Antimicrobial Activity of Lignin-Derived Polyurethane Coatings Prepared from Unmodiﬁed and Demethylated Lignins

: Due to global ecological and economic challenges that have been correlated to the transition from fossil-based to renewable resources, fundamental studies are being performed worldwide to replace fossil fuel raw materials in plastic production. One aspect of current research is the development of lignin-derived polyols to substitute expensive fossil-based polyol components for polyurethane and polyester production. This article describes the synthesis of bioactive lignin-based polyurethane coatings using unmodiﬁed and demethylated Kraft lignins. Demethylation was performed to enhance the reaction selectivity toward polyurethane formation. The antimicrobial activity was tested according to a slightly modiﬁed standard test (JIS Z 2801:2010). Besides e ﬀ ects caused by the lignins themselves, triphenylmethane derivatives (brilliant green and crystal violet) were used as additional antimicrobial substances. Results showed increased antimicrobial capacity against Staphylococcus aureus . Furthermore, the coating color could be varied from dark brown to green and blue, respectively.

In 1891, methylene blue, another TPM derivative was discovered by Paul Ehrlich to be efficient in malaria treatment, a few years later followed by the discovery of the antiseptic capacity of brilliant green (BG) [42][43][44]. For many years, malachite green was one of the most frequently used disinfectants in aquaculture due to its fungicidal effects. Due to the discovery of antibiotics and biocide polymers, antimicrobial dyes have not only been displaced in biomedicine but also other applications. Bolous et al. intensively studied the mechanisms of antimicrobial effects [45,46]. In detail, it was reported that the evidence to link the antimicrobial properties of TPM dyes, especially brilliant green, to the activity of mechanosensitive ion channel (MIC) of large conductance, which is known to be highly specific and ubiquitous in various bacterial species [47]. In 2012, Vilela et al. reported a study using methylene blue (MB) and malachite green photosensitizer microbial reduction of Staphylococcus aureus by synthesizing biofilms with it. The best results showed microbial reduction with 3000 µM of malachite green with a microbial reduction of 1.6-4.0 log10 [48]. In oral cavities, biofilm formation is considered to cause resistance to antimicrobial agents. Photodynamic therapies using phenothiazinic photosensitizers first confirmed the antimicrobial effect in biofilms. Malachite green was then compared with the phenothiazinic photosensitizers (methylene blue and toluidine blue) on Staphylococcus aureus and Escherichia coli biofilms. Noimark et al. reported the synthesis of modified photobactericidal silicones for medical applications. In detail, crystal violet and/or methylene blue were incorporated into the silicone bulk and gold nanoparticles were coated using a dipping method. The polymers showed good photostability, the photobactericidal activity was determined against Staphylococcus epidermidis and Escherichia coli. The results showed that these multidye-nanogold-polymers exhibit strong photobactericidal activity both under light and dark conditions [49]. Bartoszewicz et al. filed a patent claiming lubricious antimicrobial coatings containing silver, pyrrolidone carboxylic acid (PCA) and a TPM dye (malachite green). The coating composition of the invention provides photostability to the silver ions contained therein and is hydrophilic and antimicrobial [50]. In 2016, Santos et al. comprehensively reviewed various classes of antimicrobial polymers and discussed their bioactivity mechanisms including biocidal activity, antifungal and antibacterial capacity against numerous microorganisms (i.e., gram positive and gram negative bacteria and fungi) [51]. Table 1 summarizes literature reporting the antimicrobial activity of lignins and triphenylmethane derivatives such as malachite green, brilliant green, methylene blue and crystal violet.   Table 1).
