Ecotoxicology (2014) 23:1833–1841 DOI 10.1007/s10646-014-1314-7
Effects of enhanced UV-B radiation on the diversity and activity of soil microorganism of alpine meadow ecosystem in Qinghai– Tibet Plateau Fujun Niu • Junxia He • Gaosen Zhang • Xiaomei Liu • Wei Liu • Maoxing Dong • Fasi Wu • Yongjun Liu • Xiaojun Ma • Lizhe An Huyuan Feng
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Accepted: 9 August 2014 / Published online: 23 August 2014 Ó Springer Science+Business Media New York 2014
Abstract The effects of enhanced UV-B radiation on abundance, community composition and the total microbial activity of soil bacteria in alpine meadow ecosystem of Qinghai–Tibet Plateau were investigated. Traditional counting and 16S rRNA gene sequencing were used to investigate the culturable bacteria and their composition in soil, meanwhile the total microbial activity was measured by microcalorimetry. The population of soil culturable bacteria was slightly reduced with the enhanced UV-B radiation in both of the two depths, 2.46 9 106 CFU/g in upper layer (0–10 cm), 1.44 9 106 CFU/g in under layer (10–20 cm), comparing with the control (2.94 9 106 CFU/g in upper layer, 1.65 9 106 CFU/g in under layer), although the difference was not statistically significant (P [ 0.05). However, the bacteria diversity decreased obviously due to enhanced UV-B, the number of species for upper layer was decreased from 20 to 13, and from 16 to 13 for the lower layer. The distribution of species was also quite different between the two layers. Another obvious decrease induced by enhanced UV-B radiation was in the total soil microbial activities, which was represented by the microbial growth rate constant (k) in this study. The results indicated that the culturable bacteria community composition and the total
Fujun Niu and Junxia He contributed equally to this work. F. Niu G. Zhang State Key Laboratory of Frozen Soil Engineering, Chinese Academy of Sciences, Lanzhou 730000, China J. He X. Liu W. Liu M. Dong F. Wu Y. Liu X. Ma L. An H. Feng (&) MOE Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou 730000, China e-mail: [email protected]
activity of soil microbes have been considerably changed by the enhanced UV-B radiation. Keywords Qinghai–Tibet Plateau UV-B radiation Soil microbes Ozone depletion Microcalorimetry
Introduction In the mid 1970s, it was discovered that anthropogenic chemicals (Aucamp 2007), such as increased concentrations of chlorofluorocarbons (CFCs) and other halon gases in the upper atmosphere (Russell et al. 1996), destroy stratospheric ozone molecules. The depletion of stratospheric ozone which protects life on earth by absorbing most of the harmful ultraviolet radiation from the sun has lead to an increase in biologically harmful UV-B radiation at the ground level (Rex et al. 2004). Although the amount of UV radiation reaching the Earth’s surface comprises only a small proportion of global radiation, about 6–7 % of UVA (320–400 nm) and less than 1.0 % of UVB (280–315 nm) (Hu et al. 2008), UV radiation plays an important role in human health and the environment (Aucamp 2007). Numerous studies reported the effects of UV-B on various living organisms and the majority focused on the response of plants to UV-B radiation (Robinson et al. 2005; Ruhland et al. 2005; Searles et al. 2001; Xiong and Day 2001; Yan et al. 2012), which were grown indoor, greenhouse or chamber particularly. Recently, an increasing number of researches on animals (Robson et al. 2005; Bao et al. 2013) and microorganisms (Avery et al. 2004; Piccini et al. 2009; Rinnan et al. 2005; Zaller et al. 2002) have been reported. It is recognized that well replicated field and long-term radiation studies are highly important to realistically evaluate the effects of
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altered UV radiation. However, little is known about the change of soil microbial communities under long-term enhanced UV-B radiation in the field. Soil microorganisms play an essential role in the environment, such as recycling of nutrients, plant growth promotion/repression and soil physical chemical properties (Critter et al. 2002; Degens et al. 2000). The indirect effects of UV-B on soil microorganisms are considered to be more important and intricate than the direct effects, and in most cases, these effects are mediated through plants, but can be manifested below the ground (Caldwell et al. 2007). However, since the sunlight can hardly penetrates the surface soil (Jeffery et al. 2009), most studies investigated the microbes above-ground and fungus below-ground primarily (Rinnan et al. 2005; Zaller et al. 2002), little attention has been paid to soil bacteria, especially in alpine meadow ecosystem of Qinghai–Tibet Plateau. The Qinghai–Tibet Plateau, with an average elevation of more than 4,000 m, is the highest upland in the world (Liu et al. 2009), called the ‘world’s roof’ or ‘third polar’ (Shi et al. 2004; Zhang et al. 2007). It represents a unique environment, being a result of high elevation, low latitude and strong radiation and is one of the most sensitive regions to the global environmental changes (Shi et al. 2004). Zhou et al. demonstrated that in the past decades, total ozone level over Qinghai–Tibet Plateau has decreased continuously (Zhou et al. 1995). The ecosystem of Qinghai–Tibet Plateau would be very seriously deteriorated if the lower level of ozone persists. Previous research suggested that climatic warming has occurred in Qinghai– Tibet Plateau since the late 1980s (Liu et al. 2009). Therefore, investigations into microorganisms from this unique region in response to the changing climate, including the enhanced UV-B, are urgently needed and significant. In an alpine meadow ecosystem of Qinghai– Tibet Plateau, various responses to enhanced UV-B for several typical alpine plants were investigated in the field (Cui et al. 2003; Shi et al. 2004). Under those conditions, we hypothesized that the below ground microbes might be changed after UV-B radiation. Our objectives of this study were to determine the effects of enhanced UV-B on abundance, bacterial community composition of culturable bacteria of alpine meadow ecosystem in the Qinghai–Tibet Plateau using the traditional counting, as well as the microbial activity through the calorimetric measurements.
