Meiqun LI, Jiaoxia LU, Wensheng AI*, Ming YANG, Yong MENG, Jia TU, Xiangyun PU, Xiangbo LIU

1. Hunan Academy of Forestry, Changsha 410004, China;

2. Forestry Bureau of Yueyang County, Yueyang 414100, China

Mao bamboo is the main bamboo germplasm in Hunan Province,which has 8.87×105hm2of Mao bamboo-growing area and total 1.95×109of bamboo plants,ranking second across China[1-2].After Mao bamboo is cut down, the remaining part in forest is called as Mao bamboo stump. In bamboo forest, some bamboos and elderly, lodging and died bamboos need to be cut down every year or every two years. If the annual cutting amount is assigned as 3.3 plants/hm2, the quantity of bamboo stumps in bamboo forest will reach 16.7 plants/hm2within five years, and its density will exceed those of shootusing and shoot- and wood-using bamboos. Zheng et al[3]. surveyed the Mao bamboo forest with an area of 3.32×104hm2in Youxi County. They found that the density of bamboo stumps reached 3 952/hm2, and the bamboo stumps accounted for 10.4%of the total forest area;the natural decomposition of bamboo stumps was quite slow, and their complete decomposition required as long as 15 years with 9 years in average. If these bamboo stumps were not removed timely,the growth of young bamboo plants and yield of bamboo shoots all would be affected. At the same time, they would also bring many obstacles and insecure factors to the bamboo forest.Lin et al.[4]surveyed several bamboo forests. The results showed that the average radius of bamboo stumps was 0.29 m, the average area per bamboo stump was 0.265 m2, and the bamboo stumps represented 10.4%of the total forest area. Bamboo stumps have thick walls, widely-distributed roots and high contents of cellulose, hemicellulose and lignin,so their decomposition requires a long time. How to accelerate the decomposition of bamboo stumps in bamboo cutting is a more prominent problem[5]. Microbial flora differs greatly in bamboo stumps at different decomposition degrees. The changes of bacteria, fungi, actino-mycetes, cellulose-degrading bacteria and lignin-degrading bacteria in bamboo stumps at different decomposition degrees were studied in this study so as to provide some reference for better degradation of bamboo stumps in forest.

Material and Methods

Overview on test site

The test site was located in Wushan Forest Farm (112°42′E,27°51′ N) of Yuanjia Village, Wangcheng District, Changsha City, with altitude of 68-175 m and bamboo density of 2 595 plants/hm2. It has subtropical humid monsoon climate with wild climate, rich heat, annual average temperature of 17 ℃,annual average precipitation of 1 370 mm,sunshine hours of 1 610 h, frost-free period of 274 d,daily accumulated temperature (≥10℃)of 5 376.9 ℃,average temperature in January of 4.4 ℃and average temperature in July of 30 ℃.

Material

The average temperature in Wangcheng in October is 21 ℃. The different-decomposition degree bamboo stump samples were collected from the Wushan Farm of Yuanjia Village,Wangcheng District in October 11thand 12thbefore the bamboo harvesting. The bamboo stump samples were preserved at 4 ℃.During the determination of microorganism numbers, the bamboo stump samples were crushed, passed though 100-mesh sieve,diluted and cultured.

The used instruments and equipment included electronic balance(FA2004B, Shanghai Youke Instrument Co., Ltd.), herbal grinder(FW177, Tianjin City Taisite Instrument Co., Ltd.), drying oven on forced convection (101-E, Beijing Yongguangming Medical Equipment Factory), freezer (SC/SD-338, Nanling,Foshan Nanhai Bailing Refrigeration Equipment Factory), cleaning bench(SW-CJ-2FD, Suzhou Purification Equipment Co., Ltd.), stainless steel vertical autoclave (LDZM-40KCS,Hangzhou Huier Equipment Co., Ltd)and mold incubator (MJ-160-II,Shanghai Xinmiao Medical Devices Co.,Ltd.).

Methods

The cellulose Congo-red medium,with pH of 7.0, was composed by K2HPO40.5 g,MgSO40.25 g,cellulose powder 1.88 g,Congo Red 0.2 g,agar 16.0 g and gelatin 2.0 g. It was sterilized at 121 ℃for 20 min.The medium for lignin-decomposing bacteria, with pH of 7.0, was composed by K2HPO41.0 g, NaCl 10.5 g, MgSO4·7H2O 0.3 g, NaNO32.5 g, agar 18.0 g, FeCl30.01 g, CaCl20.1 g, soda lignin 0.3 g and distilled water to 1000 ml. It was sterilized at 121 ℃for 30 min.