In 1891, methylene blue, another TPM derivative was discovered by Paul Ehrlich to be efficient in malaria treatment, a few years later followed by the discovery of the antiseptic capacity of brilliant green (BG) [42][43][44]. For many years, malachite green was one of the most frequently used disinfectants in aquaculture due to its fungicidal effects. Due to the discovery of antibiotics and biocide polymers, antimicrobial dyes have not only been displaced in biomedicine but also other applications. Bolous et al. intensively studied the mechanisms of antimicrobial effects [45,46]. In detail, it was reported that the evidence to link the antimicrobial properties of TPM dyes, especially brilliant green, to the activity of mechanosensitive ion channel (MIC) of large conductance, which is known to be highly specific and ubiquitous in various bacterial species [47]. In 2012, Vilela et al. reported a study using methylene blue (MB) and malachite green photosensitizer microbial reduction of Staphylococcus aureus by synthesizing biofilms with it. The best results showed microbial reduction with 3000 µM of malachite green with a microbial reduction of 1.6-4.0 log 10 [48]. In oral cavities, biofilm formation is considered to cause resistance to antimicrobial agents. Photodynamic therapies using phenothiazinic photosensitizers first confirmed the antimicrobial effect in biofilms. Malachite green was then compared with the phenothiazinic photosensitizers (methylene blue and toluidine blue) on Staphylococcus aureus and Escherichia coli biofilms. Noimark et al. reported the synthesis of modified photobactericidal silicones for medical applications. In detail, crystal violet and/or methylene blue were incorporated into the silicone bulk and gold nanoparticles were coated using a dipping method. The polymers showed good photostability, the photobactericidal activity was determined against Staphylococcus epidermidis and Escherichia coli. The results showed that these multi-dye-nanogold-polymers exhibit strong photobactericidal activity both under light and dark conditions [49]. Bartoszewicz et al. filed a patent claiming lubricious antimicrobial coatings containing silver, pyrrolidone carboxylic acid (PCA) and a TPM dye (malachite green). The coating composition of the invention provides photostability to the silver ions contained therein and is hydrophilic and antimicrobial [50]. In 2016, Santos et al. comprehensively reviewed various classes of antimicrobial polymers and discussed their bioactivity mechanisms including biocidal activity, antifungal and antibacterial capacity against numerous microorganisms (i.e., gram positive and gram negative bacteria and fungi) [51]. Table 1 summarizes literature reporting the antimicrobial activity of lignins and triphenylmethane derivatives such as malachite green, brilliant green, methylene blue and crystal violet. Table 1. Literature studies regarding antimicrobial activity of lignins and triphenylmethane derivatives (i.e., malachite green, brilliant green, methylene blue and crystal violet).
Various gram positive and gram negative bacteria Evidence to link the antimicrobial properties of TPM dyes, especially brilliant green, to the activity of mechanosensitive ion channel (MIC) of large conductance, known to be highly specific/ubiquitous in various bacterial species.
S. aureus Best microbial reduction with 3000 µM malachite green with microbial reduction of 1.6-4.0 log 10 . Vilela et al. [48] TPM-based antimicrobial surfaces Antimicrobial effects of crystal violet and methylene blue.
S. epidermidis (RP62a) and E. coli (NCTC 25522) Light-activated antimicrobial surfaces with enhanced efficacy induced by a dark-activated mechanism. Noimark et al. [49] TPM-based coating additives (i.e., brilliant green, crystal violet) for polymeric substrates including PU Antimicrobial activity of photo-stable composition used for coating a variety of medical materials.

Not specified
The coating composition comprising silver and TPM dyes (malachite green) provided photostability to the silver ions and antimicrobial activity. Bartoszewicz  Antimicrobial activity of chemically modified lignins (by acetylation, epoxidation and hydroxymethylation reactions).

Bacillus aryabhattai and Klebsiella
Epoxy/lignin was found to be the most effective antibacterial among modified lignin with minimum inhibitory concentration of 90 and 200 µg/disc.

E. coli and Pseudomonas aeruginosa
Nanoparticle flash precipitation with subsequent silver ion infusion and polyelectrolyte coating including lignin.

