with those obtained by others since the degrada- In a previous study (Margesin et al. 2021), we demon- tion of lignin-derived aromatic monomers has been strated the ability of the cold-adapted strain P. aromati- generally determined under mesophilic or higher tem- civorans AR20-38 to convert 5 mM and 10 mM FA to perature conditions, i.e., at temperatures ranging from high amounts of VA (85–89% molar yield) over a tem- 28 to 37 °C (Abdelkafi et al. 2006; Ghosh et al. 2007; Lu perature range of 10–30 °C. The proposed pathway, et al. 2020; Mishra et al. 2016; Ravi et al. 2017; Upad- based on the strongly differentially expressed genes hyay et al. 2020) or even higher (up to 50 °C, Khan in the transcriptome of the strain during bioconver- et al. 2022). The assessment of the genomic degrada - sion of FA to VA, has been recently described (Poynt- tion potential of 67 Paraburkholderia strains was stud- ner et al. 2022). FA is metabolized by the activity of ied with strains grown at 20 °C (Vanwijnsberghe et al. 4-coumarate-CoA ligase to feruloyl-CoA. Subsequently, 2021). There are no data available on low-temperature hydroxycinnamoyl-CoA hydratase lyase converts feru- degradation of lignin-derived compounds by bacteria loyl CoA to vanillin, and finally, vanillin dehydrogenase and yeasts, except for our studies (Berger et al. 2021; converts vanillin to VA. Margesin et al. 2022, 2021; Poyntner et al. 2021). As L udwikowski et al. Annals of Microbiology (2023) 73:11 Page 9 of 17 Fig. 3 Eec ff t of FA concentration on growth (upper panels), FA degradation (middle panels), and VA production (lower pannels) at 10 °C (left panels) and 20 °C (right panels) by P. aromaticivorans AR20-38. Data represent mean values of three replicates (SDs < 10%) previously indicated, the ability of the studied strain In bioconversion studies, the efficient consump - for low-temperature growth and degradation of lignin- tion of high amounts of substrates with a high yield of derived aromatic compounds can be attributed to its the desired product is desirable. In our study, we could isolation source, an Alpine coniferous forest site (Franca demonstrate the remarkable tolerance of strain P. aro- et al. 2016). The presence of natural aromatic polymers maticivorans AR20-38 towards high concentrations of in forest soils, such as lignin, humic substances, and FA. Remarkably, the VA production yields obtained at tannins, favors the presence of microbial degraders 10 °C and 20 °C (82–100%) from FA concentrations up to (Vanwijnsberghe et al. 2021). Knowledge on the micro- 17.5 mM were not significantly (p ≤ 0.05) different. Yields bial contribution to the biodegradation of naturally from 20 mM FA were only lower by 10% at 10 °C (68%) occurring substances in low-temperature environments than at 20 °C (78%). The yield produced from 30 mM FA is of great ecological importance. at 20 °C was still high (62%). Thus, both FA concentration Ludwikowski et al. Annals of Microbiology (2023) 73:11 Page 10 of 17 Table 4 Eec ff t of FA concentration on growth and VA such high FA concentrations. In comparison, Ravi et al. production from FA by P. aromaticivorans AR20-38 at 10 °C and (2017) studied biodegradation and bioconversion of 20 °C 5 mM FA by Pseudomonas putida at 28 °C; the engi- neered P. putida strain degraded 10 mM FA at 30 °C with Concentration Temperature Growth Molar VA (mM FA) (°C) (OD600) yield (%) a molar VA yield of 95% (Upadhyay et al. 2020). Biocon- version of 5 mM FA by Streptomyces halstedii at 28 °C 5.0 10 0.218 a 99.1 d (Brunati et al. 2004) and by Bacillus licheniformis at 37 °C 10.0 10 0.365 b 93.4 c (Ashengroph et al. 2012) resulted in molar yields of 80% 12.5 10 0.390 b 92.0 c and 60%, respectively. Halomonas elongata converted 15.0 10 0.375 b 86.5 b 5 mM FA at 37 °C to VA with a molar yield of 86%; FA 17.5 10 0.371 b 82.3 b concentrations higher than 5 mM inhibited growth by 20.0 10 0.452 c 67.5 a 12%; 30 mM FA reduced growth by 27% (Abdelkafi et al. 5.0 20 0.232 a 99.5 g 2006). The highest molar