Lignin is a major by-product of pulp and paper industries, and is resistant to depolymerization due to its heterogeneous structure. Degradation of lignin can be achieved by the use of potential lignin-degrading bacteria. The current study was designed to evaluate the degradation efficiency of newly isolated Bacillus altitudinis SL7 from pulp and paper mill effluent. The degradation efficiency of B. altitudinis SL7 was determined by color reduction, lignin content, and ligninolytic activity from degradation medium supplemented with alkali lignin (3 g/L). B. altitudinis SL7 reduced color and lignin content by 26 and 44%, respectively, on the 5th day of incubation, as evident from the maximum laccase activity. Optimum degradation was observed at 40 °C and pH 8.0. FT-IR spectroscopy and GC-MS analysis confirmed lignin degradation by emergence of the new peaks and identification of low-molecular-weight compounds in treated samples. The identified compounds such as vanillin, 2-methyoxyhenol, 3-methyl phenol, oxalic acid and ferulic acid suggested the degradation of coniferyl and sinapyl groups of lignin. Degradation efficiency of B. altitudinis SL7 towards high lignin concentration under alkaline pH indicated the potential application of this isolate in biological treatment of the lignin-containing effluents.

  • Bacillus altitudinis SL7 isolated from effluent could efficiently degrade lignin under alkaline conditions.

  • Degradation efficiency was determined by analyzing lignin content, color reduction, and ligninolytic enzyme activity.

  • Extracellular laccase from B. altitudinis SL7 can play a significant role in the depolymerization of lignin.

  • Various low-molecular-weight lignin degradation products were determined through GC-MS.

The pulp and paper (P & P) industry is among the largest fast-growing industries in the world that utilize lignocellulosic biomass for paper production. It consumes an enormous amount of fresh water and inorganic compounds during pulping, bleaching and washing processes and generates highly contaminated wastewater (Kumar et al. 2015). The lignocellulosic biomass utilizing industries are a major threat to environmental health and the magnitude of problem is indicated by lignocellulosic waste generation from these industries: agricultural waste (200 billion tons/year), food industry (1.3 billion ton/year), P & P industry (effluent 150–200 m3/ton) and sugarcane molasses-based distilleries (effluent 15 lit/1) (Chandra et al. 2011; Kharayat 2012; Kadam et al. 2013; Ravindran & Jaiswal 2016; Taha et al. 2016). The P & P industry generates effluent that is characterized as dark brown with fluctuating pH (generally alkaline), and high chemical oxygen demand (COD) (1,110–1,272 mg/L), suspended solids (1,160–1,380 mg/L), dissolved solids (1,043–1,293 mg/L) and lignin contents (Singh 2015). The concentration of lignin in effluent depends on the type of lignocellulosic biomass used for production of pulp. Disposing off untreated effluent accounts for undesirable coloration of aquatic resources along with deterioration of aquatic flora and fauna by obstructing the passage of sunlight. Lignin derivatives such as chlorolignin affect the reproductive system of fish by causing delayed maturity, lower sex hormone, and reduction in gonad size (Singh & Chandra 2019). In the terrestrial ecosystem, these contaminants enter the food chain and have carcinogenic and genotoxic effects on humans and other animals (Savant et al. 2006).

An appropriate treatment of wastewater from P & P mills is an important issue worldwide and needs to be addressed. Different physicochemical methods such as membrane filtration, sedimentation, chemical oxidation and ozonation have been reported, but they are not suitable for the treatment of effluent due to operational cost and environmental problems. Biological treatment is an efficient, cost-effective alternative to achieve it. Intensive research has been carried out to decontaminate the effluent from P & P industries using fungi, bacteria and their enzymes (Ebanyenle et al. 2016; Mathews et al. 2016). Most of the research on detoxification of ligninocellusic waste involved brown rot and white rot fungal species (Karp et al. 2012; Karim et al. 2016; Schmidt et al. 2016), but the usage of fungal species for effluent detoxification has growth limitations. Fungi require acidic pH for the production of ligninolytic enzymes, usually, the pH of P & P mills effluent tends to be alkaline so the practice of fungal system for effluent treatment requires pH adjustment, which adds extra cost to the treatment process. Meanwhile, bacteria are gaining interest because of environmental adaptability and biochemical versatility as compared to fungi (Mathews et al. 2016). Bacteria can consume simple aromatics to produce complex lignocellulosic biomass for their growth and production of ligninolytic enzymes involved in degradation (Rinaldi et al. 2016).