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northeast of Qinghai–Tibet Plateau and south hillside of the Leng-Long-Ling which is at the east of the Qilian Mountains, 37°290 N–37°450 N, 101°120 E–101°330 E, with an altitude of approximately 3,200 m. The mean annual air temperature is -1.7 °C and the annual precipitation is 580 mm. The weather is cold but humid, belongs to Plateau continental climate. Kobresia humilis, which consists of perennial herb, is the dominated species growing in this land with alpine meadow soils. In 1998, a block with well-distributed vegetation was selected to establish the field experimentation site and subdivided into six plots arranged in a randomized complete block experimental design, consisting of two treatments, three for enhanced UV-B treatment (UV-B) and three for ambient UV-B (CK). The experimental system consisted of six 40 W fluorescent lamps (UV-B-313, Beijing Electric and Light Source Institute, PR China) which were mounted on a metal frame (2.5 m 9 1.5 m 9 0.75 m high) and suspended over the vegetation vertically. In contrast, the control system only installed metal frame without fluorescent lamps. The lamps can filter the lethality UV-C (\280 nm) with cellulose diacetate film (0.13 mm; Courtauld Specialty Plastics, Derby, UK) which allowed transmission of UV-B (280–315 nm) and UV-A (315–400 nm) radiation. Cellulose diacetate filters were replaced weekly to maintain a stable light quality. UV-B supplementation was performed from 10:00 to 16:00 daylight. The enhanced UV-B density was 15.80 kJ/m2 (Shi et al. 2004). The radiation reached the ground was a combination of the UV-B from the fluorescent lamps and the ambient UV-B radiation, simulating 14 % ozone depletion of stratosphere (Shi et al. 2004). Sampling In August 2007, all three replicate blocks of each treatment (CK and UV-B) were sampled by taking five soil cores for the upper layer and lower layer, above 10 cm (0–10 cm) and below 10 cm (10–20 cm), respectively, with the surface layer being removed to a depth of 5 mm. Then the five soil core samples of each block were mixed as one sample, and stored in a sealed plastic bag for transporting back to laboratory. Samples were stored at -20 °C prior to analyses. Determination of soil chemical properties
Materials and methods Site description and experimental design The experiment was conducted at the Haibei Alpine Meadow Ecosystem Research Station. This station is in the
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Air-dried soils were passed through a sieve in 0.074 mm diameter, then soil organic carbon and total N were determined with CHNS-analyser(Elementar Vario EL, Elementar Analysensysteme GmbH, Hanau, Germany)by combustion method at 450 and 1,250 °C, respectively (Analysis and Test Center of Lanzhou University). Total
UV-B impact soil microbe in Qinghai–Tibet Plateau field
P and available P were measured by HClO4–H2SO4 Mo–Sb colorimetric method and Olsen method, respectively. Cultivation, counting and isolation The total numbers of culturable bacteria were determined as colony forming units (CFU) on agar plates by dilution plate methods. Briefly, 3 g soil of each sample were homogenized in 27 ml of sterilized water and serially diluted. Aliquots (0.2 ml) of the diluted suspension were spread on the beef extract peptone agar plates for the enumeration of bacteria. The CFU on plates were counted after incubation for 1–3 weeksunder 25 °C. Representative colonies were purified according to their morphological appearance and color. DNA extraction, PCR and RFLP for individual bacterial colony Total genomic DNA of the isolates was extracted using the chloroform–isoamyl alcohol extraction procedures (Zhou et al. 1996). 16S rRNA genes of these isolates were amplified using the universal primer set of 27F (50 -AGAGTTT GATCCTGGCTCAG-30 ) and 1492R (50 -TACGGCTACC TTGTT ACGACTT-30 ) (Lane 1991). The reaction mixture (25 ll) consisted of 1 Unit Taq polymerase, 0.2 mM dNTP, 2.5 ll of 10 9 PCR Buffer, 2.5 mM of MgCl2, 0.2 lM of each primer, 2.5 ll (ca. 10 ng DNA) of template. The PCR amplification program was as follows: initial denaturation at 94 °C for 3 min, 30 cycles of 94 °C for 1 min, annealing at 58 °C for 1 min, and extension at 72 °C for 1:30 min, and a final extension of 10 min at 72 °C. PCR products were detected by electrophoresis in 1 % agarose gel. The PCR products were screened for similarity using restriction fragment length polymorphism (RFLP) analysis. Amplified rRNA gene products were digested with the restriction endonucleases BsuRI(GG/CC) and Hin6I(G/ CGC). The digested fragments were visualized on a 2.5 % agarose gel. Isolates were grouped together on the basis of RFLP patterns, and one isolate was chosen from each group for cloning and sequencing after purified using a quick Midi purification kit (Tiangen Co., Beijing, China).
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The suspension of expected clones was used to sequence by Shanghai Majorbio Bio-technology Company. The 30 sequences obtained, ca. 1,500 bp,were then analyzed with the National Center for Biotechnology Information (NCBI) Blast program. The most similar GenBank sequences to the clones were extracted from the GenBank. A phylogenetic neighbor-joining tree including obtained isolates and their closest relatives was constructed using MEGA 4.0. Nucleotide sequence accession numbers The obtained sequences were submitted to the GenBank database and assigned accession numbers of GU385847– GU385876. Microcalorimetric measurements The microcalorimetric measurement was performed on a TAM2277 (Thermometric, AB, Sweden) which is a commercial version of the system developed by (Suurkuusk and Wadso¨ 1982). Measurements were carried out in sterilized ampoules with Teflon sealing disks to avoid evaporation of the sample solution. All soil samples were first placed at room temperature for 24 h, then submitted to microcalorimetric measurements. For each sample, 1.0 g soil was put into the prepared ampoule before adding 0.5 ml of a solution containing 5.0 mg of glucose and 5.0 mg of ammonium sulphate. The reference ampoule was filled with 0.5 ml of distilled water instead of the nutrient solution. All measurements were run at 25 °C. Statistical analysis The means and standard deviations for three replicates were calculated and ANOVA was used to test the significant differences of UV-B radiation with software SPSS 13.0.