The bacteria were cultured in beef extract peptone agar medium at 37 ℃for 1-2 d; the actinomycetes were cultured in Gause’s No.1 agar medium at 30 ℃for 5-7 d; the fungi were cultured in PDA medium at 30 ℃for 3-5 d; the cellulose-degrading bacteria were cultured in cellulose Congo-red medium at 30 ℃for 3-7 d; the lignindegrading bacteria were cultured in the specific medium at 30 ℃for 3-7 d.The numbers of bacteria, fungi, actinomycetes, cellulose-degrading bacteria and lignin-degrading bacteria were all counted by dilution method of plate counting.There were three duplicates for each plate, and the average data was used for data statistics and analysis.

Decomposition degrees of bamboo stumps

The decomposition situation of bamboo stumps in bamboo forest was surveyed and graded(Table 1).

Results and Analysis

Changes in bacterial quantity in bamboo stumps at different decomposition degrees

Cellulose, hemicellulose and lignin are the major components forthe cell wall of bamboo. The closer to the outer bamboo wall is, the smaller the vascular bundle is. Moreover, the vascular bundle is basically composed by fibers[6].The closer to the inner bamboo wall is,the larger the vascular bundle is.The structure of the inner vascular bundle is relatively loose,so it is a part of bamboo stump that is easier to be decomposed. Fig.1 showed that the bacterial number fluctuated to some extent. In the initiallydecomposed bamboo stump,the bacterial number, of which the logarithmic value was 5.903, was relatively small.With the accelerated decomposition of bamboo stump, the bacteria grew rapidly with nutrients in bamboo stump as substrates and its number was increased, and the logarithmic value of bacterial concentration reached 6.845.With the growth of propagation of fungi and actinomycetes,the bacterial number declined. Subsequently, the bacterial number was increased and then decreased to the lowest level with logarithmic value of 5.477.Since then,the environment was more suitable for the growth and propagation of bacteria,so its number was increased.In the bamboo stump with higher decomposition degree,the logarithmic value of bacterial concentration reached 7.380.

Changes in fungal quantity in bamboo stumps at different decomposition degrees

The thorough degradation of fiber tissues in plants depends on the longterm interactions among multiple kinds of microorganisms.Among them,fungi play a dominant role.The enzyme system that is responsible for degrading cellulose consists of several components[7]. Therefore, in the study on degradation of macromolecules, such as cellulose, the synergistic effects among enzyme-producing systems in microorganisms, especially the strong interaction between bacteria and fungi, must be taken into account[8]. As shown in Fig.2, the fungal number was overall increased slowly.In the bamboo stump at grade 9 decomposition, the fungal number was decreased greatly. With the increase of decomposition grade, the fungal number was then increased slowly. At the very initial stage, the logarithmic value of fungal concentration was 5.301.Due to rich nutrients in bamboo stump, the number of fungi was increased gradually. After a slight decline, it was then increased gradually with logarithmic value reaching 6.903.In the bamboo stump at grade 6 decomposition, the fungal number was decreased slightly. Subsequently, it was increased gradually. But at grade 9 decomposition, the fungal number was decreased greatly, and the logarithmic value of fungal concentration was decreased to 5.477. At this decomposition degree, the bacteria were dominant in bamboo stump. It suggested that there was certain competition between bacteria and fungi during the decomposition of bamboo stump,and the fungal number was still maintained at a relatively high level in greatly decomposed bamboo stump.

Changes in actinomycetes quantity in bamboo stumps at different decompositions degrees

The decomposition of bamboo stump is also the process of interactions among bacteria, fungi and actinomycetes.Li et al.[9]isolated 16 stains with better cellulose-degrading or lignin-degrading ability, including 8 fungal strains,5 bacterial strains and 3 actinomycetes strains. Gong et al.[10]isolated 145 cellulose-degrading strains from composted manure, cow dung and soil samples, and after preliminary screening and re-screening, a total of 8 stains with higher cellulosedecomposing ability were screened,including 3 bacterial strains, 3 mould strains and 2 actinomycetes strains.Fang et al.[11]isolated 7 cellulose-decomposing strains from soil, including 3 fungal strains and 4 bacterial strains. It was indicated that the isolated cellulose-degrading bacteria and fungi were more than isolated actinomycetes.