Few bacterial species have been investigated for detoxification of lignin and production of ligninolytic enzymes. Bacteria such as Bacillus sp. and Paenibacillus sp. have been isolated from P & P mill sludge, their lignin degradation potential was confirmed by analysis of degradation products (Chandra et al. 2007). Laccase-producing bacteria, like Azotobacter, B. megatarium, and Serratia marcescens isolated from soil, are capable of lignin depolymerization, and their degradation activities are correlated with the production of laccase (Xu et al. 2018). Elsalam & Bahobail (2016) reported lignin degradation efficiency of B. licheniformis and B. subtilis of approximately 0.6 and 0.7 g/L of lignin in 7 days, respectively. However, the lignin reduction rates of reported bacterial species are much lower as compared to fungi, and there is also a gap in knowledge about ligninolytic enzymes involved in lignin degradation. There is a need to search for more efficient lignin-degrading bacteria as well as their characterization for maximum lignin reduction. Therefore, the current study is focused on isolation and identification of efficient lignin-degrading bacteria from P & P mill effluent, and optimization of growth conditions for lignin degradation. The degradation efficiency of newly isolated Bacillus altitudinis SL7 was analyzed for its ability to reduce lignin content and decolorize degradation medium. Furthermore, the lignin degradation products were analyzed by gas chromatography-mass spectrometry (GC-MS). Degradation efficiency of B. altitudinis SL7 towards a high concentration of lignin could attract more attention for detoxification of the lignin-contaminated sites.

Materials

Purified synthetic alkali lignin, 2-methoxyphenol (Guaiacol), and azure B dye were purchased from Sigma-Aldrich (St. Louis, MO, USA), the COD Cell Test kit (25–1,500 mg/L) was obtained from Merck Co. (Darmstadt, Germany) and a genomic DNA purification kit, pGEM-T Easy cloning vector and plasmid DNA extraction kit were purchased from Promega (Madison, WI, USA). All other chemicals used in this study were of highest commercial grades.

Collection of samples

The paper industry utilizes a sulfite process for pulping raw material and chlorine for bleaching of the pulp. To assess the pollutant load, samples were collected from different units of effluent treatment plants (ETP) from Century Paper and Board Mills Limited, Punjab, Pakistan. These units included; inlet point, aeration tank I, aeration tank II, secondary sedimentation tank, and final sedimentation tank. Effluent samples were collected carefully to make them as representative as possible of the whole water. Three grab samples with a volume of 1 L were collected, at uniform time intervals over the sampling period and mixed in 5 L sterile plastic bottles to give a composite sample of effluent. Next, sludge samples were collected in sterile zipper bags from the aeration tank of ETP. The samples were kept in an ice chest at below 4 °C and transported to the Applied, Environmental and Geomicrobiology Laboratory at Quaid-i-Azam University, Islamabad, and processed for analysis within 24 hours of collection.

Physicochemical characteristics of wastewater

All the effluent samples were subjected to physicochemical analysis and analyzed for pH, total suspended solids (TSS), total dissolved solids (TDS), nitrates (NO3) and sulphates (SO4) as per the standard methods provided by the American Public Health Association (APHA) (Rice et al. 2017) and COD was determined by COD Cell Test kit according to the instructions provided by the manufacturer. Lignin concentration and color were determined by the methods of Pearl and Benson (Brauns & Brauns 1960), and the Canadian Pulp and Paper Association (CCPA) (CPPA 1974), respectively.

Isolation and screening of lignin-degrading bacteria from sludge

The spread plate method was used to isolate potential lignin-degrading bacteria on alkali lignin adjusted mineral salt medium (AL-MSM) plates. AL-MSM was composed of (g/L): lignin 0.5; Na2HPO4, 2.4; NH4NO3, 0.1; K2HPO4, 2.0; MgSO4, 0.01; CaCl2, agar 20, in which lignin provided the sole carbon source. The sludge sample was serially diluted to 10−7, and 100 μL of diluted sample was spread on the AL-MSM agar plates and aerobically incubated at 35 °C for 7 days to recover the bacterial population that utilized lignin as a carbon source. Morphologically distinct colonies were picked and purified after multiple re-streaking on nutrient agar. Stock cultures of pure isolates were prepared in 20% glycerol and stored at −20 °C for further experiments. Primary screening was carried out on AL-MSM plates supplemented with increasing concentrations of lignin, i.e., 0.5–3 g/L. One potential bacterial isolate designated as SL7 exhibited good growth in the presence of maximum concentrations of lignin (3 g/L) and was selected for further study.