Results Soil chemical properties
Cloning and sequencing Cloning was performed with the pGM-T Vector System (Tiangen Co., Beijing, China) following the protocol of the manufacturer and the ligation product was subsequently transformed into E. coli DH-5a, which allows blue–white screening, and plated on LB medium containing ampicillin (100 mg/ml), X-Gal (20 mg/ml) and IPTG (200 mg/ml). Positive clones were identified by PCR amplification with pGM-T vector primer pairs T7/Sp6 using the same program as 16S rDNA amplification.
A summary of the main chemical properties of all soil samples is presented in Table 1. Changes of total N, total C and organic C were not significant different (P [ 0.05). However, total P and Olsen-P decreased significantly after treating with enhanced UV-B (P \ 0.05). The abundance of the culturable bacterial population The mean values of CFU of culturable bacteria for all soil samples are given in Fig. 1. In general, the number of
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Table 1 The effect of the enhanced UV-B on main chemical properties of soil samples of Qinghai–Tibet Plateau alpine meadow ecosystem Treatment
TN(mg/g)
TC(mg/g)
SOC(mg/g)
TP(mg/g)
AP(mg/kg)
CK
3.79 ± 0.11a
46.89 ± 0.94a
24.85 ± 0.99a
0.37 ± 0.00a
9.859 ± 0.112a
UV-B
3.89 ± 0.12a
49.00 ± 0.98a
25.97 ± 1.04a
0.32 ± 0.01b
7.977 ± 0.195b
TN total nitrogen, TC total carbon, SOC soil organic carbon, TP total phosphorus, AP available phosphorus. We obtained the data by amalgamating the samples of two depths of three replicates for each treatment. So the value is mean ±SD (n = 6). Values within the same column not followed by the same letter differ significantly (P \ 0.05)
was very different due to their multiplex distribution in different depths with different treatments. The total OTU number for CK and UV-B of the upper layer was 20 and 13, and 16 and 13 for CK and UV-B foe the lower layer, respectively (Fig. 2). Obviously, there was a decrease due to enhanced UV-B, compared with CK,and samples of the upper layer showed a greater change than that of the lower layer. However, some OUT (e.g., QT4, QT6) appeared while others (e.g., QT14, QT19, QT20) disappeared, and some OTUs (e.g., QT7, QT8, QT22, QT25 and QT27) were only detected in the lower layer with treatment of enhanced UV-B radiation. Fig. 1 The effect of enhanced UV-B on culturable bacterial abundance in soil of Qinghai–Tibet Plateau alpine meadow ecosystem. The values are the average of three replicates for each sample. Different letters over the bars show significant differences
bacteria obviously decreased with the increase of soil depth. As a result of enhanced UV-B, the abundance was slightly reduced, from 2.94 9 106 to 2.46 9 106 CFU/g for the upper layer and from 1.65 9 106 to 1.44 9 106 CFU/g for the lower layer, though the differences were not significant statistically between different treatments for the same soil layer (P [ 0.05). RFLP and phylogenetic analyses Through restriction fragment length polymorphism (RFLP) analysis, all 154 isolates distinguished by the morphological characteristics were clustered into 41 groups. After comparing 16S rDNA sequence of the representative strains of each group, they formed 30 groups. The recovered populations fell into four categories: Firmicutes, Actinobacteria, Proteobacteria and Bacteroidetes (Fig. 2). All samples were dominated by Firmicutes and Proteobacteria (Fig. 3), especially the Bacillus of Firmicutes and the Pseudomonas of Proteobacteria. Bacteroidetes was subordinate. Actinobacteria was the least, and reduced nearly by half (from 15 to 7.69 % for the upper layer and from 12.5 to 7.69 % for the lower layer) due to enhanced UV-B radiation (Fig. 3). The diversity in each soil sample
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Microbial activity measured by microcalorimetric method The results obtained from calorimetric determination for all soil samples are shown in Fig. 4 and Table 2. The recorded power–time curves (Fig. 4) showed evolution changes of microbial activity. The mean values of the maximum peakheat output power, Pmax (lw), the time to reach the maximum of the peak, tmax (min), and the microbial growth rate constant, k (min-1), were obtained through integration of each curve. The total heat evolution, QT (J/g), was also calculated for these samples (Table 2). The two curves of the lower layer samples displayed a longer lag phase than that of the upper layer which showed a rapid microbial growth and the curves of UV-B were lower than that of CK for the two layers (Fig. 4). The Pmax of the former were lower than the latter. Between enhanced UV-B and CK of either upper or lower layer, there were no significant differences (P [ 0.05) (Table 2). The values of k, representing microbial growth rate constant during the log phase or the exponential phase of microbial activity curves, reduced with the increase of soil depth as well as with the decrease of numbers of bacteria (Fig. 5a). Furthermore, the k of upper layer samples showed a significant difference between enhanced UV-B and CK (P \ 0.05) (Table 2). Compared to the bacterial population, the time to reach the peak, tmax, presented a reverse trend in each soil layer
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Fig. 2 Phylogenetic dendrogram based on a comparison of the 16S rDNA sequences of the 30 representative isolates from the soil samples of alpine meadow ecosystem of Qinghai–Tibet Plateau, with the treatment of enhanced UV-B, and some of their closest phylogenetic relatives. The tree created by the neighbor-joining
method. aC(a-CK) and aU(a-UV-B), the soil samples above 10 cm with the treatments of control and enhanced UV-B, respectively; bC(b-CK) and bU(b-UV-B), the soil samples below 10 cm with the two treatments, respectively. ‘‘?’’, stand for the presence of corresponding bacteria
(Fig. 5b), although there were also no significant differences between different treatments (P [ 0.05) (Table 2). The QT, the value of total heat released by soil microorganisms, negatively but implicitly correlated with the value of CFU (Fig. 5c).
