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Journal of Applied Sciences Research, 3(6): 470-475, 2007 2007, INSInet Publication

Fingerprinting for the Lignin Degrading Bacteria from the Soil


A.A. El-Hanafy and 2Hassan E. Abd-Elsalam and 3Elsayed E. Hafez


Department of Nucleic Acid Research, Department of Environmental Biotechnology, Genetic Engineering and Biotechnology Research Institute, 3 Department of plant molecular pathology, ARDI, Mubark City for Scientific Researches and Technology Application, Borg EL-Arab, Alexandria, Egypt

Abstract: Lignin is the most abundant renewable aromatic material on earth. Research on lignin biodegradation has accelerated greatly during the past 20 years, mainly because of the substantial potential applications of bioligninolytic systems in pulping, bleaching, converting lignins to useful products and treating of agricultural wastes using bacteria. Isolation and identification environmental friendly bacteria for lignin degradation becomes an essential, because all the previous researches concentrated on using fungal treatments. However, bacteria seem to play a leading role in decomposing lignin in aquatic ecosystem because wood degrading bacteria have a wider tolerance of temperature, pH and oxygen limitations than fungi. Therefore, four Egyptian types of soils were collected from different geographic areas of Egypt (Bahig, El- Etr, Abu El- Matamer and Kafr El- Dawar) and used for isolation of lignin degrading bacteria. After initial period (90 days) of incubation of soil suspensions with wheat straw as a source of lignocelluloses, soil suspension was used for reinoculation of medium containing pure lignin (Lignin, Alkali). Twenty-one bacterial isolates were selected for their ability to achieve maximum growth on pure lignin. DNA was extracted from bacteria and RAPD PCR was performed to fingerprint the obtained isolates. Key words: Egyptian soils, Lignin Alkali, RAPD, Phylogeny INTRODUCTION Cell walls of forages and roughages provide an essential source of potential energy to ruminants. However, utilization of this energy source is far from complete leaving large portion (as high as 60-70 % for some warm season grasses) to be excreted by animal. It is generally believed that lignin and cross-linking to cell wall polysaccharides are the major impediments to fiber digestion [7 ]. Lignin is formidable substrate [6 ,8 ], formed through oxidation and free radical coupling of phenyl alcohol precursors, the insoluble polymer lack stereo-regularity. In contrast to hydrolysable bonds between subunits of other wood polymers (e.g., cellulose, hemicellulose), lignin degradation requires oxidative attack on the carbon-carbon and ether inter unit bonds. Lignin biodegradation is responsible for much of the natural destruction of wood in use and it may have an important role in plant pathogenesis. On the other hand, potential application utilizing lignin microorganisms and their enzymes have become attractive, because first they may maximize the utilization of crop wastes and roughages for livestock feeding throughout biodegradation of their lig n in c o n te n t. S e c o n d , th e y m a y p r o v id e environmentally friendly technologies for pulp and paper industry via biodegradation of lignin in black liquor waste, which represents negative impact on environment. The decomposition of lignin in nature has been considered for long time to occur by the action of wood-rot fungi mostly Basidiomycete class. These microorganisms simultaneously decompose lignin and wood polysaccharides [1 1 ]. These wood decay fungi are common inhabitants of forest litter and fallen trees. The most widely studied white rot organisms, P h ane roc haete c hry sosporium , belong to the homobasidio-mycetes. Lignin degradation by the white rot fungi is a complex secondary metabolic process mediated by the action of several extra cellular enzymes, of which lignin peroxidases are the most important[3 ]. Complete degradation of the lignin model compound veratrylglycerol-beta-(o-methoxyphenyl) ether is accomplished mutualistically by a two-membered bacterial culture [1 6 ].

Corrsponding Author: Elsayed E. Hafez, Department of plant molecular pathology, ARDI, Mubarak City for Scientific Research and Technology Applications, Research Area, New Borg El-Arab city, Post Code 21934, Alexandria, Egypt. E-mail: elsayed_hafez@yahoo.com 470