VA production yield (56%) by 10.0 20 0.333 b 95.4 f Streptomyces sannanensis was obtained from 5 mM FA 12.5 20 0.317 b 90.9 ef at 28 °C; an increase in temperature (37 °C) and FA con- 15.0 20 0.307 b 86.2 de centration (7.5–10 mM) resulted in considerably lower 17.5 20 0.361 b 82.3 cd VA yields (Ghosh et al. 2007). The conversion yield of 20.0 20 0.438 c 77.5 c Paenibacillus lactis at 37 °C was very low in the presence 25.0 20 0.499 c 67.7 b of 5 mM or 7.5 mM FA compared to 2.5 mM FA (33%) 30.0 20 0.792 d 62.0 a in the presence of FA as the sole carbon source (Mishra Data represent mean values of three replicates. Different letters (a-f ) indicate et al. 2016). The comparison of these data with those statistically significant differences (LSD, p ≤ 0.05) between FA concentrations from our study demonstrates impressively that the bio- conversion efficiency of P. aromaticivorans AR20-38 (tol - and temperature had a significant effect on FA biocon - erance to high amounts of FA and high VA production version. Biomass production increased with increased yields at 10 °C and at 20 °C) is especially remarkable and FA concentrations, which demonstrated the ability of the points to its biotechnological relevance for the produc- strain to metabolize high amounts of FA. An increase in tion of the value-added product VA from FA. FA concentration and a decrease in temperature resulted Media composition is known to affect biodegradation. in a decreased bioconversion yield, which, however, could Since the biomass produced by P. aromaticivorans AR20- not be attributed to growth inhibition, since an increase 38 in MM with FA as the sole carbon source was quite in FA concentrations was accompanied at both tempera- low, we assessed the effect of medium supplementation tures by an increase in biomass production; however, with three C sources (glucose, fructose, mannose) and with longer lag phases. Increased lag phase elongation two N-rich complex compounds (named as N sources; with increasing concentrations of phenolic compounds YE, CAS) on growth and FA conversion by the stud- has also been observed in cultures of Saccharomyces cer- ied strain. MM supplementation with C and N sources evisiae (Adeboye et al. 2014). Prolonged lag phases are alone resulted in an about sixfold and 12-fold higher an indication of stressed cells and might be a defense biomass production, respectively. The combination of mechanism that allows bacteria to tolerate stress (Ber- C and N sources even enhanced growth by a factor of trand 2019). Long lag phases could also be an indication 17–19. These data demonstrate the significantly positive of the time required by the strain to repair cell damage effect of nutrient supplementation on biomass produc - and overcome inhibiting effects (Rolfe et al. 2012). In our tion. However, this was not the case for FA bioconver- study, long lag phases before growth on high amounts of sion. The molar VA yield was not significantly different FA indicated that the strain adapted successfully to sub- in the absence or presence of nutrient supplements and optimal conditions. However, once maximum biomass was always in the range of 96–100%. Nonetheless, due formation was obtained, high VA yields were achieved. to accelerated growth, the incubation time necessary to Nonetheless, the significantly lower VA yields found with achieve full bioconversion could be reduced from 3 to high amounts of FA point to an inhibitory effect on FA 2 days at 20 °C in the presence of YE, CAS, or the com- metabolization that could not be overcome by time. Fur- bination of glucose with YE or CAS. However, supple- ther studies on the metabolites produced during FA bio- mentation with YE or CAS might also have provided the conversion could elucidate the underlying biochemical strain with additional micronutrients, vitamins, and trace mechanisms. elements. To the best of our knowledge, this is the first study to Medium supplementation with nutrients to increase describe the efficient bioconversion of FA