Discussion The effects of enhanced UV-B on soil bacteria from alpine meadow ecosystem of Qinghai–Tibet Plateau were investigated. The abundance of soil culturable bacteria was
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Fig. 3 The effect of enhanced UV-B on the distribution of Firmicutes, Actinobacteria, Proteobacteria and Bacteroidetes in different soil samples of alpine meadow ecosystem of Qinghai–Tibet Plateau
Fig. 4 Power-time curves recorded microcalorimetrically from different soil samples of Qinghai–Tibet Plateau alpine meadow ecosystem with two treatments of enhanced UV-B and CK, amended with glucose and ammonium sulphate. In these curves Pmax (lw) is plotted against tmax (min)
slightly reduced due to enhanced UV-B radiation, although there were no significant statistically differences between different treatments in the same soil layer (Fig. 1). As we known, fungi and bacteria are generally more sensitive to damage by UV-B radiation in direct sunlight than are plants (Braga et al. 2001; Mu¨hlenberg and Stadler 2005; Yan et al. 2012), however, sunlight scarcely penetrates the soil surface (Aucamp 2007; Avery et al. 2004), less than 0.3 % of light is transmitted through to 2 mm soil depth (Jeffery et al. 2009). So the bacteria may receive the direct effects of UV-B radiation scarcely. Moreover, Avery et al. reported there were no significant effects of UV-B treatment on culturable counts of bacteria in undisturbed plot similar to ours. Thus we speculate that the slight changes may be mediated by the vegetation (Avery et al. 2004). Bacterial community composition, however, were altered due to the enhanced UV-B. In Greenland and Antarctica, altered solar UV-B radiation did result in qualitative changes in the soil microbial communities, this was thought to be due to altered quantity and/or quality of root exudates (Johnson et al. 2002; Rinnan et al. 2005). However, the root production was apparently unaffected by the UV-B radiation between 5 and 10 cm (Zaller et al. 2002). Hence, the number of soil bacteria species in the lower layer (below 10 cm) showed insignificant changes as well as the small changes of culturable bacterial abundance in this layer. Nevertheless, the distribution of these detected species displayed great differences in all of soil samples (Fig. 2). Some bacteria (e.g., QT4, QT6) presented in the enhanced UV-B treatment, not in CK. This may because species diversity was affected by solar UV-B radiation because of species-specific sensitivity to the radiation and another studies reported that UV-B radiation could alter the plant and its root exudates, so some of the soil bacteria were stimulated or inhibited (Kashimada et al. 1996; Piccini et al. 2009; Ulevicˇius et al. 2004). In addition, the Bacillus of Firmicutes and the Pseudomonas of Proteobacteria are dominant. As is well known,
Table 2 The effect of enhanced UV-B on microbial activity of samples in different soil depth of Qinghai–Tibet Plateau alpine meadow ecosystem Depth Above 10 cm Below 10 cm
Treatment
Pmax (lw)
tmax (min)
k (min-1)
QT (J/g)
CK
180.04 ± 18.04a
1,015.5 ± 87.60a
0.0054 ± 0.0007a
5.18 ± 0.39a
UV-B
169.31 ± 27.38ab
993.83 ± 83.93a
0.0040 ± 0.0008b
5.52 ± 0.36a
CK
136.19 ± 23.79b
1,184.83 ± 22.55ab
0.0039 ± 0.0004b
5.66 ± 0.84a
UV-B
147.26 ± 17.90ab
1,246.33 ± 207.13b
0.0030 ± 0.0003b
6.25 ± 1.46a
The data obtained from power–time curves with 1.0 g soil samples supplemented with 0.5 ml solution containing 5.0 mg of glucose and 5.0 mg of ammonium sulphate. Pmax (lw), the maximum peak-heat output power; tmax (min), the time to reach the maximum of the peak; k (min-1), the microbial growth rate constant; QT (J/g), the total heat evolution. The value is mean ±SD (n = 3). Values within the same column not followed by the same letter differ significantly (P \ 0.05)
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other groups also presented with special pigment, e.g., some of QT24 and QT28 groups were lemon yellow, creamy yellow or orange, even some species of Firmicutes produce pigments, e.g., some of QT3 group were bright mauve, and some of QT11 were pastel pink. Pigment plays an important role in resisting UV for bacteria (Sandmann et al. 1998; Sundin and Murillo 1999), UV-B radiation sensitivity was inversely correlated with pigmentation and well pigmented species were considered to be tolerant of UV-B radiation corresponding to solar UV-B radiation with substantially depleted ozone (Aucamp 2007). Thus, the result that Bacillus and Pseudomonas are better to grow in the conditions like Qinghai–Tibet Plateau is creditable. The fact that Bacteroidetes was subordinate in these detected species may also because of its ability of producing pigments, e.g., some OUT of QT1 and QT2 produce yellow or orange, and some of QT16 produce purple. Soil microbial activity
Fig. 5 Log of CFU against growth rate, k (a), peak time, tmax (b), and total heat, QT(c), respectively, for soil samples treated with enhanced UV-B. With the decrease of bacterial quantity (Log of CFU), the value of k (a) decreased, the value of tmax increased (b) and QT (c) showed an increasing tendency
Bacillus are easily to mass culture with its lower nutritional requirements and tenacious viability, and some species of Pseudomonas produce pigments, e.g., some species of QT17 group which belong to Pseudomonas in this work produce beige by morphology observing, some of QT5 and QT23 groups produce grayish blue. Besides Pseudomonas,
Microcalorimetric technique is sensitive enough to detect very low heat rates and can provide qualitative and quantitative indicators of soil microbial activity(Barros et al. 1997; Nu´n˜ez-Regueira et al. 2006b). This method had been applied in soil environments subjected to various factors such as land-use, heavy metal and pesticides (Barros et al. 2006; Nu´n˜ez-Regueira et al. 2006a; Prado and Airoldi 2002; Zheng et al. 2009). In this research, it is the first time that microcalorimetric technique was applied to soil microbial activity subjected to enhanced UV-B radiation and the results demonstrated its importance. It is clear from the foregoing results that k (min-1) showed a visible lower growth rate of microbe in UV-B treatment, while the number of culturable bacteria was also less than in control (Fig. 5a). This result indicated that the number of soil bacteria reduced slightly due to the enhanced UV-B radiation, and the growth of soil microbe become slowly, accordingly. Similar results also can be found in Zheng’s study(Zheng et al. 2009). In upper layer, Pmax (lw) of samples treated by elevated UV-B which had lower bacterial numbers displayed a lower value and tmax (min) showed a reverse trend on the whole (Fig. 5b). These results were consistent with previous studies which suggested that shorter lag phases, lower peak times (tmax), higher Pmax and greater growth rate constant (k) were all good indicators of the high microbial metabolic activity in a microcalorimetric analysis (Barros et al. 1997; Nu´n˜ez-Regueira et al. 2006b; Zheng et al. 2009; Zheng et al. 2007). This indicated that enhanced UVB radiation would change the quantity and quality of soil microbes to a certain extent and prefer to inhibition. In addition, Zheng et al. indicated that the deficiency of available P can limit microbial activity (Zheng et al. 2009).