As shown in Fig.3,the logarithmic value of fungal actinomycetes concentration was increased slowly from 5.740 at the very initial stage. At the grade 7 decomposition, the fungal number was decreased sharply. At grade 8 and grade 9 decompositions,the fungal number was also very small, indicating the actinomycetes were propagated slowly and their cellulose-degrading and lignin-degrading abilities were weaker than that of fungi.However, under unfavorable conditions, spores could be formed in actinomycetes, so actinomycetes were more resistant to high temperature and highly acidic environment. The logarithmic value of actinomycetes concentration was then increased slowlyand reached the highest level (7.000)at the grade 11 decomposition. At the initial stage,actinomycetes utilized the abundant nutrients in bamboo stumps to grow and reproduce, so their number was increased; at the degrade 7 decomposition,the numbers of actinomycetes and bacteria all declined to the lowest levels, but the fungal number was remained at a relatively high level; at the grade 8 decomposition,the bacterial number was gradually increased,the fungal number was slightly increased, but the actinomycetes number was still very low;at the grade 9 decomposition, the bacterial number was increased continuously,the fungal number declined to the lowest level,and the actinomycetes number also declined to the lowest level with logarithmic value of 5.000, which was lower than those of all the microorganisms at the very initial stage; subsequently,the numbers of bacteria, fungi and actinomycetes were all increased, and the actinomycetes were prone to die.

During the decomposition of bamboo stumps,there is certain competition among bacteria, fungi and actinomycetes.At the initial stage,nutrients are abundant, so all the microorganisms grow rapidly; with the proceeding of the decomposition, nutrients are reduced gradually,competition for substrates appears among microorganisms,resulting in affected and even inhibited growth of microorganism; at the later stage, the substances near bamboo wall are started to be decomposed, and some substrates will be provided, ensuring the growth of all the microorganism.

Changes in cellulose-degrading bacterial quantity in bamboo stumps at different decompositions degrees

Mao bamboo stumps are rich in cellulose. The closer to the bamboo wall is,the higher the cellulose content is. But the cellulose near the bamboo wall is most difficult to be degraded.In general, although the structures of bamboo inner skin and bamboo shoot are completely destroyed, the structure near the bamboo wall is still very hard. As shown in Fig.4, the logarithmic value of cellulose-degrading bacteria was relatively low (4.301) at the very initial stage; subsequently, it was increased to the highest level (6.477);it was then decreased sharply, and at the grade 6 decomposition, it declined to the lowest level (4.176); the logarithmic value of cellulose-decomposing bacteria was increased again and then maintained at a relatively stable level;at the later stage, it was increased slightly, but decreased finally. At final,cellulose-degrading bacteria also trended to die.

At the initial stage, cellulose-degrading bacteria utilized substrates in bamboo stumps to grow and reproduce,and degraded difficultly-degraded cellulose into polysaccharides,cellobiose,monosaccharides and other small molecules, which could be better utilized by other microorganisms. However, when the other microorganisms grew rapidly, the growth of cellulose-degrading bacteria was affected or inhibited, so its number was decreased rapidly. When the decomposed substances were almost consumed off, the growth of the other microorganisms was affected, but the number of cellulose-degrading bacteria was started to be increased. In the later period, the cellulose near bamboo walls was decomposed, so the number of cellulose-degrading bacteria was maintained at a relatively stable level. But when the cellulose near bamboo walls was completely degraded,the cellulose-degrading bacteria slowly trended to decline and fall.

Changes in lignin-degrading bacterial quantity in bamboo stumps at different decompositions degree

Lignin exists in wood tissues, andits content is only second to that of cellulose.Lignin mainly locates among cellulose fibers. It hardens cell wall through forming into interwoven network,playing the role of compression.

Fig.5 showed that,at the very initial stage, the logarithmic value of lignin-decomposing bacterial concentration was lowest (4.415),and it was increased to the highest level (6.799)at grade 7 decomposition. Compared with those of the other microorganisms, the number of lignin-degrading bacteria changed more fluctuantly,and it showed four times of rise and fall.It suggested that environment and the other microorganisms had great impact on lignin-degrading bacteria.At the very initial stage, substrates were abundant,and lignin-degrading bacteria grew rapidly. When the number of the other microorganisms was large,the number of lignin-degrading bacteria was small. When the decomposed substances were almost consumed off, the growth of the other microorganisms was affected, but the number of lignin-degrading bacteria was increased then, which was consistent with the change rules of cellulose-degrading bacteria. In the later period,the number of cellulose-degrading bacteria was maintained stable, but the number of lignin-degrading bacteria was reduced sharply and the low level was maintained for a long time,indicating weaker competition of lignindegrading bacteria. At the later stage,the tissues near bamboo wall were also need to be degraded, so the number of lignin-degrading bacteria was increased. At final, the lignin-degrading bacteria also showed a tendency to decline.