J. Appl. Sci. Res., 3(6): 470-475, 2007 By contrast, research on prokaryotes has been primarily concerned with obtaining good evidence for existence of ligninolytic activity. Several bacterial strains were found to degrade and assimilate lignin [1 1 ,1 3 ,2 ,1 0 ]. Lignin induced peroxidases, both extracellular and cell-associated, were identified and characterized in Streptomyces spp. [1 4 ]. Perestelo et al. [1 2 ] and Morii et al.[1 6 ] have studied lignin degradation with unicellular bacteria. Bacteria isolated from compost soil, viz. Azotobacter, Bacillus megatarium and Serratia marcescens, were capable of decolourizing, or solubilizing lignin [9 ] . S. marcescens produced laccase and its activity correlated positively with lignin mineralization and solublization [1 2 ]. The contributions of bacteria have also been reported to the utilization of low-molecular weight lignin oligomers as the sole source of carbon and energy that produce enzymes catalyzing cleavage of intermonomeric linkages [1 5 ]. However, the contribution of bacteria to the complete biodegradation of lignin in natural environment where fungi are also present is not much known. However, bacteria seem to play a leading role in decomposing lignin in aquatic ecosystem because wood degrading bacteria have a wider tolerance of temperature, pH and oxygen limitations than fungi[5 ]. The importance of ligninolytic bacteria raised, because lignin-degrading bacteria have wider tolerance of temperature, pH and oxygen limitation than fungi[5 ] . Thus, bacterial degradation of lignin can be observed when the growth of fungi in extreme environment, or substrate condition. In addition, the application of fungi in bioleaching of raw pulp is not feasible due to its structural hindrance caused by fungal filament. Therefore, identification of bacteria having lignin oxidizing enzymes would be of significant importance. The aim of this study is to substitution the usage of fungi in lignin degradation by bacteria, which push us to isolate and fingerprint new bacterial strains capable to utilize lignin from local Egyptian soils. In the future, identification and maximized the bacterial lignin degradation and justification to use these isolates in industrial and agricultural applications. M ATERIALS AND M ETHODS Isolation of Lignin Degrading Bacteria: Four Egyptian soils from different areas of Egypt (Bahig, ElEtr, Abu El-Matamer and K afr El-Dawar) were used in preparation of soil suspensions (0.5%) in mineral salt media (MSM). A total one gram of wheat straw was added to 50 ml of different soil suspensions in 100 ml rubber stopper flasks and incubate at 30C with shaking for approximately 90 days.
Table 1: prim ers sequence used in RAPD study Prim er Sequence bacR 5`-CGT GTT GTA GCC CAG GTC ATA-3` C15 5`-GGY GGY TGG AAT GAR GG-3` EzA1A13 5`-CAG GCC CTT CCA GCA CCCAC-3` R2 5`-GAG CCA SG C SG T CCA RTC SG G CCA CCA-3`

Respirometery: For respirometery carbon dioxide was measured at different intervals as described by [1 7 ] until minimum CO 2 was achieved. At the time of minimum CO 2 production from different soil types (soluble carbohydrates and readily digestible fiber were consumed) aliquots from these suspensions were spread on liquid M SM solidified with purified agar (15 g l-1 ) supplemented with 1% wheat straw in Petri dishes and incubated at 30C for 48 hours. Lignin as a Sole Carbon Source: Bacterial colonies from solidified media were selected according to their shape and growth and used to inoculate LB enriched medium and incubated at 30C for 72 hours to achieve maximum growth rate. After 72 hours, bacterial cultures were diluted to 5% in M SM for 24 hours to allow consumption of nutrients. Aliquots from diluted culture were used to inoculate culture tubes contain sterilized MSM supplemented with 0.3% lignin (Lignin Alkali, Sigma) and were incubated at 30C with agitation for 40 days. After 40 days, aliquots from tubes were diluted to 1/100 and 1/1000 with sterilized water and examined for growth by nutrient agar inoculation. Selection of Lignin Degrading Strains: After growth on nutrient agar for 24 hours, plates were counted and colonies were selected for heavy growth. Twenty-one bacterial isolates presented for the four soil types were selected and LB media was used for enrichment of this isolates for further DNA studies DNA Extraction and RAPD-PCR Analysis: DNA was extracted from different bacterial isolates according to the method described by Araujo et al. [1 ]. RAPD PCR was performed on bacterial DNA samples using four different primers (Table1) according to the method described by Williams et al.[1 8 ]. The thermal cycle profile was as follow: 4 min initial denaturation at 95C,44 cycles of 1 min at 92C, 1 min at 30C, 1 min at 72C, followed by a final extension at 72C for 10 min. PCR product were analyzed in 2% agarose gel stained with ethidium b r o m i d e . G e ls w e re p h o to g r a p h e d b y G e l Documentation system (Alpha Imager TM1220, Documentation & Analysis system, Canada). The phylogeny tree of bacterial isolates was made according to statistical program analysis (Statistica Version 5). 471

J. Appl. Sci. Res., 3(6): 470-475, 2007 RESULTS AND DISCUSSIONS Respiration Study: It is obvious from figure 1 that there is variation between soil types in respect to CO 2 production with maximum production achieved in Kafr El-Dawar followed by El-Etr then Abu El-Matamer while the lowest CO 2 production was in Bahig soil. These differences would reflect the microbial population density in these soils. In addition, the CO 2 production in the different soil types followed a decreasing pattern with time until it reached a steady state at the end of incubation time (isolation time).The decrease in CO 2 production with time may be attributed to consumption of readily soluble organic compound in the beginning of the period followed by consumption of complex insoluble compound at the end of time. This pattern is in accordance with the pattern previously described by Wright and Reddy [1 9 ] in which CO2 production in soils showed two distinct phases: 1) an initial rapid phase involving decomposition of labile soluble organic compounds; and 2) a slower rate of decomposition of polymeric organic compounds. The large reductions in colour, lignin and total substrate after 6 d observed for the mixed culture (data is not showed) are similar to those reported by [4 ]. Isolation and Screening of Lignin-Degrading Bacteria: In order to obtain possible collection of lignin-degrading bacteria, colonies achieved maximum growth on wheat straw were used to inoculate pure lignin media (3g l-1 ) as a sole source of carbon. After 40 days of incubation, diluted aliquots (1/10 and 1/100) from the lignin medium were tested for growth on nutrient agar and up to 33 bacterial isolates were selected for maximum growth. Diluted aliquots (1/1000) from lignin medium were finally tested on nutrient agar and 21 out of 33 isolates were reselected (S1 - S9 from Bahig, S10-S13 from El- Etr, S14 S16 from Abu El- Matamer and S17 - S21 from Kafr ElDawar).

