方法对纸浆厂污水流和培养微生物的生物降解能力的表征促进微生物生长
Summary
Industrial wastes can be collected and modified to analyze microbial growth. Lignocellulose extraction techniques provide components to analyze specific biodegradation ability. Gas chromatography-mass spectrometry identifies fermentation products of microorganisms grown on pulping waste. These methods determine the metabolic capacity of microorganisms to degrade pulping waste.
Abstract
硫酸盐处理应用于木屑对木质素从多糖中分离木素纤维素纸浆，将产生一个高质量的纸张。黑液是由牛皮纸工艺，具有潜在的下游生物转化产生的制浆废水。然而，木质纤维素资源，这占了大部分内黑液可用的有机碳，以及其碱性pH存在挑战，这浪费材料的微生物降解及其化学衍生物的顽抗性质。方法黑液微生物生长的收集和修改的目的是利用这种纸浆废液中的木质素，有机酸和多糖降解副产物转化成有价值的化学物质。介绍了木质纤维素的提取技术制备木质纤维素底物的增长理解为培养微生物的代谢能力，提供了一个可重复的方法。使用燃气通道的romatography – 质谱法使之从微生物对制浆废水增长所带来的发酵产物的鉴定和定量。这些方法一起使用时可以促进微生物的潜在生产发酵产品，将提供更大的价值，制浆系统，减少污水垃圾，从而增加潜在纸铣利润，并提供用于黑液的其他用途的代谢活动的决心。
Introduction
The pulping of wood is a chemically intensive process that has been optimized over many years to create a system with minimal waste. However, some outputs of this process could be used to produce higher value product(s). Black liquor is one such example. It is generated from the kraft process, which is the dominant chemical pulping method, representing 85% of world lignin production1. The kraft process (Figure 1) uses temperature (160-200 °C), pressure (120 psig), and the chemicals contained in white liquor (sodium hydroxide and sodium sulfide) to dissolve the lignin from the wood fibers2,3. Black liquor contains lignin, organic acids, and polysaccharide degradation byproducts4. It is incinerated to produce steam and recover chemicals in the recovery boiler that provides thermal energy for downstream paper making and pulping processes. The volume of black liquor generated by pulping can exceed the amount that the recovery boiler can effectively process. Disposing of the black liquor as effluent negatively affects aquatic flora and fauna and thus is not an option. Application of microbial organisms that could use black liquor for growth would be beneficial in terms of increasing chemical recovery and generation of value-added product(s) that would improve the overall life cycle analysis of the pulping system. Chemical and biological conversion of lignin derived monomers has successfully produced vanillin and cinnamic acid for use as food sweeteners and fragrance additives, phenol used for plastic and resins, and cyclohexane, which could be used for fuel5.
Previous work on biodegradation of this pulp waste has been focused on lignin depolymerization. The International Lignin Institute (ILI) reports that between 40-50 million tons of lignin is produced each year (http://www.ili-lignin.com/aboutlignin.php). Only 1.5% of that lignin is used for commercial industrial processes6. Lignin depolymerization by laccase and peroxidase enzymes produced by white rot fungi of the Phanerochaete and Trametes genera has been studied at length7. Soil bacteria known to degrade aromatic compounds such as Nocardia and Rhodococcus8, Pseudomonas putida mt-29, and Streptomyces viridosporus T7A10 have also been shown to be capable of lignin degradation. Bacterial degradation of pulping waste is promising because some bacteria can thrive in the saline and alkaline (pH 10-14) conditions that characterize the pulping waste effluents11. While lignin is the main component of black liquor, microorganisms may also degrade the other components that make up black liquor. These techniques do not exclusively identify lignin degrading microorganisms, but serve to identify microorganisms that can be applied directly to the pulping waste black liquor instead of its further processed constituents.
Black liquor was collected and modified for microbial growth through neutralization and filter-sterilization. Microbial growth requirements were identified for an environmental microbial isolate by minimal media growth experiments on lignocellulosic components produced by a novel lignocellulose extraction protocol. Growth media were analyzed by gas chromatography-mass spectrometry (GC-MS) to determine the metabolic products of the environmental microbial isolate when grown on black liquor as the sole carbon source. The combination of these techniques provides an assessment tool to determine the metabolic capacity of a microorganism when grown on pulp mill wastes such as black liquor. Use of such techniques also offers insight into the value of application of specific microorganisms to pulp mill waste for the generation of byproducts.
Protocol
Representative Results
Discussion
This protocol describes a combination of techniques that aims to identify microorganisms that can degrade pulping waste, the carbon sources utilized during growth on pulping waste, and the microbial metabolic products produced when grown on pulping waste. We have shown the success of this protocol with the microbial environmental isolate: a facultative anaerobe that can grow on 10% black liquor and use the lignocellulose extraction components as sole carbon sources for growth. This protocol could be used to determine the…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We would like to thank Jim McMurray for providing the black liquor and Dr. David Tilotta and August Meng for their help with GC-MS. Support for Stephanie L. Mathews was provided by a USDA National Needs Fellowship (award number 2010-38420-20399).