Screening of ligninolytic activity on agar plate-test

The isolate SL7 was screened for laccase and lignin peroxidase activity on substrate adjusted nutrient agar plates. Nutrient agar was sterilized by autoclaving at 121 °C for 20 mins and then poured into glass Petri plates supplemented with filter-sterilized guaiacol and azure B at the final concentrations of 0.02% and 0.01%, respectively. The bacterial isolate SL7 was spot-inoculated on plates and then observed for the appearance of activity zones after incubation at 35 °C for 7 days.

Identification of ligninolytic bacterial strain SL7

Isolate SL7 was identified by colony morphology, microscopic depiction and 16S rRNA gene sequencing. The isolate was grown overnight in 50 ml nutrient broth at 37 °C and 120 rpm. Bacterial cells were harvested through centrifugation in the late exponential phase and genomic DNA was extracted using a commercially available DNA extraction kit, according to the manufacturer's instructions. The purity and quantity of extracted DNA was analyzed by using a NanoDrop spectrophotometer (Fisher Scientific) and observed as 10 μg/mL. The 16S rRNA gene was amplified by polymerase chain reaction (PCR) using universal primers: 27F (5′-ATT CTA GAG TTT GAT CAT GGC TCA -3′) and 1492R (5′-ATG GTA CCG TGT G ACG GGC GGT GTG TA-3′). The reaction mixtures contained 0.5 μl of each primer, 12.5 μl of DreamTaq master mix, 10.5 μl of deionized water, and 1 μl DNA template (10 ng/μl). PCR was carried out under the following conditions: denaturation at 94 °C for 2 min, 30 cycles of denaturation at 95 °C for 1 min, annealing at 55 °C for 1 min, extension at 72 °C for 1 min, and a final extension step at 72 °C for 10 min. 16S rRNA gene amplification was confirmed by gel electrophoresis using 1% agarose gel containing 0.5 μg/ml ethidium bromide and analyzed using a Bio-Rad Gel Doc imaging system. The amplified 16S rRNA gene product was cloned into the pGEM-T Easy cloning vector and sequenced by the Macrogen Company (The Netherlands). The obtained sequence was subjected to BLAST analysis using the BLASTn program (http://www.ncbi.nlm.nih.gov/BLAST/). All the sequences were aligned, and a neighbor-joining phylogenetic tree was constructed using MEGA-X software (Kumar et al. 2018).

Culture conditions for alkali lignin degradation by bacterial isolate SL7

A degradation experiment was carried out in Erlenmeyer flasks (500 mL) containing 200 mL AL-MSM. The composition of the medium was (g/L): lignin, 3; Na2HPO4, 2.4; CaCl2, 0.01; NH4NO3, 0.1; MgSO4, 0.01; K2HPO4, 2.0; peptone (0.25%, w/v) and glucose (0.5%, w/v). The flasks were inoculated with 2 ml of overnight grown culture with an OD600 of 0.6. The effect of temperature and pH on lignin degradation by isolate SL7 was investigated by running the experiment at various ranges of temperature (20–50 °C) and pH (6.0–12.0). The experiment was run in triplicates and uninoculated flasks were used as a control. The samples were withdrawn at 24 h intervals up to a maximum of 7 days and processed for measurement of lignin degradation.

Lignin degradation by strain SL7

Color reduction assay

The color of the degradation medium was determined according to the CPPA standard methods (CPPA 1974). Here, 5 mL sample was collected at 24 h intervals and centrifuged at 8,000 rpm for 30 min. The pH of the supernatant was adjusted to 7.6 with 2M NaOH and its absorbance was measured at 465 nm by UV-visible spectrophotometer. Absorbance values were converted into color units (CU) by using the following formula:
formula
where A1 is the absorbance of standard solution (platinum–cobalt), and A2 is the absorbance of the sample.

Lignin reduction assay

The residual lignin contents were estimated by the method as previously described by Chandra et al. (2007). The pH of the supernatant was adjusted to 7.6 by phosphate buffer and its absorbance was measured at 280 nm by UV-visible light spectrophotometer. Degradation efficiency (%) was calculated as follows:
formula

The growth rate of strain SL7 was determined by measuring optical density at 600 nm (OD600) using a UV-visible light spectrophotometer.