to VA from bacterial biomass formation and FA bioconversion has L udwikowski et al. Annals of Microbiology (2023) 73:11 Page 11 of 17 Fig. 4 Eec ff t of temperature (10 °C and 20 °C) on VA production from various FA concentrations (10–20 mM) by P. aromaticivorans AR20-38. Data represent mean values of three replicates. Different letters (a, b) indicate statistically significant differences (LSD, P ≤ 0.05) between temperatures Fig. 5 Eec ff t of various concentrations (0.05–0.3% (w/v)) of monosaccharides (glucose, GLU; fructose, FRU; mannose, MAN), casamino acids (CAS), and yeast extract (YE) on biomass production by P. aromaticivorans AR20-38 at 20 °C. Data represent mean values of three replicates (SDs < 10%) been reported before. Mishra et al. (Mishra et al. 2016) 2019; Ravi et al. 2018; Upadhyay et al. 2020). The anal - observed that the addition of YE (0.05% w/v) increased ysis of the genome of strain P. aromaticivorans AR20- the molar yield (57.3 vs. 33%) from 2.5 mM FA and 38 demonstrated the presence of two genes encoding reduced the time required for VA production by Paeni- for the enzyme involved in the vanillin production bacillus lactis, whereas the addition of an additional C from FA (ferulolyl-CoA synthase) and the degrada- source (glucose 0.1% w/v) did not increase VA concentra- tion of vanillin to VA was evidenced by the presence tion. Low amounts of YE (0.001% w/v) also increased the of a gene encoding for vanillin dehydrogenase (vanil- yield of VA production and acceleration production time lin I funneling pathway) (eLignin Microbial Database, by the transposon mutant Pseudomonas fluorescens (Civ -www. elign indat abase. com; Margesin et al. 2021). How- olani et al. 2000). Glucose supplementation (0.1% w/v) ever, in all our biodegradation experiments the accu- resulted in an increased production of VA by a factor of mulation of the intermediate vanillin could never be 2 (however, still only 11.6% molar yield from 10 mM FA) detected. Also, Muheim and Lerch (1999) could not by the fungus Paecilomyces variotii, while a decrease in detect vanillin accumulation during FA conversion to VA accumulation was observed in the presence of the VA by Pseudomonas putida. This might be attributed cheaper substrate starch (Ghosh et al. 2006). to the toxic effect of the phenolic aldehyde vanillin on During FA biodegradation to VA, bacteria produce microorganisms and its rapid degradation or conver- the intermediate vanillin (Lu et al. 2020; Lubbers et al. sion to non- or less toxic compounds like VA (Banerjee Ludwikowski et al. Annals of Microbiology (2023) 73:11 Page 12 of 17 Fig. 6 Eec ff t of supplementation of MM with (0.3% w/v) glucose (GLU), fructose (FRU), mannose (MAN), yeast extract (YE), and casamino acids (CAS) (right panels); YE in combination with monosaccharides (middle panels), or CAS in combination with monosaccharides (right panels) on growth (top panels), 5 mM FA degradation (middle panels), and VA production (bottom panels) by P. aromaticivorans AR20-38 at 20 °C. Data represent mean values of three replicates (SDs < 10%) Fig. 7 Eec ff t of incubation time on molar VA yield (%) produced from 5 mM FA by P. aromaticivorans AR20-38 at 20 °C in MM supplemented with various nutrients and Chattopadhyay 2019; Kaur et al. 2013; Lubbers et al. average, only a concentration of 5 mM vanillin resulted 2019; Luziatelli et al. 2019; Ravi et al. 2017). in comparatively slight growth inhibition (11%) at 20 °C. Therefore, we studied the effect of vanillin on the u Th s, a decrease in temperature and an increase in vanil - growth of strain P. aromaticivorans AR20-38 at two lin concentration resulted in reduced performance, par- temperatures (10 °C and 20 °C). The growth-inhibiting alleled by longer lag phases (the same effect was noted effect of vanillin was significantly influenced by tempera - with increased FA concentrations, see above). Longer ture and vanillin concentration. While concentrations exposure times with the membrane-active compound of 2–5 mM vanillin