Materials
| 1,4-Dioxane (Certified ACS) | Fisher | D111 | Flammable, preoxidizable chemical |
| Black Liquor | Department of Forest Biomaterials at North Carolina State University | N/A | pH 12.72, 13.67% solids |
| Ethanol (microbiology grade) | Fisher | BP2818 | |
| Ethyl Acetate (ACS reagent grade) | EMD | EX0240-9 | Flammable |
| Glacial acetic acid (Certified ACS) | EMD | AX0073 | Corrosive |
| Guaiacol | Sigma Aldrich | PHR1136 | Harmful by ingestion, corrosive |
| HP-5 capillary column | Agilent | 19091J-577 | 60 m x 0.18 mm internal diameter, 0.18 μm thickness |
| Hydrochloric acid (certified ACS) | EMD | HX0603P | |
| N,O-Bis(trimethylsilyl)trifluoroacetamide with trimethylcholorsilane 99:1% (BSTFA) | Fluka | 15238 | Flammable, causes skin burns and eye damage with contact |
| Na2SO4 (FCC grade) | VWR | BDH8026 | |
| NaClO2 (80%) | Sigma Aldrich | 244155 | Flammable, toxic |
| NaOH (certified ACS) | EMD | 1.06498.1000 | Corrosive |
| Alkacid pH test ribbons | Fisher | A979 | |
| Phosphoric Acid (certified ACS) | Fisher | A242 | |
| Polaris Q mass spectrometer | Thermo Electron Corporation | ||
| Pyridine (99%) | Alfa Aesar | A12005 | Flammable, toxic in contact with skin |
| Switchgrass | Cherry Research Farm Goldsboro, NC | N/A | Harvested August 2011 |
| Thermo Finnigan trace gas chromatograph | Thermo Electron Corporation | ||
| Whatman no. 1 filter paper | Whatman | 1001-150 |
References
- Tejado, A., Pena, C., Labidi, J., Echeverria, J. M., Mondragon, I. Physico-chemical characterization of lignin from different sources for use in phenol-formaldehyde resin synthesis. Bioresource Technol. 98, 1655-1663 (2007).
- Brannvall, E., Elk, M., Gellerstedt, G., Henriksson, G. Overview of pulp and paper processes. Pulp and paper chemistry and technology. 2, 1-12 (2009).
- Biermann, C. J. Pulping fundamentals. Handbook of pulping and papermaking. , 55-100 (1996).
- Sjöström, E. . Wood chemistry: fundamentals and applications. , (1924).
- Philbrook, A., Alissandratos, A., Easton, C. J. Biochemical processes for generating fuels and commodity chemicals from lignocellulosic biomass. Environ. Biotechnol. , 39-63 (2013).
- Sanchez, R., Ferrer, A., Serrano, L., Toledano, A., Labidi, J., Rodriguez, A. Hesperaloafunifera as a raw material for integral utilization of its components. BioResources. 6 (1), 3-21 (2011).
- Font, X., Caminal, G., Gabarrel, X., Romero, S., Vicent, M. T. Black liquor detoxification by laccase of Trametesversicolorpellets. J. Chemical Tech. Biotech. 78, 548-554 (2003).
- Zimmerman, W. Degradation of lignin by bacteria. J. Biotechnol. 13, 119-130 (1990).
- Ahamad, M., Taylor, C. R., Pink, D., Burton, K., Eastwood, D., Bending, G. R., Bugg, T. D. H. Development of novel assay for lignin degradation: comparative analysis of bacterial and fungal lignin degraders. Mol. Biosystems. 6, 815-821 (2010).
- Ramchandra, M., Crawford, D. L., Hertel, G. Characterization of an extracellular lignin peroxidase of the lignocellulolyticactinomycete Streptomyces viridosporus. Appl. Environ. Micro. 54, 3057-3063 (1988).
- Mishra, M., Thakur, I. S., Satyanarayana, T., Johri, B. N., Prakash, A. . Microorganisms in environmental management: microbes and the environment. , (2012).
- Karaaslan, A. M., Tshabalala, M. A., Wood Bushcle-Diller, G. hemicellulose chitosan-based semi-interpenetrating network hydrogels: mechanical, swelling and controlled drug release properties. BioResources. 5 (2), 1036-1054 (2010).
- Lech, K., Brent, R., Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., Kevin, S. . Short protocols in molecular biology. , (1992).
- Raj, A., Krishna Reddy, M. M., Chandra, R. Identification of low molecular weight aromatic compounds by gas chromatography-mass spectrometry (GC-MS) from kraft lignin degradation by three Bacillus sp. Int. Biodeterior. Biodegradation. 59, 292-296 (2007).
- Chandra, R., Raj, A., Purohit, H. J., Kapley, A. Biodegradation of kraft lignin by three pure and mixed aerobic bacterial cultures isolated from pulp and paper sludge. , (2005).
- Chen, Y., Chai, L., Tang, C., Yang, Z., Zheng, Y., Shi, Y., Zhang, H. Kraft lignin biodegradation by Novosphingobium sp. B-7 and analysis of the degradation process. Bioresource Technol. 123, 682-685 (2012).
- Bandounas, L., Wierckx, N. J., de Winde, J. H., Ruijssenaars, H. J. Isolation and characterization of novel bacterial strains exhibiting ligninolytic potential. BMC Biotechnol. 11 (94), (2011).
- Chandra, R., Abhishek, A., Sankhwar, M. Bacterial decolorization and detoxification of black liquor from rayon grade pulp manufacturing industry and detection of their metabolic products. Bioresource Technol. 102, 6429-6436 (2011).
- Gellerdtedt, G., Ek, M., Gellerstedt, G., Henriksson, G. Chemistry of Bleaching of Chemical Pulp. Pulp and Paper Chemistry and Technology. 2, 201-237 (2009).
- Kringstad, K. P., Lindstrom, K. Spent liquors from pulp bleaching. Environ. Sci. Tech. 18, 236-247 (1984).