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In this work, available P reduced due to enhanced UV-B (Table 1) and microbial activity for samples treating with enhanced UV-B was relatively lower than control (Fig. 4). It showed that enhanced UV-B can change the soil chemical properties and then change microbial activity. The QT (J/g) negatively correlated with culturable bacterial abundance in this work (Fig. 5c). The highest bacterial numbers corresponded to the lowest QT. In fact, besides UV-B manipulation, soil microbial metabolism is mainly associated with many factors such as soil pH, nutrient, temperature, community structure and so on. So QT is probably associated with microbial diversity in soils since enhanced UV-B had little effects on microbial abundance but altered microbial community composition obviously (Rinnan et al. 2005). Additionally, in contrast to other microorganisms, bacteria are numerically dominant, but the other microorganisms in soils, including actinomycetes, fungi, protozoa and algae, developed multiple cooperative and competitive interactions as well as their relations with heat evolution of microbial activity (Zheng et al. 2007). Still less the majority of bacteria are uncultivable organisms. What’s more, compared to the calorimetric method which measured the total interaction of all microorganism growth in the system, plate count operation which reflected a specific growth of bacteria did not distinguish between active and dormant bacteria. If an inactive bacterial was spread on a culture medium, it will germinate and produce a colony that will be counted, but the inactive unit in the calorimetric system is not measured by this method (Critter et al. 2002). Therefore, the particularly change of QT, whose change was opposite from the CFU of culturable bacteria and the growth rate constant (k) of microbes in soil, may be due to these facts. On the other hand, our result that samples with the greatest value of QT showed the smallest value of k (Table 2), was consistent with previous research (Barros et al. 1997), which suggested that samples with the greatest percentages of organic matter showed the greatest values of QT and the smallest values of k. This may be due to a bigger availability of substrate sensible to microbial attack and organic matter suffers a slower decomposition, or may be due to the different vegetation distribution of sampling sites (Zheng et al. 2007). Therefore, there was a possible correlation between QT and organic matter or vegetation quantity. In conclusion, the results illustrated that the enhancement in 14 % of UV-B irradiation led to the change of soil microbial communities in alpine meadow ecosystem of Qinghai–Tibet Plateau subjected to nearly consecutive 10 years. Despite detrimental effects of enhanced UV-B on some species and activating effects on some others, an evidently inhibition of the diversity and activity was observed, especially in the upper soil layer. Since sunlight
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scarcely penetrated the soil surface, changes in microbial metabolic potential were likely to be plant-mediated. Thus, the changes that soil microbe manifested possibly due to the alterations in the quality and/or quantity of root exudation induced by UV-B. Although responses of soil culturable bacteria abundance to ambient even enhanced UVB were relatively slight in present, the bacteria community composition and the total activity of soil microbe have been changed considerably and there is no doubt that a possible future increase of UV-B levels will be occur more seriously due to the aggravating circumstances. Therefore, we cannot rule out the possibility that these increases would change the below-ground function of alpine meadow ecosystem. Acknowledgments This research was supported by National Basic Research Program (2012CB026105), National Natural Science Foundation (31170482, 31370450), PhD Programs Foundation of Ministry of Education (2010021111002, 20110211110021), The Fundamental Research Funds for the Central Universities (LZUJBKY-2013-92) in China, and State Key Laboratory of Frozen Soil Engineering, Chinese Academy of Sciences (SKLFSE200901). We are grateful to Dr Yantian Ma for his help. Conflict of interest of interest.
The authors declare that they have no conflict
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UV-B impact soil microbe in Qinghai–Tibet Plateau field Degens BP, Schipper LA, Sparling GP, Vojvodic-Vukovic M (2000) Decreases in organic C reserves in soils can reduce the catabolic diversity of soil microbial communities. Soil Biol Biochem 32(2):189–196 Hu B, Wang Y, Liu G (2008) Influences of the clearness index on UV solar radiation for two locations in the Tibetan Plateau-Lhasa and Haibei. Adv Atmos Sci 25(5):885–896 Jeffery S, Harris JA, Rickson RJ, Ritz K (2009) The spectral quality of light influences the temporal development of the microbial phenotype at the arable soil surface. Soil Biol Biochem 41(3):553–560 Johnson D, Campbell CD, Lee JA, Callaghan TV, Gwynn-Jones D (2002) Arctic microorganisms respond more to elevated UV-B radiation than CO2. Nature 416(6876):82–83 Kashimada K, Kamiko N, Yamamoto K, Ohgaki S (1996) Assessment of photoreactivation following ultraviolet light disinfection. Water Sci Technol 33(10):261–269 Lane DJ (1991) 16S/23S rRNA sequencing. In: Stackebrandt E, Goodfellow M (eds) Nucleic acid techniques in bacterial systematics. Wiley, New York, pp 115–148 Liu J, Wang S, Yu S, Yang D, Zhang L (2009) Climate warming and growth of high-elevation inland lakes on the Tibetan Plateau. Global