inhibited growth at 10 °C by 30% on vanillin (due to longer lag phases) might have resulted in L udwikowski et al. Annals of Microbiology (2023) 73:11 Page 13 of 17 Table 5 Eec ff t of vanillin concentration (0–5 mM) on relative a short lag phase and high amounts of biomass produced growth inhibition of P. aromaticivorans AR20-38 in MM with in the presence of nutrients. glucose (0.2% w/v) at 10 °C (100%: OD600 = 2.74 ± 0.02) and 20 °C (100%: OD600 = 2.03 ± 0.04) Conclusion The data reported in this study demonstrate a number of Vanillin conc. (mM) Temperature (°C) Relative growth (%) remarkable degradation properties of the cold-adapted 0 10 100.0 c strain Paraburkholderia aromaticivorans AR20-38. Its 1 10 91.8 b ability to accumulate VA by FA bioconversion has been 2 10 69.3 a described before (Margesin et al. 2021). In this study, the 3 10 69.2 a variation of culture conditions led to the following find - 4 10 70.1 a ings and production improvements: 5 10 70.0 a 0 20 100.0 b – Agitation favors growth and FA bioconversion; how- 1 20 103.3 b ever, the studied strain is not sensitive to the agita- 2 20 102.0 b tion speed (150–250 rpm), which facilitates its han- 3 20 98.5 b dling. 4 20 100.0 b – The strain has an excellent performance to com - 5 20 89.4 a pletely convert 5 mM FA over a broad temperature Data represent mean values of three replicates. Different letters (a-c) range of 5–30 °C, with the fastest activity at 15–30 °C indicate statistically significant differences (LSD, p ≤ 0.05) between vanillin (3–4 days), followed by 10 °C (7 days) and 5 °C concentrations (16 days). This allows application in environments with fluctuating temperature conditions. negative interactions of vanillin with the cell membrane – The strain is able to handle very high concentrations of the studied strain. The antibacterial activity of vanillin (up to 30 mM) of FA. Despite delayed growth and bio- was found to be dependent on the exposure time, con- degradation at 10 °C compared to 20 °C, there were no centration, and the target microorganisms (Fitzgerald significant (p ≤ 0.05) differences between molar VA et al. 2004). yields produced from 5 to 17.5 mM FA at 10 °C and Since the ability of strain P. aromaticivorans AR20-38 20 °C. This demonstrates the excellent performance of to produce the intermediate vanillin during FA biodeg- the strain at low and moderate temperatures. radation was evident from genomic data (Margesin et al. – The presence of additional nutrient supplements in 2021), we assessed vanillin biodegradation in the absence the MM resulted in accelerated and full bioconver- and presence of FA as well as in the absence and presence sion of 5 mM FA. Interestingly, the strain does not of the nutrient supplements glucose and YE. Indeed, VA require a specific nutrient, which also facilitates VA production from vanillin and from FA occurred at both production by saving time. Nonetheless, nutrient temperatures tested (10 °C and 20 °C). Full bioconver- supplementation did not influence the molar yield, sion of vanillin, independent of the presence of FA, was which points to the robustness of the strain. obtained in the presence of an additional nutrient sup- – Biomass production could be significantly increased plement (glucose or YE). Significantly accelerated vanil - with higher FA concentrations and with nutrient lin consumption occurred, however, only in the presence supplementation. However, biomass increase did of glucose at 20 °C. VA was even produced from 5 mM not influence VA yield. This is of advantage when vanillin in the absence or presence of FA without nutrient VA production should be obtained (from natural supplementation; however, no full bioconversion could sources) under varying nutrient or environmental be obtained since, in contrast to nutrient-supplemented conditions. assays, the stationary growth phase was not reached – Our studies proved the ability of the strain to utilize during the studied incubation period. Our data show and convert vanillin, the