Novel thermo-alkali-stable cellulase-producing Serratia sp. AXJ-M cooperates with Arthrobacter sp. AXJ-M1 to improve degradation of cellulose in papermaking black liquor

https://doi.org/10.1016/j.jhazmat.2021.126811Get rights and content

Highlight

  • The first report on cellulose degradation in black liquor by bacterial co-cultures.

  • Novel thermo-alkali-stable cellulases were observed in co-cultures.

  • The purified bcsZ displayed remarkable thermostability and alkali resistance.

  • Co-cultures led to noticeable pollution load reduction and toxicity reduction effect.

  • Co-cultures are suitable for the bioremediation of papermaking black liquor.

Abstract

There is an urgent requirement to treat cellulose present in papermaking black liquor since it induces severe economic wastes and causes environmental pollution. We characterized cellulase activity at different temperatures and pH to seek thermo-alkali-stable cellulase-producing bacteria, a natural consortium of Serratia sp. AXJ-M and Arthrobacter sp. AXJ-M1 was used to improve the degradation of cellulose. Notably, the enzyme activities and the degradation rate of cellulose were increased by 30%−70% and 30% after co-culture, respectively. In addition, the addition of cosubstrates increased the degradation rate of cellulose beyond 30%. The thermo-alkali-stable endoglucanase (bcsZ) gene was derived from the strain AXJ-M and was cloned and expressed. The purified bcsZ displayed the maximum activity at 70 °C and pH 9. Mn2+, Ca2+, Mg2+ and Tween-20 had beneficial effects on the enzyme activity. Structurally, bcsZ potentially catalyzed the degradation of cellulose. The co-culture with ligninolytic activities significantly decreased target the parameters (cellulose 45% and COD 95%) while using the immobilized fluidized bed reactors (FBRs). Finally, toxicological tests and antioxidant enzyme activities indicated that the co-culture had a detoxifying effect on black liquor. Our study showed that Serratia sp. AXJ-M acts synergistically with Arthrobacter sp. AXJ-M1 may be potentially useful for bioremediation for black liquor.

Introduction

With the rapid development of the global pulp and paper manufacturing industry, the resultant environmental pollution has become a severe problem that needs to be solved (Velagapudi et al., 2017). China contributes to the greatest paper yield and consumption. The pulp and paper industry ranks 41st and the wastewater amount discharged from it accounts for 13.0% of the overall volume discharged from the various industries in China (An et al., 2021b). The production of pulp from plant materials utilizes diverse chemicals and great volumes of water and generates intense-colored and toxic wastewater in large amounts (Haq et al., 2016). Generally, the produced wastewater displays strong color, chemical/biochemical oxygen demand (COD/BOD). Besides, it contains poisonous chlorinated compounds, lignins, tannins, phenols, sulfur compounds, resin acids, hemicelluloses, and cellulose. Such materials are blended to produce the dark black effluents referred to as black liquor, which occupies about 10–15% of the overall wastewater amount in the pulp and paper mill industry; the organic pollution load takes up about 90–95% of the overall wastewater pollution load (Haq et al., 2016). As a result, an efficient and complete black liquor treatment is a critical step for reducing environmental pollution induced by wastewater from papermaking. Due to the huge pollution load, black liquor has caused serious soil and aquatic pollution. Additionally, numerous articles suggest that black liquor is significantly environmentally toxic (Rybczyńska-Tkaczyk and Korniłłowicz-Kowalska, 2017). Consequently, treating black liquor before disposal is of great necessity. Cellulose accounts for an important impenetrable pollutant in the produced black liquor and takes up 33% of the overall organic pollutants. It is one of the main sources of COD and BOD in black liquor (Azadi Aghdam et al., 2016). Thus, the efficient treatment of cellulose is very important to reduce the high pollution load of black liquor.