Planet Change 67(3):209–217 Mu¨hlenberg E, Stadler B (2005) Effects of altitude on aphid-mediated processes in the canopy of Norway spruce. Agric For Entomol 7(2):133–143 Nu´n˜ez-Regueira L, Proupı´n-Castin˜eiras J, Rodrı´guez-An˜o´n J, Villanueva-Lo´pez M, Nu´n˜ez-Ferna´ndez O (2006a) Design of an experimental procedure to assess soil health state. J Therm Anal Calorim 85(2):271–277 Nu´n˜ez-Regueira L, Rodrı´guez-An˜o´n JA, Proupı´n-Castin˜eiras J, Nu´n˜ez-Ferna´ndez O (2006b) Microcalorimetric study of changes in the microbial activity in a humic Cambisol after reforestation with eucalyptus in Galicia (NW Spain). Soil Biol Biochem 38(1):115–124 Piccini C, Conde D, Pernthaler J, Sommaruga R (2009) Alteration of chromophoric dissolved organic matter by solar UV radiation causes rapid changes in bacterial community composition. Photochem Photobiol Sci 8(9):1321–1328 Prado AG, Airoldi C (2002) The toxic effect on soil microbial activity caused by the free or immobilized pesticide diuron. Thermochim Acta 394(1):155–162 Rex M, Salawitch R, von der Gathen P, Harris N, Chipperfield M, Naujokat B (2004) Arctic ozone loss and climate change. Geophys Res Lett 31(4):L04116. doi:10.1029/2003GL018844 Rinnan R, Keina¨nen M, Kasurinen A, Asikainen J, Kekki T, Holopainen T, Ro-Poulsen H, Mikkelsen TN, Michelsen A (2005) Ambient ultraviolet radiation in the Arctic reduces root biomass and alters microbial community composition but has no effects on microbial biomass. Glob Change Biol 11(4):564–574 Robinson SA, Turnbull JD, Lovelock CE (2005) Impact of changes in natural ultraviolet radiation on pigment composition, physiological and morphological characteristics of the Antarctic moss. Grimmia antarctici. Global Change Biology 11(3):476–489 Robson TM, Pancotto VA, Scopel AL, Flint SD, Caldwell MM (2005) Solar UV-B influences microfaunal community
1841 composition in a Tierra del Fuego peatland. Soil Biol Biochem 37(12):2205–2215 Ruhland CT, Xiong FS, Clark WD, Day TA (2005) The influence of ultraviolet-B radiation on growth, hydroxycinnamic acids and flavonoids of deschampsia antarctica during springtime ozone depletion in Antarctica. Photochem Photobiol 81(5):1086–1093 Russell JM, Luo MZ, Cicerone RJ, Deaver LE (1996) Satellite confirmation of the dominance of chlorofluorocarbons in the global stratospheric chlorine budget. Nature 379:526–529 Sandmann G, Kuhn S, Bo¨ger P (1998) Evaluation of Structurally Different Carotenoids inEscherichia coli Transformants as Protectants against UV-B Radiation. Appl Environ Microbiol 64(5):1972–1974 Searles PS, Flint SD, Caldwell MM (2001) A meta-analysis of plant field studies simulating stratospheric ozone depletion. Oecologia 127(1):1–10 Shi S-B, Zhu W-Y, Li H-M, Zhou D-W, Han F, Zhao X-Q, Tang Y-H (2004) Photosynthesis of Saussurea superba and Gentiana straminea is not reduced after long-term enhancement of UVB radiation. Environ Exp Bot 51(1):75–83 Sundin GW, Murillo J (1999) Functional analysis of the Pseudomonas syringae rulAB determinant in tolerance to ultraviolet B (290–320 nm) radiation and distribution of rulAB among P. syringae pathovars. Environ Microbiol 1(1):75–87 Suurkuusk J, Wadso¨ I (1982) A multichannel microcalorimetry system. Chem Scr 20:155–163 Ulevicˇius V, Pecˇiulyt_e D, Lugauskas A, Andriejauskien_e J (2004) Field study on changes in viability of airborne fungal propagules exposed to UV radiation. Environ Toxicol 19(4):437–441 Xiong FS, Day TA (2001) Effect of solar ultraviolet-B radiation during springtime ozone depletion on photosynthesis and biomass production of Antarctic vascular plants. Plant Physiol 125(2):738–751 Yan A, Pan J, An L, Gan Y, Feng H (2012) The responses of trichome mutants to enhanced ultraviolet-B radiation in Arabidopsis thaliana. J Photochem Photobiol, B 113:29–35 Zaller JG, Caldwell MM, Flint S D, Scopel AL, Salo OE, Ballare´ CL (2002) Solar UV-B radiation affects below-ground parameters in a fen ecosystem in Tierra del Fuego, Argentina: implications of stratospheric ozone depletion. Glob Change Biol 8(9):867–871 Zhang G, Ma X, Niu F, Dong M, Feng H, An L, Cheng G (2007) Diversity and distribution of alkaliphilic psychrotolerant bacteria in the Qinghai–Tibet Plateau permafrost region. Extremophiles 11(3):415–424 Zheng S, Yao J, Zhao B, Yu Z (2007) Influence of agricultural practices on soil microbial activity measured by microcalorimetry. Eur J soil biol 43(3):151–157 Zheng S, Hu J, Chen K, Yao J, Yu Z, Lin X (2009) Soil microbial activity measured by microcalorimetry in response to long-term fertilization regimes and available phosphorous on heat evolution. Soil Biol Biochem 41(10):2094–2099 Zhou J, Bruns M, Tiedje J (1996) DNA recovery from soils of diverse composition. Appl Environ Microbiol 62:316–322 Zhou X, Luo C, Li W, Shi J (1995) Changes of total ozone in whole China and its low contents center in Qing-Zang Plateau regions. Chin Sci Bull 40:1396–1398
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Effects of enhanced UV-B radiation on the diversity and activity of soil microorganism of alpine meadow ecosystem in Qinghai– Tibet Plateau Fujun Niu • Junxia He • Gaosen Zhang • Xiaomei Liu • Wei Liu • Maoxing Dong • Fasi Wu • Yongjun Liu • Xiaojun Ma • Lizhe An Huyuan Feng
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Accepted: 9 August 2014 / Published online: 23 August 2014 Ó Springer Science+Business Media New York 2014