intermediate produced dur- clearly (1) that strain P. aromaticivorans AR20-38 con- ing bacterial FA degradation, both at 10 °C and 20 °C. verted both FA and vanillin to VA, which confirmed our Vanillin was always consumed first. genomic data (Margesin et al. 2021), and (2) that vanil- lin was always consumed first (i.e., before FA). This dem - Our study adds knowledge on the utilization of lignin- onstrates the bacterial response to the toxic compound derived aromatic compounds by the bacterial genus vanillin by its quick elimination, as described before Paraburkholderia under varying environmental condi- (Muheim and Lerch 1999; Ravi et al. 2017). The elimina - tions, especially temperature, substrate concentration, tion by degradation and conversion to VA was favored by and nutrient availability, which is of importance for Ludwikowski et al. Annals of Microbiology (2023) 73:11 Page 14 of 17 Fig. 8 A Eec ff t of glucose (0.3% w/v) and YE (0.3% w/v) on growth and bioconversion of 5 mM vanillin to VA in the absence (top panels) or presence (bottom panels) of 5 mM FA by P. aromaticivorans AR20-38 at 10 °C. Data represent mean values of three replicates (SDs < 10%). B Eec ff t of glucose (0.3% w/v) and YE (0.3% w/v) on bioconversion of 5 mM vanillin to VA in the absence (top panels) or presence (bottom panels) of 5 mM FA by P. aromaticivorans AR20-38 at 20 °C. Data represent mean values of three replicates (SDs < 10%). C Bioconversion of 5 mM vanillin to VA after 32 days of incubation in the absence or presence of 5 mM FA (in the absence of nutrient supplements) by P. aromaticivorans AR20-38 at 10 °C (left panel) and 20 °C (right panel). Data represent mean values of three replicates (SDs < 10%) L udwikowski et al. Annals of Microbiology (2023) 73:11 Page 15 of 17 Table 6 VA production from 5 mM vanillin in the absence or presence of 5 mM FA and nutrient supplements by P. aromaticivorans AR20-38 at 10 °C and 20 °C. Data represent mean values of three replicates Vanillin (mM) FA (mM) Nutrient supplement Temperature Incubation Growth (OD600) Molar VA yield (%) from (°C) time (days) 5 mM vanillin 5 mM vanillin + 5 mM FA 5 0 Without 10 11 0.047 22.7 a 5 0 Yeast extract 10 10 1.043 90 b 5 0 Glucose 10 10 3.167 100.5 c 5 5 Without 10 11 0.048 15.0 a 32 0.052 31.1 b 5 5 Yeast extract 10 10 1.163 96.1 c 5 5 Glucose 10 10 3.357 95.6 c 5 0 Without 20 8 0.048 30.6 a 5 0 Yeast extract 20 8 0.855 101.6 b 5 0 Glucose 20 3 2.560 101.5 b 5 5 without 20 8 0.051 19.9 a 32 0.051 67.7 b 5 5 Yeast extract 20 8 1.130 97.9 c 5 5 Glucose 20 3 2.800 99.1 c Different letters (a-c) indicate statistically significant differences (LSD, p ≤ 0.05) between vanillin concentrations Consent for publication potential future application. Our study adds also knowl- All of the authors have read and approved the manuscript. This work has not edge on the capability and potential of cold-adapted been published previously, nor is it being considered by any other peer- microorganisms (Collins and Margesin 2019) for the for- reviewed journal. mation of value-added products from natural resources Competing interests like lignin. It has to be emphasized that the remark- The authors declare that they have no competing interests. able biodegradation capacity described in this study was obtained with a native and not with an engineered strain. Received: 30 November 2022 Accepted: 3 February 2023 Further transcriptomic and in vitro studies with resting cells and cell-free enzyme extracts should elucidate the expression and activity of enzymes involved in vanillin and FA bioconversion. FA bioconversion in agricultural References residues will also be examined. Abdelkafi S, Sayadi S, Gam ZBA, Casalot L, Labat M (2006) Bioconversion of ferulic acid to vanillic acid by Halomonas elongata isolated from table- olive fermentation. FEMS Microbiol Lett 262(1):115–120. https:// doi. org/ Acknowledgements 10. 1111/j. 1574- 6968. 2006. 