E. coli and S. aureus
Lignin particles exhibit antibacterial effect against E. coli and S. aureus in the same order of magnitude as other antibacterial agents such as Bronopol ® and Chlorohexidine ® .
Gregorova et al. 2011 [13] Coatings 2019, 9, 494 5 of 16 In a recently published study, chitosan/hydroxypropylmethylcellulose (HPMC) composites with varying ratio up to 30% of Kraft lignins (isolated from black liquor and purified via solvent extraction) were prepared and tested against spoilage bacteria that grow at low temperatures. The results revealed the activity against both B. thermosphacta and P. fluorescens for samples with 30% lignin. In HPMC/lignin/chitosan films, the 5% addition exhibited activity against both B. thermosphacta and P. fluorescens [52]. Currently, these lignin-derived composites are studied regarding their applications as scaffold component for mesenchymal stem cell differentiation and bone regeneration [55]. To do so, lignin as feedstock component has to be specified including protocols for quality control using novel chemometric data analysis methods [56,57].
In the present study, lignins isolated from black liquor at different pH values were used to explore the potential of these compounds as an antimicrobial component in polyurethane coatings. First, the extraction conditions that favored high lignin yields were optimized. Unmodified and demethylated lignins were used to prepare the lignin-polyurethane (LPU) coatings. The last part of the study aimed to correlate the antimicrobial properties with extraction conditions (i.e., pH value) and molecular structures (unmodified versus demethylated lignins). Furthermore, the influence of additional antimicrobial dyes (brilliant green and crystal violet) on the LPU coating bioactivity, color and morphology was studied.

Extraction of Kraft Lignin (KL) and Organosolv Lignin (OL)
The Kraft lignin (KL) was extracted through the acidic precipitation from black liquor according to a procedure reported by Garcia et al. [58]. First, about 450 mL of black liquor was filtered with a vacuum filter. The filter cake was rejected. Of the filtrate, 400 mL was heated to 50-60 • C. Sulfuric acid (160 mL, 25 vol.%) was added while stirring. The mixture was stirred for another hour at room temperature and then vacuum filtered. The filter cake was washed with distilled water and sulfuric acid (25 vol.%) until the requested pH value was reached (pH 2 to pH 5). Finally, the precipitated lignin was dried in a freeze dryer for 48 h. The organosolv lignin (OL) was isolated according to a procedure recently reported [10].

Synthesis of Demethylated Kraft Lignin
For the demethylation, a procedure reported by Li et al. was used and slightly modified [33]. The sample (1 g), 0.1 g of Na 2 SO 3 as the demethylating reagent and 6 g of 2.5 mol NaOH solution were introduced into a 15 mL rolled rim glass on an analytical balance and homogenized. The solution was heated with stirring to 90 or 72 • C and stirred for 1 h at this temperature. After cooling to room temperature (RT), the pH was adjusted to pH 2 by means of 1% HCl. The demethylated lignin precipitated as a brown solid. The suspension was transferred to a 45 mL tube and centrifuged for 10 min at 3000 rpm to separate the demethylated lignin from the aqueous solution. The lignin was washed with distilled water and the pH adjusted to pH 7 with 2.5 molar NaOH solution. It was again centrifuged (for 30 min at 4000 rpm) to separate the aqueous phase from the demethylated lignin. The product was first stored at 40 • C in a drying oven and then freeze-dried at 80 • C and 0.10 mbar. Subsequently, the samples were homogenized and transferred for storage in rolled edge glasses, which were closed with snap lids. Furthermore, the samples were protected against UV radiation.

Size Exclusion Chromatography
Size exclusion chromatography was used to determine the number-average (M n ) and weight-average (M w ) molecular weights of lignins and their polydispersities, analogue to recently reported methods [29,30]. A PSS SECurity 2 GPC System was used with tetrahydrofuran as the mobile phase, a run time of 30 min and an injection volume of 100 µL. The system was calibrated using polystyrene standards at different molecular weights.

Determination of Hydroxyl Groups
The content of hydroxyl groups was determined via two different methods. ISO 14900:2001(E) developed for polyether polyols with steric hindrance was recently reported [29]. Shortly, each lignin sample was boiled under reflux in 25 mL of acetylation reagent solution with a blank sample simultaneously under the same conditions. After three hours at reflux, the flasks were left to cool down to room temperature. Twenty-five milliliters of sample and blank, respectively, were filled up with water to 100 mL and were titrated with sodium hydroxide (0.5 M). The split up of the acetylated samples and blanks allowed a triple determination via titration. Different amounts of sample and blank were needed. The differences were used to determine the total hydroxyl content.