Abstract The effects of enhanced UV-B radiation on abundance, community composition and the total microbial activity of soil bacteria in alpine meadow ecosystem of Qinghai–Tibet Plateau were investigated. Traditional counting and 16S rRNA gene sequencing were used to investigate the culturable bacteria and their composition in soil, meanwhile the total microbial activity was measured by microcalorimetry. The population of soil culturable bacteria was slightly reduced with the enhanced UV-B radiation in both of the two depths, 2.46 9 106 CFU/g in upper layer (0–10 cm), 1.44 9 106 CFU/g in under layer (10–20 cm), comparing with the control (2.94 9 106 CFU/g in upper layer, 1.65 9 106 CFU/g in under layer), although the difference was not statistically significant (P [ 0.05). However, the bacteria diversity decreased obviously due to enhanced UV-B, the number of species for upper layer was decreased from 20 to 13, and from 16 to 13 for the lower layer. The distribution of species was also quite different between the two layers. Another obvious decrease induced by enhanced UV-B radiation was in the total soil microbial activities, which was represented by the microbial growth rate constant (k) in this study. The results indicated that the culturable bacteria community composition and the total
Fujun Niu and Junxia He contributed equally to this work. F. Niu G. Zhang State Key Laboratory of Frozen Soil Engineering, Chinese Academy of Sciences, Lanzhou 730000, China J. He X. Liu W. Liu M. Dong F. Wu Y. Liu X. Ma L. An H. Feng (&) MOE Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou 730000, China e-mail: [email protected]
activity of soil microbes have been considerably changed by the enhanced UV-B radiation. Keywords Qinghai–Tibet Plateau UV-B radiation Soil microbes Ozone depletion Microcalorimetry
Introduction In the mid 1970s, it was discovered that anthropogenic chemicals (Aucamp 2007), such as increased concentrations of chlorofluorocarbons (CFCs) and other halon gases in the upper atmosphere (Russell et al. 1996), destroy stratospheric ozone molecules. The depletion of stratospheric ozone which protects life on earth by absorbing most of the harmful ultraviolet radiation from the sun has lead to an increase in biologically harmful UV-B radiation at the ground level (Rex et al. 2004). Although the amount of UV radiation reaching the Earth’s surface comprises only a small proportion of global radiation, about 6–7 % of UVA (320–400 nm) and less than 1.0 % of UVB (280–315 nm) (Hu et al. 2008), UV radiation plays an important role in human health and the environment (Aucamp 2007). Numerous studies reported the effects of UV-B on various living organisms and the majority focused on the response of plants to UV-B radiation (Robinson et al. 2005; Ruhland et al. 2005; Searles et al. 2001; Xiong and Day 2001; Yan et al. 2012), which were grown indoor, greenhouse or chamber particularly. Recently, an increasing number of researches on animals (Robson et al. 2005; Bao et al. 2013) and microorganisms (Avery et al. 2004; Piccini et al. 2009; Rinnan et al. 2005; Zaller et al. 2002) have been reported. It is recognized that well replicated field and long-term radiation studies are highly important to realistically evaluate the effects of
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altered UV radiation. However, little is known about the change of soil microbial communities under long-term enhanced UV-B radiation in the field. Soil microorganisms play an essential role in the environment, such as recycling of nutrients, plant growth promotion/repression and soil physical chemical properties (Critter et al. 2002; Degens et al. 2000). The indirect effects of UV-B on soil microorganisms are considered to be more important and intricate than the direct effects, and in most cases, these effects are mediated through plants, but can be manifested below the ground (Caldwell et al. 2007). However, since the sunlight can hardly penetrates the surface soil (Jeffery et al. 2009), most studies investigated the microbes above-ground and fungus below-ground primarily (Rinnan et al. 2005; Zaller et al. 2002), little attention has been paid to soil bacteria, especially in alpine meadow ecosystem of Qinghai–Tibet Plateau. The Qinghai–Tibet Plateau, with an average elevation of more than 4,000 m, is the highest upland in the world (Liu et al. 2009), called the ‘world’s roof’ or ‘third polar’ (Shi et al. 2004; Zhang et al. 2007). It represents a unique environment, being a result of high elevation, low latitude and strong radiation and is one of the most sensitive regions to the global environmental changes (Shi et al. 2004). Zhou et al. demonstrated that in the past decades, total ozone level over Qinghai–Tibet Plateau has decreased continuously (Zhou et al. 1995). The ecosystem of Qinghai–Tibet Plateau would be very seriously deteriorated if the lower level of ozone persists. Previous research suggested that climatic warming has occurred in Qinghai– Tibet Plateau since the late 1980s (Liu et al. 2009). Therefore, investigations into microorganisms from this unique region in response to the changing climate, including the enhanced UV-B, are urgently needed and significant. In an alpine meadow ecosystem of Qinghai– Tibet Plateau, various responses to enhanced UV-B for several typical alpine plants were investigated in the field (Cui et al. 2003; Shi et al. 2004). Under those conditions, we hypothesized that the below ground microbes might be changed after UV-B radiation. Our objectives of this study were to determine the effects of enhanced UV-B on abundance, bacterial community composition of culturable bacteria of alpine meadow ecosystem in the Qinghai–Tibet Plateau using the traditional counting, as well as the microbial activity through the calorimetric measurements.