00381.x. We thank P. Thurnbichler for skillful technical assistance. The authors thank the Adeboye PT, Bettiga M, Olsson L (2014) The chemical nature of phenolic publishing fund of the Universität Innsbruck for financial support. compounds determines their toxicity and induces distinct physiological responses in Saccharomyces cerevisiae in lignocellulose hydrolysates. Authors’ contributions AMB Express 4:46. https:// doi. org/ 10. 1186/ s13568- 014- 0046-7. R.M. conceptualized the study, designed the degradation experiments, and Ashengroph M, Nahvi I, Zarkesh-Esfahani H, Momenbeik F (2012) Novel strain wrote the manuscript. T.M.L. performed and analyzed the degradation experi- of SHL1 with potential converting ferulic acid into vanillic acid. Ann ments. A.O.W. designed and performed the HPLC analyses and analyzed the Microbiol 62(2):553–558. https:// doi. org/ 10. 1007/ s13213- 011- 0291-9. data. All authors contributed significantly to the manuscript, read, and agreed Banerjee G, Chattopadhyay P (2019) Vanillin biotechnology: the perspectives and to the published version of the manuscript. future. J Sci Food Agric 99(2):499–506. https:// doi. org/ 10. 1002/ jsfa. 9303. Baniahmad B, Safaeian L, Vaseghi G, Rabbani M, Mohammadi B (2020) Cardio- Funding protective effect of vanillic acid against doxorubicin-induced cardiotoxic- Open-access funding is provided by the University of Innsbruck. ity in rat. Res Pharm Sci 15(1):87–96. https:// doi. org/ 10. 4103/ 1735- 5362. Availability of data and materials Berger T, Poyntner C, Margesin R (2021) Culturable bacteria from an Alpine All data generated and analyzed during this study are included in this article. coniferous forest site: biodegradation potential of organic polymers and pollutants. Folia Microbiol 66(1):87–98. https:// doi. org/ 10. 1007/ Declarations s12223- 020- 00825-1. Bertrand RL (2019) Lag phase is a dynamic, organized, adaptive, and Ethics approval and consent to participate evolvable period that prepares bacteria for cell division. J Bacteriol Not applicable. 201(7):e00697-e718. https:// doi. org/ 10. 1128/ JB. 00697- 18. Ludwikowski et al. Annals of Microbiology (2023) 73:11 Page 16 of 17 Brink DP, Ravi K, Liden G, Gorwa-Grauslund MF (2019) Mapping the diversity cold-adapted yeast Rhodosporidiobolus colostri isolated from Alpine forest of microbial lignin catabolism: experiences from the eLignin database. soil. Microorganisms 10(3) doi:https:// doi. org/ 10. 3390/ micro organ isms1 Appl Microbiol Biotechnol 103(10):3979–4002. https:// doi. org/ 10. 1007/ 00305 15. s00253- 019- 09692-4. Margesin R, Schinner F (1997) Bioremediation of diesel-oil-contaminated Brunati M, Marinelli F, Bertolini C, Gandolfi R, Daffonchio D, Molinari F (2004) alpine soils at low temperatures. Appl Microbiol Biotechnol 47(4):462– Biotransformations of cinnamic and ferulic acid with actinomycetes. 468. https:// doi. org/ 10. 1007/ s0025 30050 957. Enzyme Microb Technol 34(1):3–9. https:// doi. org/ 10. 1016/j. enzmi ctec. Margesin R, Volgger G, Wagner AO, Zhang DC, Poyntner C (2021) Biodegrada- 2003. 04. 001. tion of lignin monomers and bioconversion of ferulic acid to vanillic acid Bugg TDH, Ahmad M, Hardiman EM, Rahmanpour R (2011) Pathways for deg- by Paraburkholderia aromaticivorans AR20-38 isolated from Alpine forest radation of lignin in bacteria and fungi. Nat Prod Rep 28(12):1883–1896. soil. Appl Microbiol Biotechnol 105(7):2967–2977. https:// doi. org/ 10. https:// doi. org/ 10. 1039/ c1np0 0042j.1007/ s00253- 021- 11215-z. Chauhan PS (2020) Role of various bacterial enzymes in complete depolymeri- Mishra S, Kullu M, Sachan A, Vidyarthi AS, Sachan SG (2016) Bioconversion of zation of lignin: a review. Biocatal Agric Biotechnol 23 doi:https:// doi. org/ ferulic acid to vanillic acid by Paenibacillus lactis SAMS-2001. Ann Micro- 10. 1016/j. bcab. 2020. 101498. biol 66(2):875–882. https:// doi. org/ 10. 