Antibacterial Activity of Lignin
The antimicrobial activity of the lignin powders samples was analyzed in a quantitative way by modifying the test for antimicrobial activity and efficacy (JIS Z 2801:2010) of liquid samples [59]. The JIS is based on a comparison of bacteria counts in saline solution on reference and sample materials after a defined incubation temperature and time. Staphylococcus aureus (DSM No. 799) was applied as the test organism. The inoculum was prepared in the same way as described above. According to the McFarland-standard the inoculum was adjusted in physiological saline solution with tryptone (Blank, Vörstetten, Germany; VWR International, Darmstadt, Germany) to a concentration of 108 cfu mL −1 . This inoculum suspension was diluted in physiological saline solution with tryptone (Blank, Vörstetten, Germany; VWR International, Darmstadt, Germany) to a final concentration of 105 cfu mL −1 . Lignin powder was added into tubes with 5 mL physiological saline solution with tryptone to a final concentration of 0.1, 0.01 and 0.001 g mL −1 . Each tube was inoculated with 50 µL of the inoculum. The same measurements were done in nutrient broth instead of physiological saline solution. The measurements were carried out in triplicates.
The inoculum (1 mL) was incubated at 37 • C for 24 h in a mixture of 9 mL nutrient broth (Merck KGaA, Darmstadt, Germany) and 1 mL of sample or reference. Afterwards viable counts were determined by counting the colonies on plate-count agar after incubation at 37 • C for 24 h.
The value of antimicrobial activity was calculated by subtracting the logarithmic value of viable counts of the sample from the logarithmic value of reference material after inoculation and incubation: where as c gew (reference) = arithmetic mean of bacterial counts of reference 24 h after inoculation, and c gew (sample) = arithmetic mean of bacterial counts of sample material 24 h after inoculation. According to the JIS Z 2801:2010 a material can be characterized as antimicrobial, if the calculated log 10 -reduction is ≥2.0 after 24 h at 37 • C [59].

Hemmhoff Test
The antimicrobial activity of the lignin was tested according to the disk diffusion test of the National Committee for Clinical Laboratory Standards (NCCLS) standard method. The disk diffusion test is based on the diffusion of the sampling material in agar. If the bacterium is sensitive to the tested substance, the growth of the bacterium is inhibited and a visible inhibition zone arises. The inhibition zone is the defined area between the punched out area and the beginning of the grown bacterium. If there is no inhibition zone, the bacterium is not sensitive to the tested substance.
Staphylococcus aureus (DSM No. 799) was used as a test organism. The inoculum was prepared by transferring a frozen culture to 10 mL of nutrient broth (Merck KGaA, Darmstadt, Germany). The nutrient broth with the inoculum was incubated at 37 • C for 24 h. According to the McFarland-standard the inoculum was adjusted in physiological saline solution with tryptone (Blank, Vörstetten, Germany; VWR International, Darmstadt, Germany) to a final concentration of 108 cfu mL −1 . In each Petri dish (Sarstedt AG, Nümbrecht, Germany) 100 µL of the inoculum was spatulated on Mueller-Hinton agar (VWR International, Darmstadt, Germany) which were impregnated with the different lignins and blank filter papers as references and were put on the inoculated agar plates.
The agar plates were incubated at 37 • C for 24 h. Afterwards, the diameter of the inhibition zone was measured with a digital caliper (Traceable Digital Caliper 6, VWR International, Darmstadt, Germany).