Ligninolytic enzymes assay from strain SL7 during the time course of degradation

Laccase assay

The extracellular laccase activity was determined by measuring the oxidation of guaiacol by the method as previously reported by Barapatre et al. (2017). The culture-free supernatant was obtained by centrifugation of samples at 8,000 rpm for 10 min. The assay mixture contained: 1 mL supernatant; 10 mM sodium acetate buffer (pH 4.5); and 2 mM guaiacol, incubated for 15 min at 30 °C. The laccase activity was measured at 450 nm using a UV-visible light spectrophotometer. The activity was expressed in terms of the International Unit (IU) and described as the amount of laccase required to oxidize 1 μM of guaiacol per minute under standard assay conditions. The laccase activity in U/ml was calculated using the following formula:
formula
where: E.A = Enzyme activity, A = Absorbance, V = Total mixture volume (ml), v = enzyme volume (ml), t = incubation time, e = extinction coefficient for guaiacol (0.6740 μmol/cm).

Lignin peroxidase assay

The lignin peroxidase activity was determined by monitoring the decolorization of azure B using the method as previously described by Archibald (Menon & Hartz-Karp 2019). The reaction mixture consisted of 500 μL supernatant; 100 mM sodium tartrate buffer (pH 3.0); 32 μM azure B; and the reaction was initiated by adding 1 mM H2O2. The enzyme activity was measured at 651 nm using a UV-visible light spectrophotometer. Lignin peroxidase activity was expressed in terms of IU and described as the amount of lignin peroxidase required to oxidize 1 μM of azure B per minute under standard assay conditions.

Analysis of lignin degradation

Determination of structural modification by FT-IR

Fourier transform infra-red (FT-IR) spectroscopy was performed to analyze changes in the functional group of heteropolymeric lignin during the degradation experiment. 2 mg of vacuum-dried sample was mixed with 200 mg of potassium bromide (KBr), the mixture was homogenized and compressed under continuous pressure of 40 MPa to form thin pellets. These pellets were analyzed by FT-IR spectra (Perkin Elmer Spectrum One FT-IR, Waltham, USA) within the range 4,000 to 400 cm−1, and the time for analysis of each sample was set as 60 sec.

Determination of lignin degradation products by GC-MS

Here, 100 mL of test and control samples were centrifuged at 10,000 rpm for 15 min. The cell-free supernatant was acidified to pH 1.0–2.0 by adding concentrated HCl and the acidified supernatant was thoroughly extracted with three volumes of ethyl acetate. The organic layer was collected and dehydrated over anhydrous Na2SO4, the residues were dried in a rotary vacuum evaporator. The dried residues were derivatized by adding dioxane (100 μL) and pyridine (10 μL), followed by silylation with 50 μL trimethylsilyl (TMS). The silylated mixture was heated at 60 °C for 15 min with periodic shaking to dissolve the residues. 5 μL of silylated sample was injected into the GC-MS injector port (Shimadzu Corporation, GC-2014C, Japan), equipped with DB-FFAP capillary column (30 m × 0.25 μm × 0.25 mm) (Agilent Technologies, Wilmington, DE, USA). The column temperature was set to 120–280 °C (10 °C per min increase) with a flow rate of 1.5 mL per min. A solvent removal time was set to 3.0 min and electron ionization mass spectra in the range of 50–750 (m/z). The detection of lignin degradation products was accomplished by comparing the retention time and mass spectra of products in the test sample with available mass spectra in the National Institute of Standards and Technology (NIST) library.

Physicochemical characteristics of effluent

The characteristic features of wastewater are good indicators of the toxicity level in it. The effluent sample was dark brown and alkaline in nature (pH 8.0) with high COD (994 mg/L), lignin (7,416 mg/L), TDS (440 mg/L), TSS (600 mg/L), sulphate (440 mg/L), nitrates (144 mg/L) and color (954 CU). The effluent was treated through an aerated lagoon system at an industrial site that reduced the pollutant concentration but still it remained beyond the permissible limits as recommended by the EPA (US-EPA 2002) (Table 1). Lignin is recalcitrant in nature and the major constituent of lignocellulosic biomass, the presence of lignin and its derivatives possibly contributed to the dark brown color of the effluent and the high concentration of COD (Rice et al. 2017). The source of sulphate in the effluent might be sodium sulfite, which is used during the pulping process (Singhal & Thakur 2009).