Fig. 1:

Respiration rate (as mg CO 2 /g soil) of diffrent soil types and wheat straw incubated at 30C with time (hours).

Fig. 2:

RAPD profiles obtained with primer BacR from the D NA of the 21 isolates from different soils. M: 1Kb DNA ladder (Biotools-Biotechnological & Medical Laboratories).


J. Appl. Sci. Res., 3(6): 470-475, 2007

Fig. 3:

RAPD profiles obtained with primer C15 from the DNA of the 21 isolates from different soils. M : 1Kb DNA ladder (Biotools-Biotechnological & Medical Laboratories).

Fig. 4:

RAPD profiles obtained with primer EzA1A13 from the DNA of the 21 isolates from different soils. M: 1Kb DNA ladder (Biotools-Biotechnological & Medical Laboratories).

Fig. 5:

RAPD profiles obtained with primer R2 from the DNA of the 21 isolates from different soils. M: 1Kb DNA ladder (Biotools-Biotechnological & Medical Laboratories). 473

J. Appl. Sci. Res., 3(6): 470-475, 2007

Fig. 6:

Phylogeny tree is showing the relationships among the different isolate of bacteria strains obtained by RAPD analysis using four different primers. subdivided into 18 groups at distance linkage percent 15% . W henever, at distance linkage 7% there is no similarity had been observed. The obtained results will pushing us to use these material on the industrial and agricultural levels. REFERENCES 1. Araujo, W .L., D.A. Angellis and J.L. Azevedo, 2004. Direct RAPD evaluation of bacteria with out conventional DNA extraction. Brazilian Archives of Biology and Technology, 47: 375-380. Ball, A.S., W .B. Betts and A.G. McCarthy, 1989. Degradation of lignin-related compounds by Actinomycetes. Applied and Environmental Microbiology, 55: 1642-1646. Buswell, J.A. and E. Odier, 1987. Lignin biodegradation. Crit. Rev. Biotechnol., 6: 1-60. Chandra, R., A. Raj, H.J. Purohit and A. Kapley, 2007. Characterisation and optimization of three potential aerobic bacterial strains for kraft lignin degradation from pulp paper waste. Chemosphere, 67: 839846. Daniel, G. and T. Nilsson, 1998. Developments in the study of soft rot and bacterial decay. In: Bruce, A., Palfreyman, J.W . (Eds.), Forest Products Biotechnology. Taylor & Francis, London, UK, pp: 3762. H iguchi, T ., 1990. Lignin biochemistry: biosynthesis and biodegradation. W ood Science Technol., 24: 23-63. Jung, H.G., D. Buxton, R. Hatfield, D. M ertens, J. Ralph and P. W eimer, 1996. Improving forage fiber digestibility. Feed Mix, 4: 30-34.

RAPD Fingerprinting of Bacterial Isolates DNA: All the isolated bacteria were subjected to REP-PCR using four primers: bacR, C15, EzA1A13 and R2. The band patterns obtained by the each primer showed a great differentiation between the examined isolates. The BacR primer gave approximately band pattern ranged from one to three with different molecular weights (250-750 bp); in addition, primer C15 gave approximately from 2 to 13 bands with molecular weights (less than 250 up to 750 bp). However, primer EzA1A13 showed high polymorphism between the examined isolates with molecular weight (less than 250 to 2500 bp), also primer R2 indicated that there are approximately 4 18 bands with molecular weights (less than 250 up to 2000 bp); The data presented in figures 2, 3, 4 and 5. The phylogeny tree (Figure 6) obtained from statistical analysis of the RAPD-PCR band pattern (Figure 2, 3, 4 and 5) showed that, there are only two main groups branched from one ancestor. These two groups branched in linkage distance 35% into seven subgroups and these seven groups are divided into 18 groups at distance linkage percent 15%. W henever, at distance linkage 7% there is no similarity had been observed. Conclusion: Approximately 21 out 33 isolates were selected based on their ability to degrade the lignin. In addition, most of the bacteria showed ability to utilize the lignin as a sole of carbon source these bacteria which isolated from Bahig and Kafr El- Dawar areas in successive manner. The phylogeny tree revealed that there are only two main groups branched from one ancestor. These two groups 474


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