Synthesis of Lignin-Based Polyurethane Coatings
PEG400 was obtained from Sigma-Aldrich (Steinheim, Germany). 4,4-Diphenylmethane diisocyanate (MDI, for synthesis) was purchased from Merck in Darmstadt and triethylamine (TEA, for synthesis) was received from Carl Roth GmbH in Karlsruhe. All chemicals were used without further purification. PEG400 was mixed with lignin to obtain 1 g of polyol blend. Coatings prepared from lignins isolated at different pH values were produced analogously to the previously described procedure, with the MDI amount adapted to the hydroxyl number of the lignin and the resulting polyol blend. Lignin-based PU coatings were prepared using unmodified and demethylated lignins, respectively, and 4,4-diphenylmethandiisocyanate (MDI). The NCO:OH ratio was 1.7. The calculation was performed according to literature reference [60,61]: where w MDI , w L and w P are the weights (g) of MDI, lignin and polyol, respectively.
[OH] L and [OH] P are the molar contents of total hydroxyl groups in the lignin and the polyol, respectively. Masses of lignin and polyol were kept constant. Thus, 1 g of lignin was dissolved in 6 mL THF under constant stirring. MDI was added and the mixture was transferred on a polyethylene (PE) transparency and dried for 1 h at room temperature. Finally, the pre-films were cured at 37 • C for 3 h to obtain the final lignin PU films. The synthesis of lignin-modified PU coatings with brilliant green (BG) and crystal violet (CV) followed the same procedure, using 0.8% (w/v) of the corresponding triphenylmethane derivative. Analogously, 1 g of demethylated lignin was dissolved in 6 mL of THF under constant stirring to prepare the LPU coatings. MDI was added and the mixture was transferred onto a PE-transparency and dried for 1 h at room temperature. Finally, the pre-films were cured at 35 • C for 3 h to obtain the final lignin-derived PU films.

Antimicrobial Activity of the LPU Coatings
The antimicrobial activity of the coatings was analyzed based on the Japanese Industrial Standard (JIS) Z 2801:2010 [59]. The JIS is based on the comparison of bacteria counts on sample coating/surface and reference material after a defined storage temperature and time (35 • C, 24 h). The reduction of bacteria counts were calculated and represented as log 10 -reduction. The log 10 -reduction is a measure for the antimicrobial activity and effectiveness of the coatings. According to the JIS a material is called antimicrobial when the log 10 -reduction is ≥2 log 10 .
Staphylococcus aureus (DSM No. 799) and Listeria monocytogenes were used as test organisms. The inoculum was prepared by transferring a frozen culture to 10 mL of nutrient broth (Merck KGaA, Darmstadt, Germany). The nutrient broth with the inoculum was incubated at 37 • C for 24 h. According to the McFarland-standard the inoculum was adjusted in physiological saline solution with tryptone (Blank, Vörstetten, Germany; VWR International, Darmstadt, Germany) to a final concentration of 108 cfu mL −1 . This inoculum suspension was diluted in physiological saline solution with tryptone to a final concentration of 105 cfu mL −1 .
The coatings and references were inoculated with 400 µL of the inoculum suspension. To enlarge the contact area of the coatings with the inoculum, the inoculum was covered with a sterile foil (Interscience, Saint-Nom-la-Bretèche, France). The plates were incubated at 37 • C for 24 h. After incubation the inoculated suspension was washed out with 10 mL soybean casein lecithin polysorbate 80 broth (SCDLP) solution (Merck KGaA, Darmstadt, Germany). This served as the first solution stage and was used for further decimal solution series. The bacteria counts were determined by using the drop-plate-technique and counting the colonies on plate-count agar (Merck KGaA, Darmstadt, Germany) after incubation at 37 • C for 24 h.
The value of antimicrobial activity was calculated by subtracting the logarithmic value of viable counts of the sample from the logarithmic value of reference material after inoculation and incubation: where c gew (reference) = arithmetic mean of bacterial counts of reference 24 h after inoculation, and c gew (sample) = arithmetic mean of bacterial counts of sample material 24 h after inoculation. According to the JIS Z 2801:2010 a material can be characterized as antimicrobial, if the calculated log 10 -reduction is ≥2.0 after 24 h at 37 • C.

Thermogravimetric Analysis
TGA measurements were performed with about 10 mg of lignin using a Netzsch (Selb, Germany) TGA 209 F1 with a heating rate of 10 • C min −1 under a nitrogen atmosphere. The temperature ranged from ambient to 800 • C.