1007/ s13213- 015- 1175-1. Civolani C, Barghini P, Roncetti AR, Ruzzi M, Schiesser A (2000) Bioconversion of Muheim A, Lerch K (1999) Towards a high-yield bioconversion of ferulic acid ferulic acid into vanillic acid by means of a vanillate-negative mutant of to vanillin. Appl Microbiol Biotechnol 51(4):456–461. https:// doi. org/ 10. Pseudomonas fluorescens strain BF13. Appl Environ Microbiol 66(6):2311–1007/ s0025 30051 416. 2317. https:// doi. org/ 10. 1128/ aem. 66.6. 2311- 2317. 2000. Oddou J, Stentelaire C, Lesage-Meessen L, Asther M, Ceccaldi BC (1999) Collins T, Margesin R (2019) Psychrophilic lifestyles: mechanisms of adaptation Improvement of ferulic acid bioconversion into vanillin by use of high- and biotechnological tools. Appl Microbiol Biotechnol 103(7):2857–2871. density cultures of Pycnoporus cinnabarinus. Appl Microbiol Biotechnol https:// doi. org/ 10. 1007/ s00253- 019- 09659-5. 53(1):1–6. https:// doi. org/ 10. 1007/ s0025 30051 605. dos Santos OAL, Goncalves TA, Sodre V, Vilela N, Tomazetto G, Squina FM, Gar- Poyntner C, Kutzner A, Margesin R (2021) Biodegradation potential and puta- cia W (2022) Recombinant expression, purification and characterization tive catabolic genes of culturable bacteria from an Alpine deciduous of an active bacterial feruloyl-CoA synthase with potential for application forest site. Microorganisms 9(9) doi:https:// doi. org/ 10. 3390/ micro organ in vanillin production. Protein Expr Purif 197 doi:https:// doi. org/ 10. 1016/j. isms9 091920. pep. 2022. 106109. Poyntner C, Ludwikowski TM, Wagner AO, Margesin R (2022) Transcriptome Fitzgerald DJ, Stratford M, Gasson MJ, Ueckert J, Bos A, Narbad A (2004) Mode profiling of Paraburkholderia aromaticivorans AR20-38 during ferulic of antimicrobial action of vanillin against Escherichia coli, Lactobacillus acid bioconversion. AMB Express 12:148 https:// doi. org/ 10. 1186/ plantarum and Listeria innocua. J Appl Microbiol 97(1):104–113. https:// s13568- 022- 01487-7. doi. org/ 10. 1111/j. 1365- 2672. 2004. 02275.x. Poyntner C, Zhang DC, Margesin R (2020) Draft genome sequence of the Franca L, Sannino C, Turchetti B, Buzzini P, Margesin R (2016) Seasonal and bacterium Paraburkholderia aromaticivorans AR20–38, a Gram-Negative, altitudinal changes of culturable bacterial and yeast diversity in Alpine cold-adapted degrader of aromatic compounds. Microbiol Resour forest soils. Extremophiles 20(6):855–873. https:// doi. org/ 10. 1007/ Announc 9(27) doi:https:// doi. org/ 10. 1128/ mra. 00463- 20. s00792- 016- 0874-2. Ravi K, Garcia-Hidalgo J, Gorwa-Grauslund MF, Liden G (2017) Conversion of Ganewatta MS, Lokupitiya HN, Tang CB (2019) Lignin biopolymers in the age lignin model compounds by Pseudomonas putida KT2440 and isolates of controlled polymerization. Polymers 11(7) doi:https:// doi. org/ 10. 3390/ from compost. Appl Microbiol Biotechnol 101(12):5059–5070. https:// doi. polym 11071 176.org/ 10. 1007/ s00253- 017- 8211-y. Ghosh S, Sachan A, Mitra A (2006) Formation of vanillic acid from ferulic acid Ravi K, Garcia-Hidalgo J, Nobel M, Gorwa-Grauslund MF, Liden G (2018) Biolog- by Paecilomyces variotii MTCC 6581. Curr Sci 90(6):825–829. ical conversion of aromatic monolignol compounds by a Pseudomonas Ghosh S, Sachan A, Sen SK, Mitra A (2007) Microbial transformation of isolate from sediments of the Baltic Sea. AMB Express 8 doi:https:// doi. ferulic acid to vanillic acid by Streptomyces sannanensis MTCC 6637. org/ 10. 1186/ s13568- 018- 0563-x. J Ind Microbiol Biotechnol 34(2):131–138. https:// doi. org/ 10. 1007/ Rolfe MD, Rice CJ, Lucchini S, Pin C, Thompson A, Cameron ADS, Alston M, s10295- 006- 0177-1. Stringer MF, Betts RP, Baranyi J, Peck MW, Hinton JCD (2012) Lag phase is Kaur B, Chakraborty D (2013) Biotechnological and molecular approaches for a distinct growth phase that prepares bacteria for exponential growth vanillin production: a review. Appl Biochem Biotechnol 169(4):1353–1372. and involves transient metal accumulation. J Bacteriol 194(3):686–701 https:// doi. org/ 10. 1007/ s12010- 012- 0066-1. doi/https:// doi. org/ 10. 