The traditional treatment of papermaking black liquor can be accomplished using a surface aeration tank and conventional activated sludge plants; however, these methods are not effective in decreasing color and phenolics (Costa et al., 2017). In most situations, untreated or treated wastewater is directly emitted into the rivers, streams or other water bodies, resulting in high COD and BOD, which cause serious environmental pollution. Water bodies that might be mixed with industrial effluents are used by farmers to irrigate crop plants in many developing countries, which may lead to the risk of bioaccumulation of toxic substances up the entire food chain (An et al., 2021b). Therefore, the treatment of this industrial wastewater before its final emission is very pressing. To date, the alkali recovery process has been proved to be the most effective method for the treatment of black liquor. Organic substances in wastewater were burned by this way, and alkali was recovered (Xiao et al., 2017). China has a few huge papermaking companies, on the contrary, there are many medium and small-scale paper mills in China. For the black liquor produced by these factories, especially the black liquor produced by using grass fiber as the raw material for papermaking, the content of silicon is high, and the viscosity of the black liquor is also very high. Therefore, it is difficult to concentrate it to the requirement suitable for the combustion in an alkali recovery furnace, and the ideal treatment effect is hard to achieve. Moreover, the investment in the alkali recovery process is huge, and the operating cost is relatively high. Generally, larger paper mills are suitable for the alkali recovery method. However, the medium and small-scale paper mills cannot afford such a large investment. Similarly, large consumption of mineral acids and secondary pollution of chlorine and sulfur make the acid precipitation technology unsatisfactory (An et al., 2021b). It is therefore exigent to seek more practical, energy-saving, lowcost and environmentally friendly means of remediation for small and medium scale industries.

Microorganisms are nature’s original recyclers, converting toxic organic compounds to harmless products. Also, intensive studies have been carried out to explore the microbial diversity, particularly of contaminated areas in search of organisms that can degrade a wide range of pollutants at a high pollution load. In this field, fungi have received increasing attention due to their powerful lignin-degrading enzyme system (Paliwal et al., 2016). Amriani et al. (2017) evaluated black and brownish liquor decolorization using the isolated fungal strain from Japan Trametes versicolor U 80 and Phanerochaete chrysosporium. The results showed that T. versicolor U 80 was able to decolorize brownish liquor by 51.5% after 21 days of incubation and 68.6% black liquor at 15 days incubation. Costa et al. (2017) explored the lignin-degrading ability of white-rot fungi, as B. adusta and P. crysosporium, and results have confirmed the great biotechnological potential of these two strains for complete lignin removal in industrial wastewater and can open the way to next industrial applications on a large scale. Rybczyńska-Tkaczyk and Korniłłowicz-Kowalska (2017) identified optimized parameters for decolorization of industrial alkali lignin in black liquor by the microscopic fungi Haematonectria haematococca BwIII43, K37 and Trichoderma harzianum BsIII33. The results showed that the phenolic compounds, phytotoxicity and ecotoxicity decreased. Although several studies have been done with fungi for lignin degradation due to their highly developed nonspecific ligninolytic system, the pH of black liquor is strongly alkaline (9.0–11.0) with a high temperature (40–50 ℃), and fungal activity is unstable under such extreme environmental and physiological stress conditions. Thus, the fungal application is restricted in the treatment of black liquor. By contrast, bacteria have a high growth ratio, broad tolerance range, presence of synergy in a complicated enzyme system, and great feasibility and compatibility to genetic engineering. At present, several bacterial strains isolated from different ecological niches have been evaluated for removal of the pulp and paper mill effluent color and toxicity (Haq et al., 2016, Paliwal et al., 2016). Haq et al. (2016) reported that the strain Serratia liquefaciens LD-5 effectively reduce pollution parameters (color 72%, lignin 58%, COD 85% and phenol 95%) of real effluent after 144 h of treatment at 30 ℃, pH 7.6 and 120 rpm. Paliwal et al. (2016) studied the optimization of biodegradation process of black liquor performed by novel bacterial consortium which consist of two indigenous bacterial strains viz., Bacillus megaterium ETLB-1 and Pseudomonas plecoglossicida ETLB-3. However, the adaptation temperature of these bacteria is mostly medium temperature, and the pH adaptation range is narrow. These strains are not suitable for the treatment of actual papermaking wastewater with extreme environmental characteristics. To date, bacterium possessing thermophilic and alkali tolerance simultaneously that can be used for black liquor treatment is scarce. Thermophilic bacteria have been identified as favorable candidates in biotreatment methodologies since they utilize many lignocellulosic feedstocks and exhibit high acidic/saline/alkali tolerance. Therefore, cellulase-producing thermostable bacteria have bioremediation potential to decolorize the industrial effluent containing cellulose. However, bacterial cellulases display low activity, and no study has reported the degradation of cellulose by thermo-alkali-stable cellulase-producing bacteria within black liquor from the papermaking industry.