Optical Contact Angle
Static optical contact angle (OCA) measurements were performed on the PU films at room temperature using an OCA device equipped with a charge-coupled device (CCD) photocamera (DataPhysics Instruments, Filderstadt, Germany). A 40 µL volume of distilled water was used to dispense liquid droplets.

Scanning Electron Microscopy
Scanning electron microscopy (SEM) from ThermoFischer was combined with X-ray analysis (SEM-EDX). Characterization of the texture, phases and the thin LPU layer were determined by SEM-EDX microscopy using an ESEM Quanta FEG 250 FEI with Apollo XL30 EDX (Thermo Fisher Scientific Inc., Huntsville, AL, USA).

Antibacterial Activity of Kraft Lignin
Kraft lignins were demethylated (DL) and characterized regarding their molecular weight and hydroxyl content ( Table 2). In addition, Table 2 shows Kraft lignins isolated at different pH values [29].
Results for both solutions (NaCl, NB) clearly showed an increase in antimicrobial activity against Staphylococcus aureus for the lignins isolated at different pH values (pH 2 to pH 5) with the highest activity (log 10 reduction of 7.0) for the pH 5 samples. This tendency could also be confirmed for the corresponding LPU coatings prepared from the different lignin samples (see next paragraph). Due to the measurement procedure, the study started using the highest concentrations (0.1 mol/L), then the concentration decreased down to 0.001 mol/L. Obviously, the lowest concentrations were sufficient for the observed antimicrobial effects. Similar results could be observed for the HPMC/lignin coatings recently reported [52] and also for organosolv lignins (not yet published). Further studies are required to clarify the correlation of concentration and antimicrobial activity.  Studies of the antimicrobial activity were performed following procedures reported to investigate intrinsically antimicrobial polymers based on poly((tertbutyl-amino)-methyl-styrene) [62][63][64][65][66] and coatings based on HPMC/lignin/chitosan [52]. Two different nutritions were used: sodium chloride (NaCl) and physiological saline solution (NB) of different concentrations ( Figure 2). Results for both solutions (NaCl, NB) clearly showed an increase in antimicrobial activity against Staphylococcus aureus for the lignins isolated at different pH values (pH 2 to pH 5) with the highest activity (log10 reduction of 7.0) for the pH 5 samples. This tendency could also be confirmed for the corresponding LPU coatings prepared from the different lignin samples (see next paragraph). Due to the measurement procedure, the study started using the highest concentrations (0.1 mol/L), then the concentration decreased down to 0.001 mol/L. Obviously, the lowest concentrations were sufficient for the observed antimicrobial effects. Similar results could be observed for the HPMC/lignin coatings recently reported [52] and also for organosolv lignins (not yet published). Further studies are required to clarify the correlation of concentration and antimicrobial activity.

Antibacterial Activity of LPU Coatings
For the comparability of subsequent investigations, first the antimicrobial effect on different reference surfaces was tested according to Japanese Industrial Standard Z 2801:2000 [59]. The results are shown in Table 3 and Figure 3.