1128/ JB. 06112- 11. Kaur B, Chakraborty D, Kumar B (2013) Phenolic biotransformations during Rosazza JPN, Huang Z, Dostal L, Volm T, Rousseau B (1995) Review: biocatalytic conversion of ferulic acid to vanillin by lactic acid bacteria. Biomed Res Int transformations of ferulic acid: An abundant aromatic natural product. J 2013 doi:https:// doi. org/ 10. 1155/ 2013/ 590359. Ind Microbiol 15(6):457–471. https:// doi. org/ 10. 1007/ bf015 70016. Khan SI, Zarin A, Ahmed S, Hasan F, Belduz AO, Canakci S, Khan S, Badshah M, Schiefelbein S, Frohlich A, John GT, Beutler F, Wittmann C, Becker J (2013) Farman M, Shah AA (2022) Degradation of lignin by Bacillus altitudinis SL7 Oxygen supply in disposable shake-flasks: prediction of oxygen transfer isolated from pulp and paper mill effluent. Water Sci Technol 85(1):420– rate, oxygen saturation and maximum cell concentration during aerobic 432. https:// doi. org/ 10. 2166/ wst. 2021. 610. growth. Biotechnol Lett 35(8):1223–1230. https:// doi. org/ 10. 1007/ Lee Y, Lee Y, Jeon CO (2019) Biodegradation of naphthalene, BTEX, and s10529- 013- 1203-9. aliphatic hydrocarbons by Paraburkholderia aromaticivorans BN5 isolated Sharma A, Kumar R, Rathi M, Bhatia D, Malik DK (2018) Lignocellulose biodeg- from petroleum-contaminated soil. Sci Rep 9 doi:https:// doi. org/ 10. 1038/ radation: an advance technology for sustainable environment. Biosci s41598- 018- 36165-x. Biotechnol Res Commun 11(4):634–637. https:// doi. org/ 10. 21786/ bbrc/ Lu P, Wang WN, Zhang GX, Li W, Jiang AJ, Cao MJ, Zhang XY, Xing K, Peng X, 11.4/ 14. Yuan B, Feng ZZ (2020) Isolation and characterization marine bacteria Upadhyay P, Singh NK, Tupe R, Odenath A, Lali A (2020) Biotransformation capable of degrading lignin-derived compounds. Plos One 15(10) of corn bran derived ferulic acid to vanillic acid using engineered Pseu- doi:https:// doi. org/ 10. 1371/ journ al. pone. 02401 87. domonas putida KT2440. Prep Biochem Biotechnol 50(4):341–348. https:// Lubbers RJM, Dilokpimol A, Visser J, Makela MR, Hilden KS, de Vries RP (2019) A doi. org/ 10. 1080/ 10826 068. 2019. 16979 35. comparison between the homocyclic aromatic metabolic pathways from Vanwijnsberghe S, Peeters C, De Ridder E, Dumolin C, Wieme AD, Boon N, plant-derived compounds by bacteria and fungi. Biotechnol Adv 37(7) Vandamme P (2021) Genomic aromatic compound degradation poten- doi:https:// doi. org/ 10. 1016/j. biote chadv. 2019. 05. 002. tial of novel Paraburkholderia Species: Paraburkholderia domus sp. nov., Luziatelli F, Brunetti L, Ficca AG, Ruzzi M (2019) Maximizing the efficiency of Paraburkholderia haematera sp. nov. and Paraburkholderia nemoris sp. nov. vanillin production by biocatalyst enhancement and process optimiza- Int J Mol Sci 22(13) doi:https:// doi. org/ 10. 3390/ ijms2 21370 03. tion. Front Bioeng Biotechnol 7 doi:https:// doi. org/ 10. 3389/ fbioe. 2019. Wagner AO, Markt R, Puempel T, Illmer P, Insam H, Ebner C (2017) Sample 00279. preparation, preservation, and storage for volatile fatty acid quantifica- Margesin R, Ludwikowski TM, Kutzner A, Wagner AO (2022) Low-temperature tion in biogas plants. Eng Life Sci 17(2):132–139. https:// doi. org/ 10. 1002/ biodegradation of lignin-derived aromatic model monomers by the elsc. 20160 0095. L udwikowski et al. Annals of Microbiology (2023) 73:11 Page 17 of 17 Wang HL, Pu YQ, Ragauskas A, Yang B (2019) From lignin to valuable products- strategies, challenges, and prospects. Biores Technol 271:449–461. https:// doi. org/ 10. 1016/j. biort ech. 2018. 09. 072. Xu ZX, Lei P, Zhai R, Wen ZQ, Jin MJ (2019) Recent advances in lignin valoriza- tion with bacterial cultures: microorganisms, metabolic pathways, and bio-products. Biotechnol Biofuels 12 doi:https:// doi. org/ 10. 1186/ s13068- 019- 1376-0. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Re Read ady y to to submit y submit your our re researc search h ? Choose BMC and benefit fr ? 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Journal
Annals of Microbiology – Springer Journals
Published: Mar 13, 2023
Keywords: Lignin-derived aromatic monomers; Ferulic acid; Vanillin; Vanillic acid; Bioconversion; Cold-adapted Paraburkholderia