Table 1

Physicochemical characteristics of wastewater from effluent treatment plant

ParametersInlet pointAeration tank (1)Aeration tank (2)Secondary sedimentation tankFinal sedimentation tankPermissible limit (US-EPA, 2002)
pH 8.0 7.3 7.3 7.4 7.6 5–9 
COD (mg/L) 994 997 962 650 407 120 
Sulphate (mg/L) 440 540 670 620 740 252 
TDS (mg/L) 440 439 440 426 408 – 
TSS (mg/L) 600 700 700 400 200 – 
Color (CU) 1,954 2,636 2,272 2,136 1,909 – 
Nitrates (mg/L) 144 200 188 224 64 10 
Lignin (mg/L) 7,416 7,958 5,916 7,790 279 0.05 
ParametersInlet pointAeration tank (1)Aeration tank (2)Secondary sedimentation tankFinal sedimentation tankPermissible limit (US-EPA, 2002)
pH 8.0 7.3 7.3 7.4 7.6 5–9 
COD (mg/L) 994 997 962 650 407 120 
Sulphate (mg/L) 440 540 670 620 740 252 
TDS (mg/L) 440 439 440 426 408 – 
TSS (mg/L) 600 700 700 400 200 – 
Color (CU) 1,954 2,636 2,272 2,136 1,909 – 
Nitrates (mg/L) 144 200 188 224 64 10 
Lignin (mg/L) 7,416 7,958 5,916 7,790 279 0.05 

(–): not specified.

Isolation and screening of lignin-degrading bacteria

The sludge samples were collected from the ETP of a P & P mill. The area for collecting the samples was selected by assuming the presence of potential lignin-degraders in the area that contains lignin and other toxic chemicals. Several researchers have reported the isolation of lignin-degrading bacteria from lignin-contaminated sites that were successfully applied for remediation of P & P mill effluent (Barapatre et al. 2017; Menon & Hartz-Karp 2019). In this study, eight bacterial strains designated as strains S1–S8 were isolated from sludge samples on L-MSM agar plates, but only strain SL7 showed a marked ability to grow well in the presence of a high concentration of lignin (3 g/L), utilizing lignin as the sole carbon source (Table 2). Only potent bacteria can survive in the presence of a high concentration of lignin because lignin-derived aromatics have harmful effects and cause cell death by membrane disruption, DNA damage, and enzyme inhibition (Zeng et al. 2014). Strain SL7 was further screened for ligninolytic activity (laccase and peroxidase) on nutrient agar plates adjusted with respective substrates. Strain SL7 was found to be an efficient laccase producer, indicated by the appearance of a brown color around bacterial growth on the guaiacol agar plates (Figure 1(a)). The oxidation of guaiacol is one of the most suitable tests for laccase assays produced by bacteria. Hence, the formation of the brown color was induced by laccase due to the oxidative polymerization of guaiacol (Kumar et al. 2020). Peroxidase activity was not detected, as no decolorization zone around the bacterial growth was observed on azure B agar plates (results are not shown).

Table 2

Screening of tolerance pattern of bacterial growth on different concentrations of lignin

IsolatesConcentration of Lignin (g/L)
0.51.01.52.02.53.0
SL1 + + + + + + + + − − 
SL2 + + + + + + + + + + − 
SL3 + + + + + + + + − 
SL4 + + + + + + + + + + + + + + 
SL5 + + + + + + + + + + + − 
SL6 + + + + + + + + + + + 
SL7 + + + + + + + + + + + + + + + ++ 
SL8 + + + + + + + + − − 
IsolatesConcentration of Lignin (g/L)
0.51.01.52.02.53.0
SL1 + + + + + + + + − − 
SL2 + + + + + + + + + + − 
SL3 + + + + + + + + − 
SL4 + + + + + + + + + + + + + + 
SL5 + + + + + + + + + + + − 
SL6 + + + + + + + + + + + 
SL7 + + + + + + + + + + + + + + + ++ 
SL8 + + + + + + + + − − 

(+) indicating slow growth; (+ +) indicating moderate growth; (+ + +) indicating fast growth.

Figure 1

Screening of bacterial strains from a P & P mill for laccase activity on a guaiacol agar plate. Strain SL7 was found to produce laccase enzyme as indicated by appearance of brown color around bacterial growth.

Figure 1

Screening of bacterial strains from a P & P mill for laccase activity on a guaiacol agar plate. Strain SL7 was found to produce laccase enzyme as indicated by appearance of brown color around bacterial growth.