To overcome the poor activity of bacterial cellulases, the present work established the microbial co-culture system. Generally, a microbial co-culture conducts several complicated processes efficiently and demonstrates higher production ratios than monocultures (Ghosh et al., 2016). Moreover, microbial co-cultures can remarkably promote microbial characteristics. For instance, Bacillus cereus can enhance the Ketogulonicigenium vulgare growth and acid production in the co-culture process (Mandlaa et al., 2018). Besides, fungal co-cultures are proved to degrade lignocelluloses, yet fungal cultures may not be expanded, which also show high environmental sensitivity (Sperandio and Ferreira, 2019). Consequently, establishing a bacterial co-culture system for the effective degradation of lignocellulose is of great importance to treat black liquor. Till now, no article has reported the promotion of lignocellulosic degradation by utilizing a co-culture in black liquor treatment. The present work is the first study to investigate the utilization of co-culture of thermo-alkali-stable cellulase-producing bacteria for cellulose degradation within black liquor.

The aim of this study was to screen out bacteria producing thermo-alkali-stable cellulase from the previously lignocellulosic bacteria and improve the cellulose degradation rate within black liquor derived from papermaking using a microbial co-culture. The optimal pH, reaction temperature, and thermostability parameters of cellulase were characterized to evaluate its potential for bioremediation. Further, the effect of co-existing substrates on cellulose degradation was explored. Next, an Serratia sp. AXJ-M endoglucanase were cloned, recombinantly expressed, purified, and characterizated, which was used to identify its industrial application value. Subsequently, the treatment of high pollution load was attempted from the black liquor. Finally, the degree of detoxification of black liquor was evaluated by phytotoxicity and genotoxicity studies. Overall, the co-culture of the thermostable and alkali-stable cellulase-producing Serratia sp. AXJ-M in synergy with the Arthrobacter sp. AXJ-M1 offered a novel and efficient method for cellulose degradation within black liquor.

Section snippets

Chemicals, culture medium and black liquor

Isopropyl-β-D-thiogalactopyranoside (IPTG) and carboxymethyl cellulose sodium (CAS no. 9004–32–4) were procured from Sigma-Aldrich Co. Ltd (Poole, UK). Kanamycin was obtained from Sigma and utilized in selection at 50 µg/mL. β-1,4-D-glucan from barley, amylase from potato, D-galacto-D-mannan from Ceratonia siliqua, and chitin from shrimp shells were procured from Sigma-Aldrich (St. Louis, MO, USA). Sucrose was obtained from Solarbio Science & Technology Co., Ltd. (Beijing, China).

Isolation and identification of cellulase-producing bacteria

Some studies have reported the use of agar medium containing CMC or cellulose to screen cellulase-producing bacteria from the formation of the hydrolysis zone (Islam and Roy, 2018, Yin et al., 2010). Here, cellulose-degrading bacteria were screened using an enrichment culture where CMC-Na was the unique carbon source. The strains able to degrade CMC-Na were selected by enzymatic activity and Congo-red staining. Based on the above preliminary screening steps, four cellulose-degrading isolates

Conclusions

Bacterial mediated remediation of papermaking black liquid offers a great opportunity for the restoration of black liquid contaminated environments in an ecologically acceptable manner. Especially, the co-culture rather than as individual pure cultures are well efficient for complete mineralization of any pollutants. Thus, in the present study, four strains of lignocellulosic degrading bacteria collected from Harbin were enriched with CMC-Na as the unique carbon source to evaluate cellulase

CRediT authorship contribution statement

Xuejiao An: Experiment, Writing − review & editing, Project administration, Supervision. Zhengbin Zong: Software analysis. Qinghua Zhang: Polish the language. Zhimin Li: Software analysis. Min Zhong: Data analysis. Haozhi Long: Data analysis. Changzhi Cai: Software analysis. Xiaoming Tan: Software analysis.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This study was supported by the National Natural Science Foundation of China (42007220), the Science and Technology Research Project of Education Department of Jiangxi Province, China (J90182), Open Funding Project of the State Key Laboratory of Biocatalysis and Enzyme Engineering (SKLBEE2019015).

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