Antibacterial Activity of LPU Coatings
For the comparability of subsequent investigations, first the antimicrobial effect on different reference surfaces was tested according to Japanese Industrial Standard Z 2801:2000 [59]. The results are shown in Table 3 and Figure 3.  As suggested, the results showed normal bacterial growth on blank surfaces: untreated glass, plastic sheets (polypropylene, PP), transparent polystyrene films (PS) and stainless steel (Figure 3). Notable was an emerging germ resistance on the untreated stainless-steel surfaces which underlines the natural antimicrobial effect of stainless-steel surfaces for different bacteria, known as oligodynamic effect. The oligodynamic effects describes the damaging effect of various metal ions on different bacteria, viruses and fungi, most probably due stainless-steel alloy formation initiated by different metal cations [64].
Furthermore, lignin-modified PU coatings prepared from demethylated lignins were applied to various surfaces and analyzed for their antimicrobial action. The results are listed below in Table 4 and Figure 4.  As suggested, the results showed normal bacterial growth on blank surfaces: untreated glass, plastic sheets (polypropylene, PP), transparent polystyrene films (PS) and stainless steel (Figure 3). Notable was an emerging germ resistance on the untreated stainless-steel surfaces which underlines the natural antimicrobial effect of stainless-steel surfaces for different bacteria, known as oligodynamic effect. The oligodynamic effects describes the damaging effect of various metal ions on different bacteria, viruses and fungi, most probably due stainless-steel alloy formation initiated by different metal cations [64].
Furthermore, lignin-modified PU coatings prepared from demethylated lignins were applied to various surfaces and analyzed for their antimicrobial action. The results are listed below in Table 4 and Figure 4. The results showed significant microbial reduction against S. aureus for the PU coatings synthesized from demethylated lignins. It is also noticeable that the germ reduction can be correlated to the pH value for lignin isolation: lignins isolated at pH 3, 4 and 5 showed a higher germ reduction and antimicrobial activity, respectively, than the reference (blind value: polypropylene glycol (PPG) as polyol without lignin). One reason for this could be the improved homogeneity of the coatings, caused by higher crosslinking density of the LPU due to high OH numbers, analogous to the correlation of OH number and antioxidant activity of LPU coatings [29,30]. Besides LPU coatings, it was recently reported that the antimicrobial activity of various lignin-derived cellulose and cellulose/chitosan composites against S. aureus and E. coli (Table 1) [52]. A comparison of hydroxypropylmethyl cellulose/lignin films were blended with Kraft lignin in different amounts up to 30 wt.%. Comparing both systems (HPMC versus PU), the capacity against S. aureus was highest for the lignin isolated at pH 5 ( Table 5). As supposed, the addition of triphenylmethane derivatives (BG, crystal violet (CV)) resulted in increased antimicrobial activity against S. aureus. The results showed significant microbial reduction against S. aureus for the PU coatings synthesized from demethylated lignins. It is also noticeable that the germ reduction can be correlated to the pH value for lignin isolation: lignins isolated at pH 3, 4 and 5 showed a higher germ reduction and antimicrobial activity, respectively, than the reference (blind value: polypropylene glycol (PPG) as polyol without lignin). One reason for this could be the improved homogeneity of the coatings, caused by higher crosslinking density of the LPU due to high OH numbers, analogous to the correlation of OH number and antioxidant activity of LPU coatings [29,30]. Besides LPU coatings, it was recently reported that the antimicrobial activity of various lignin-derived cellulose and cellulose/chitosan composites against S. aureus and E. coli (Table 1) [52]. A comparison of hydroxypropylmethyl cellulose/lignin films were blended with Kraft lignin in different amounts up to 30 wt.%. Comparing both systems (HPMC versus PU), the capacity against S. aureus was highest for the lignin isolated at pH 5 ( Table 5). As supposed, the addition of triphenylmethane derivatives (BG, crystal violet (CV)) resulted in increased antimicrobial activity against S. aureus.

Thermal Properties (TGA)
TGA measurements were performed to describe and evaluate the thermal stability of the corresponding LPU coatings with antimicrobial additives and the coatings prepared from demethylated lignins ( Figure 5).

Thermal Properties (TGA)
TGA measurements were performed to describe and evaluate the thermal stability of the corresponding LPU coatings with antimicrobial additives and the coatings prepared from demethylated lignins ( Figure 5). TGA results showed thermal stability between 143-165 °C which are reasonable temperature stabilities for applications in construction and packaging (Table 6). Coatings with CV as antimicrobial additive showed the highest temperature stability with 165 °C in contrast to LPU coatings prepared with BG with a stability of 146 °C. The residual mass of both LPU coatings was between 20%-21.5%. TGA results showed thermal stability between 143-165 • C which are reasonable temperature stabilities for applications in construction and packaging (Table 6). Coatings with CV as antimicrobial additive showed the highest temperature stability with 165 • C in contrast to LPU coatings prepared with BG with a stability of 146 • C. The residual mass of both LPU coatings was between 20%-21.5%. Obviously, decomposition temperature and residuals are influenced not only by the pulping process used for lignin isolation (Kraft versus organosolv), but also by demethylation and added antimicrobial triphenylmethane derivatives (brilliant green and crystal violet). Further quantification of these effects by DSC measurements is under investigation.