Identification of bacterial isolate SL7

Strain SL7 was an aerobic, motile, Gram-positive and rod-shaped bacterium. The DNA fragment of 1,500 bp was cloned into the pGEM-T Easy vector and sequenced. The sequencing results were analyzed by comparing the nucleotide sequences obtained from the National Center for Biotechnology Information (NCBI) database and a phylogenetic tree was constructed. The 16S rRNA gene sequence of strain SL7 exhibited 100% similarity with Bacillus altitudinis strain SGAir0031 (CP022319) (Figure 2), thus the bacterium was identified as Bacillus altitudinis strain SL7. The nucleotide sequence reported here can be obtained from the NCBI nucleotide database under accession number MZ400969.

Figure 2

Phylogenetic tree showing the position of strain SL7 constructed by neighbor-joining method using Mega-X software.

Figure 2

Phylogenetic tree showing the position of strain SL7 constructed by neighbor-joining method using Mega-X software.

Effect of temperature and pH on lignin degradation by B. altitudinis strain SL7

The effect of temperature and pH on lignin degradation by B. altitudinis strain SL7 was determined in a shaking flask experiment. The experiment was run in triplicate and a control with each experiment was set up in a separate flask without inoculum. The maximum reduction in lignin content (44.2%) was observed at a temperature 40 °C and pH 8.0 (Figure 3(a) and 3(b)). Strain SL7 showed the ability to degrade lignin under alkaline pH 7.0–11.0, with optimum at pH 8.0. In a lignin degradation system, the pH value has been reported to be an important factor. Pulp and paper industries utilize alkaline chemicals during pulping to dissolve lignocellulosic biomass; hence the effluent released from industries with a high pH (8.0–12.0). Therefore, bacterial strains that could degrade lignin under alkaline conditions would be the suitable choice for detoxification of such effluent (Fang et al. 2018). Several researchers have reported bacteria such as Aneurinibacillus aneurinilyticus, Comamonas sp, Bacillus strain, Rhodococcus pyridinivorans and Streptomyces strain with optimum degradation pH ranges from 7.0–7.5 (Chen et al. 2012; Shi et al. 2013; Chong et al. 2018). The lignin degradation potential of Bacillus altitudinis SL7 under alkaline pH (7.0–11.0) could make it a suitable candidate for application on sites with lignin contamination in soil and water.

Figure 3

Effect of temperature (20–50 °C) and pH (6.0–12.0) on lignin degradation by B. altitudinis SL7 in MSM after 7 days of incubation. (a) Effect of temperature. (b) Effect of pH.

Figure 3

Effect of temperature (20–50 °C) and pH (6.0–12.0) on lignin degradation by B. altitudinis SL7 in MSM after 7 days of incubation. (a) Effect of temperature. (b) Effect of pH.

Bacterial growth and lignin degradation

The relationship between the growth and lignin degradation by B. altitudinis SL7 was investigated during the degradation experiment. Figure 4(a) shows the growth curve of isolate SL7 during lignin degradation over 7 days of incubation. B. altitudinis SL7 grew at a fast rate during the initial 2 days of incubation and achieved maximum growth at day 5. A significant reduction in lignin was observed after 3 days of incubation with maximum reduction (44%) on day 5. In contrast, the color reduction started after 24 h of incubation, while maximum reduction (26%) achieved on day 5 followed by a gradual decrease up to day 7 (16%) (Figure 4(b)). Due to fast bacterial growth during the initial days of incubation, a significant reduction in color and lignin contents was achieved on day 5, which indicates the phenomenon of co-metabolism adopted by B. altitudinis SL7. It could be possible that bacteria utilized glucose and peptone as carbon and nitrogen sources to initiate its growth followed by the utilization of lignin as a co-substrate. Similar to this study, co-metabolism in bacteria and fungi for lignin degradation has also been reported by various authors (Singhal & Thakur 2009; Singh & Chandra 2019). While considering the initial concentration of lignin, the degradation efficiency of B. altitudinis SL7 was higher than the previous reports (Table 3).