Conclusions and Discussion

The degradation of plant tissue fibers is the process of growth, reproduction and decline and fall of fungi,bacteria and actinomycetes, and it is also the result of interactions among a variety of enzymes. In Mao bamboo stumps at different decompositions degrees,the logarithmic values of bacterial concentrations ranged from 5.477 to 7.380;the logarithmic values of fungal concentrations ranged from 5.301 to 6.903;the logarithmic values of actinomycetes concentrations ranged from 5.740 to 7.000; the logarithmic values of cellulose-degrading bacterial concentrations ranged from 4.301 to 6.447; the logarithmic values of lignindegrading bacterial concentrations ranged from 4.415 to 6.799.During the decomposition of bamboo stumps,there is certain competition among bacteria, fungi and actinomycetes. At the very initial stage, nutrients are relatively abundant, all the microorganisms grow rapidly; with the proceeding of the decomposition, the substrates are reduced gradually, competition for substrates appears among the microorganisms,resulting in affected and even inhibited growth of microorganisms; at the later stage, the substances near bamboo wall are degraded by cellulose-degrading and lignin-degrading bacteria, and some substrates are generated, promoting the growth of all the microorganisms;finally, substrates in bamboo stumps are basically consumed off, the numbers of all the microorganism are reduced gradually.

[1]DING XX (丁新新), HONG W (洪偉),CHEN HZ(陳建忠),et al.Comparison of soil water physical properties in different Phyllostachys pubescens management model(不同經(jīng)營模式下毛竹林土壤水分物理性質比較)[J].Research of Soil and Water Conservation (水土保持研究),2009,16(3):74-78.

[2]AI WS (艾文勝),YANG M (楊明),LI DJ( 李典軍), et al. Studies on material properties of Phyllostachys heterocycla cv pubescens(毛竹材性研究)[J].Hunan Forestry Science Technology(湖南林業(yè)科技),2009,36(4):1-5.

[3]ZHENG RY (鄭瑞鈺). Effects of Phyllostachys pubescens forest bamboo pockets fertilization on bamboo pockets rot and the bamboo shoot emergence(毛竹林竹蔸施肥對竹蔸腐爛及出筍的影響)[J].Journal of Fujian Forestry Science and Technology (福建林業(yè)科技),2011,38(1):72-74.

[4]LIN LG (林連光).A preliminary study of distribution and rotting speed of bamboo stump in Phyllostachys heterocycla var.pubescens forest (毛竹林內(nèi)竹蔸分布及其促腐情況的調查分析)[J]. World Bamboo and Rattan (世界竹藤通訊),2011,9(3):57-59.

[5]WENG FJ(翁甫金),WANG KH(汪奎宏),HE QJ (何奇江),et al.Research on experiment of fast rotten technology on bamboo stake (毛竹伐樁快速腐爛技術試驗研究)[J]. Journal of Bamboo Research(竹子研究匯刊),2001,20(4):47-51.

[6]ZHENG YS (鄭郁善),HONG W (洪偉).Business Administration of Phyllostachys heterocycla cv pubescens(毛竹經(jīng)營學)[M].Xiamen:Xiamen University Press (廈門: 廈門大學出版社),1999.

[7]NI QL (倪啟亮). Study on screening of lignocellulose-degrading bacteria and degradation of straw(木質纖維素降解菌的篩選及秸稈降解的研究)[D]. Guilin:Guilin Institute of Technology (桂林: 桂林工學院),2008.

[8]ZHAO XR (趙小蓉),LIN QM (林啟美),SUN YX (孫炎鑫),et al.Decomposition of different cellulose materials by some cellulose-degrading microbes(纖維素分解菌對不同纖維素類物質的分解作用)[J].Journal of Microbiology(微生物學雜志),2000,20(3):12-14.

[9]LI C(李超), LI LB(李潞濱), YANG K(楊凱), et al. Isolation and screening of microorganisms for decomposing bamboo stump (竹伐樁促腐微生物的分離篩選)[J]. Forest Research (林業(yè)科學研究),2008,21(2):253-257.

[10]GONG YS (宮玉勝), LI YC (李玉成),WU WB (吳旺寶), et al. Research on screening and mixed fermentation of thermophilic cellulose-decomposing microorganisms(嗜熱纖維素分解菌的篩選及混合發(fā)酵研究)[J]. Jiangsu Agricultural Sciences (江蘇農(nóng)業(yè)科學),2010,3:394-396.

[11]FANG XT (房興堂), CHEN H (陳宏),ZHAO XF (趙雪鋒),et al.Determination of enzyme activity of straw cellulose-decomposing microorganisms(秸稈纖維素分解菌的酶活力測定)[J].Letters in Biotechnology (生物技術通訊),2007,18(4):628-630.