Contact Angle of LPU Coatings
The wettability properties of the surfaces of all lignin-based PUs were investigated by means of static contact angle measurements against water ( Table 6). The LPU coatings possessed a rather hydrophobic character with water contact angles θH 2 O up to 92 degrees, higher than literature data for LPUs reported by Jia et al. prepared from organosolv lignin (61 • ). The PU coatings with demethylated lignins showed a contact angle of 84.22 ± 0.51 • . Table 7 summarizes the contact angle data of all LPU with antimicrobial additives. The results revealed that the LPU with brilliant green had a better wettability (87.36 ± 0.15 • ) compared to the LPU with crystal violet (67.40 ± 0.18 • ).

Morphology of the LPU Coatings
To get a first idea of the homogeneity of the lignin-derived coatings, the coatings were observed via reflected light microscopy showing that homogeneous coatings could be obtained using lignins of number-average molecular weight (M n ) < 500 g/mol (equivalent weight-average (M w ) < 1570 g/mol) and OH content above 5 mmol/g (samples isolated at pH 4 and pH 5). Using scanning electron microscopy (SEM), the thickness of the casted films was determined to range between 150-160 µm [29]. Figure 6 shows the prepared LPU coatings: (a) with 0.8% brilliant green added resulted in homogeneous films of greenish color and smooth surface; (b) with 0.8% crystal violet added also Coatings 2019, 9,494 13 of 16 resulted in homogeneous films of smooth surface colored in dark blue. Reference coatings are shown containing PU/CV (no lignin) and PU (no lignin, no crystal violet). The antimicrobial activities were determined according to Japanese Industrial Standard (JIS) Z 2801:2010 [59]. Figure 6c shows coatings on different surfaces: stainless steel, wood, plastic (polypropylene). On all surfaces, the coatings showed smooth homogeneous surfaces. In ongoing studies, the adhesion strength will be quantified.
To get a first idea of the homogeneity of the lignin-derived coatings, the coatings were observed via reflected light microscopy showing that homogeneous coatings could be obtained using lignins of number-average molecular weight (Mn) < 500 g/mol (equivalent weight-average (Mw) < 1570 g/mol) and OH content above 5 mmol/g (samples isolated at pH 4 and pH 5). Using scanning electron microscopy (SEM), the thickness of the casted films was determined to range between 150-160 µm [29]. Figure 6 shows the prepared LPU coatings: (a) with 0.8% brilliant green added resulted in homogeneous films of greenish color and smooth surface; (b) with 0.8% crystal violet added also resulted in homogeneous films of smooth surface colored in dark blue. Reference coatings are shown containing PU/CV (no lignin) and PU (no lignin, no crystal violet). The antimicrobial activities were determined according to Japanese Industrial Standard (JIS) Z 2801:2010 [59]. Figure 6c shows coatings on different surfaces: stainless steel, wood, plastic (polypropylene). On all surfaces, the coatings showed smooth homogeneous surfaces. In ongoing studies, the adhesion strength will be quantified.

Conclusions
The results of the antimicrobial activity study of lignin-based polyurethane coatings confirmed the capacity of Kraft lignin against special microorganisms such as S. aureus. Triphenylmethane derivatives (brilliant green, crystal violet) significantly increased this antimicrobial effect. The coating color changed from dark brown to green (in case of BG) and blue (in case of CV). Wettability tests using contact angle measurements confirmed the hydrophobic character of the lignin-derived PU coatings.
Author Contributions: S.E.K. mainly contributed to the manuscript, performed the experiments and analyzed the data; A.A. contributed analytical data regarding the lignins extracted from organic solvents; J.R. contributed in PU coating preparation. I.K. and J.K. contributed antimicrobial analyses; M.S. conceived and designed the experimental studies and contributed in writing the manuscript.