Table 3

Summary of lignin degradation by bacterial strains

Bacteria strainLignin load (g/L)pHDegradation time (days)Degradation (%)References
Bacillus altitudinis SL7 44 This study 
Rhodococcus pyridinivorans 0.06 30 Chong et al. (2018)  
Bacillus sp. strains CS-1 & CS-2 0.05 61 Chang et al. (2014)  
Citrobacter freundii (FJ581026) 0.6 49 Chandra & Bharagava (2013)  
Novosphingobium sp. B-7 30 Chen et al. (2012)  
Streptomyces sp. F-6 12 37 Yang et al. (2012)  
Bacillus sp. 81 Abd-Elsalam & El-Hanafy (2009)  
Aneurinibacillus aneurinilyticus 0.25 7.6 43 Raj et al. (2007)  
Bacteria strainLignin load (g/L)pHDegradation time (days)Degradation (%)References
Bacillus altitudinis SL7 44 This study 
Rhodococcus pyridinivorans 0.06 30 Chong et al. (2018)  
Bacillus sp. strains CS-1 & CS-2 0.05 61 Chang et al. (2014)  
Citrobacter freundii (FJ581026) 0.6 49 Chandra & Bharagava (2013)  
Novosphingobium sp. B-7 30 Chen et al. (2012)  
Streptomyces sp. F-6 12 37 Yang et al. (2012)  
Bacillus sp. 81 Abd-Elsalam & El-Hanafy (2009)  
Aneurinibacillus aneurinilyticus 0.25 7.6 43 Raj et al. (2007)  
Figure 4

Time course of bacterial growth and lignin degradation at 40 °C, over 7 days of incubation. Data represented as a means of triplicate experiments. (a) B. altitudinis SL7 growth and lignin degradation (b) Color reduction.

Figure 4

Time course of bacterial growth and lignin degradation at 40 °C, over 7 days of incubation. Data represented as a means of triplicate experiments. (a) B. altitudinis SL7 growth and lignin degradation (b) Color reduction.

Figure 5

FT-IR spectra of lignin after incubation in degradation medium with Bacillus altitudinis SL7 for 7 days. (a) Control; (b) test sample.

Figure 5

FT-IR spectra of lignin after incubation in degradation medium with Bacillus altitudinis SL7 for 7 days. (a) Control; (b) test sample.

Ligninolytic enzymes produced by B. altitudinis SL7 during degradation

Laccase assay

The enzyme laccase activity was measured during the course of lignin degradation by B. altitudinis SL7. A gradual rise in laccase activity was observed from day 2 and reached up to the maximum (1.3 U/mL) at day 5, followed by a decrease in activity. Lignin degradation is mainly related to enzymes secreted by Bacillus (Mei et al. 2020). Laccase is a copper-dependent enzyme that can oxidize lignin phenolic hydroxyl groups, thereby destroying the stability of the aromatic ring (Niu et al. 2021). Previously, laccase-producing Bacillus megatarium and Serratia marcescens have been reported for lignin degradation (Xu et al. 2018). B. altitudinis SL7 did not produce lignin peroxidase, as the enzyme activity was not detected in the degradation medium.

FT-IR spectroscopy

The samples were analyzed for changes in the polymeric structure of lignin after treatment with B. altitudinis SL7 through FT-IR spectroscopy (Figure 5). The spectra of test samples showed a decrease in absorbance around wavenumber 3,500–3,000 cm−1 that was attributed to the stretching frequency of –OH bonds of alcohol and phenol in lignin, which indicates lignin degradation. B. altitudinis SL7 not only caused the oxidation of side chains but also transformed and degraded the aromatic skeleton of lignin. There was a marked difference observed in the fingerprint region between 1,800 and 600 cm−1. The absorbencies and shapes of the peaks in the region 1,600–1,400 cm−1 was changed, and corresponded to the stretching of C=C bonds in the aromatic skeleton of lignin, meaning that aromatic ring was destroyed during degradation (Wang et al. 2021). The ligninolytic enzymes specifically attack C=C bonds in the aromatic skeleton of lignin that led to enzymatic depolymerization of lignin structure (Zeng et al. 2014). Two new peaks appeared at 1,079 and 1,045 cm−1 while some peaks disappeared from the spectrum around 1,375–1,260 cm−1 indicating C–O stretching. These new bands indicate that the guaiacyl (G) and syringyl (S) groups were transformed and converted into simpler compounds, such as ethers, phenol and alcohols (Kumar et al. 2015). Increase in intensity of peaks represents the production of high amounts of lignin degradation products (Sonkar et al. 2019). It could be inferred from the findings that B. altitudinis SL7 chemically modified and degraded lignin.

Analysis of lignin degradation products by GC-MS

The degradation of alkali lignin by Bacillus altitudinis SL7 was determined by analyzing degradation products through GC-MS. Figure 6 shows a total ion chromatograph (TIC) of low-molecular-weight compounds extracted with ethyl acetate from acidic supernatant of both test and control samples. The compounds were identified based on their retention time (RT) and mass to charge ratio (Table 4). About nine low-molecular-weight products were detected in test samples, identified as glutamic acid (RT-2.27), oxalic acid (RT-2.53), succinic acid (RT-2.65), vanillin (RT-3.62), 3-methylphenol (RT-6.38), 2-methyoxyphenol (RT-8.12), ethyl vanillin (RT-10.15), ferulic acid (RT-10.32) and styrene (RT-12.21). Detection of phenolic compounds was a clear indication of lignin degradation, because these compounds were considered as the basic structural units of the lignin polymer. The presence of various phenolic and chlorinated compounds in lignin degradation medium was previously reported by several researchers (Sonkar et al. 2019). Formation of acidic compounds during lignin degradation was possibly due to the enzymatic degradation of phenolic side chain into ketones, which were further degraded through Cα–Cβ cleavage and formed acids (Yang et al. 2012). Raj et al. (2007) reported the identification of vanillin, guaiacol, gallic and ferulic acid during lignin degradation by Bacillus sp. These results confirmed the oxidation of coniferyl (G) and sinapyl (S) groups of lignin polymer. Previously, it has been reported that, during fungal degradation of lignosulfonate and bacterial degradation of lignin, the above degradation compounds were also detected (Jiang et al. 2020). The peak at RT 5.09 was observed both in test and control samples, and could be due to lignin that was not detected on the mass spectrometer because of its high molecular weight and out of the range of the m/z ratio.

Table 4

List of compounds detected as TMS derivatives in ethyl acetate extract from Bacillus altitudinis SL7 degraded lignin sample

Retention time (min)Product nameProduct structurem/z
2.27 Glutamic acid  147 
2.53 Oxalic acid  90 
2.65 Succinic acid  118 
3.62 Vanillin  152 
6.38 3-Methylphenol  108 
8.12 2-Methyoxyphenol  124 
10.15 Ethyl vanillin  166 
10.32 Ferulic acid  194 
12.2 Styrene  104 
Retention time (min)Product nameProduct structurem/z
2.27 Glutamic acid  147 
2.53 Oxalic acid  90 
2.65 Succinic acid  118 
3.62 Vanillin  152 
6.38 3-Methylphenol  108 
8.12 2-Methyoxyphenol  124 
10.15 Ethyl vanillin  166 
10.32 Ferulic acid  194 
12.2 Styrene  104 
Figure 6

GC chromatographs of control and lignin degraded sample. Degradation products were identified according to the retention time and mass spectra at specific retention time. (a) Control. (b) Lignin degraded sample by Bacillus altitudinis SL7.

Figure 6

GC chromatographs of control and lignin degraded sample. Degradation products were identified according to the retention time and mass spectra at specific retention time. (a) Control. (b) Lignin degraded sample by Bacillus altitudinis SL7.

The study was aimed to explore the potential of lignin-degrading bacteria from the sludge of P & P mills. The present study confirmed the degradation and decolorization of lignin by Bacillus altitudinis SL7 at mesophilic temperature and alkaline pH. FT-IR analysis confirmed the changes in functional groups of lignin treated with bacteria. GC-MS analysis of control and bacterial treated samples showed the metabolization and transformation of alkaline lignin into low-molecular-weight compounds such as glutamic acid, oxalic acid, succinic acid, vanillin, methyoxyphenol and ferulic acid. B. altitudinis SL7 has the potential to degrade lignin under alkaline pH ranges from 7.0–10.0 as compared with other reported lignin-degrading bacterial species, therefore no additional adjustment of pH was required during lignin degradation. The presence of extracellular laccase enzyme favors the idea of enzymatic degradation of lignin. It is finally concluded that Bacillus altitudinis SL7 could be of interest for the degradation of high loads of lignin from P & P mill effluent.

The present study was funded by the Higher Education Commission of Pakistan under the ‘Pak Turk Researchers’ Mobility Grant Program 2017’ in collaboration with Quaid-i-Azam University, Islamabad Pakistan and Karadeniz Technical University, Trabzon, Turkey.

I hereby confirm that this manuscript is our original work. It has neither been published before in any form nor is under consideration by another journal at the same time as in this journal. All the authors have no financial as well as commercial conflict of interest regarding this manuscript and have approved its submission to Water Science and Technology.

All relevant data are included in the paper or its Supplementary Information.

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