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northeast of Qinghai–Tibet Plateau and south hillside of the Leng-Long-Ling which is at the east of the Qilian Mountains, 37°290 N–37°450 N, 101°120 E–101°330 E, with an altitude of approximately 3,200 m. The mean annual air temperature is -1.7 °C and the annual precipitation is 580 mm. The weather is cold but humid, belongs to Plateau continental climate. Kobresia humilis, which consists of perennial herb, is the dominated species growing in this land with alpine meadow soils. In 1998, a block with well-distributed vegetation was selected to establish the field experimentation site and subdivided into six plots arranged in a randomized complete block experimental design, consisting of two treatments, three for enhanced UV-B treatment (UV-B) and three for ambient UV-B (CK). The experimental system consisted of six 40 W fluorescent lamps (UV-B-313, Beijing Electric and Light Source Institute, PR China) which were mounted on a metal frame (2.5 m 9 1.5 m 9 0.75 m high) and suspended over the vegetation vertically. In contrast, the control system only installed metal frame without fluorescent lamps. The lamps can filter the lethality UV-C (\280 nm) with cellulose diacetate film (0.13 mm; Courtauld Specialty Plastics, Derby, UK) which allowed transmission of UV-B (280–315 nm) and UV-A (315–400 nm) radiation. Cellulose diacetate filters were replaced weekly to maintain a stable light quality. UV-B supplementation was performed from 10:00 to 16:00 daylight. The enhanced UV-B density was 15.80 kJ/m2 (Shi et al. 2004). The radiation reached the ground was a combination of the UV-B from the fluorescent lamps and the ambient UV-B radiation, simulating 14 % ozone depletion of stratosphere (Shi et al. 2004). Sampling In August 2007, all three replicate blocks of each treatment (CK and UV-B) were sampled by taking five soil cores for the upper layer and lower layer, above 10 cm (0–10 cm) and below 10 cm (10–20 cm), respectively, with the surface layer being removed to a depth of 5 mm. Then the five soil core samples of each block were mixed as one sample, and stored in a sealed plastic bag for transporting back to laboratory. Samples were stored at -20 °C prior to analyses. Determination of soil chemical properties
Materials and methods Site description and experimental design The experiment was conducted at the Haibei Alpine Meadow Ecosystem Research Station. This station is in the
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Air-dried soils were passed through a sieve in 0.074 mm diameter, then soil organic carbon and total N were determined with CHNS-analyser(Elementar Vario EL, Elementar Analysensysteme GmbH, Hanau, Germany)by combustion method at 450 and 1,250 °C, respectively (Analysis and Test Center of Lanzhou University). Total
UV-B impact soil microbe in Qinghai–Tibet Plateau field
P and available P were measured by HClO4–H2SO4 Mo–Sb colorimetric method and Olsen method, respectively. Cultivation, counting and isolation The total numbers of culturable bacteria were determined as colony forming units (CFU) on agar plates by dilution plate methods. Briefly, 3 g soil of each sample were homogenized in 27 ml of sterilized water and serially diluted. Aliquots (0.2 ml) of the diluted suspension were spread on the beef extract peptone agar plates for the enumeration of bacteria. The CFU on plates were counted after incubation for 1–3 weeksunder 25 °C. Representative colonies were purified according to their morphological appearance and color. DNA extraction, PCR and RFLP for individual bacterial colony Total genomic DNA of the isolates was extracted using the chloroform–isoamyl alcohol extraction procedures (Zhou et al. 1996). 16S rRNA genes of these isolates were amplified using the universal primer set of 27F (50 -AGAGTTT GATCCTGGCTCAG-30 ) and 1492R (50 -TACGGCTACC TTGTT ACGACTT-30 ) (Lane 1991). The reaction mixture (25 ll) consisted of 1 Unit Taq polymerase, 0.2 mM dNTP, 2.5 ll of 10 9 PCR Buffer, 2.5 mM of MgCl2, 0.2 lM of each primer, 2.5 ll (ca. 10 ng DNA) of template. The PCR amplification program was as follows: initial denaturation at 94 °C for 3 min, 30 cycles of 94 °C for 1 min, annealing at 58 °C for 1 min, and extension at 72 °C for 1:30 min, and a final extension of 10 min at 72 °C. PCR products were detected by electrophoresis in 1 % agarose gel. The PCR products were screened for similarity using restriction fragment length polymorphism (RFLP) analysis. Amplified rRNA gene products were digested with the restriction endonucleases BsuRI(GG/CC) and Hin6I(G/ CGC). The digested fragments were visualized on a 2.5 % agarose gel. Isolates were grouped together on the basis of RFLP patterns, and one isolate was chosen from each group for cloning and sequencing after purified using a quick Midi purification kit (Tiangen Co., Beijing, China).
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The suspension of expected clones was used to sequence by Shanghai Majorbio Bio-technology Company. The 30 sequences obtained, ca. 1,500 bp,were then analyzed with the National Center for Biotechnology Information (NCBI) Blast program. The most similar GenBank sequences to the clones were extracted from the GenBank. A phylogenetic neighbor-joining tree including obtained isolates and their closest relatives was constructed using MEGA 4.0. Nucleotide sequence accession numbers The obtained sequences were submitted to the GenBank database and assigned accession numbers of GU385847– GU385876. Microcalorimetric measurements The microcalorimetric measurement was performed on a TAM2277 (Thermometric, AB, Sweden) which is a commercial version of the system developed by (Suurkuusk and Wadso¨ 1982). Measurements were carried out in sterilized ampoules with Teflon sealing disks to avoid evaporation of the sample solution. All soil samples were first placed at room temperature for 24 h, then submitted to microcalorimetric measurements. For each sample, 1.0 g soil was put into the prepared ampoule before adding 0.5 ml of a solution containing 5.0 mg of glucose and 5.0 mg of ammonium sulphate. The reference ampoule was filled with 0.5 ml of distilled water instead of the nutrient solution. All measurements were run at 25 °C. Statistical analysis The means and standard deviations for three replicates were calculated and ANOVA was used to test the significant differences of UV-B radiation with software SPSS 13.0.
Results Soil chemical properties
Cloning and sequencing Cloning was performed with the pGM-T Vector System (Tiangen Co., Beijing, China) following the protocol of the manufacturer and the ligation product was subsequently transformed into E. coli DH-5a, which allows blue–white screening, and plated on LB medium containing ampicillin (100 mg/ml), X-Gal (20 mg/ml) and IPTG (200 mg/ml). Positive clones were identified by PCR amplification with pGM-T vector primer pairs T7/Sp6 using the same program as 16S rDNA amplification.
A summary of the main chemical properties of all soil samples is presented in Table 1. Changes of total N, total C and organic C were not significant different (P [ 0.05). However, total P and Olsen-P decreased significantly after treating with enhanced UV-B (P \ 0.05). The abundance of the culturable bacterial population The mean values of CFU of culturable bacteria for all soil samples are given in Fig. 1. In general, the number of
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Table 1 The effect of the enhanced UV-B on main chemical properties of soil samples of Qinghai–Tibet Plateau alpine meadow ecosystem Treatment
TN(mg/g)
TC(mg/g)
SOC(mg/g)
TP(mg/g)
AP(mg/kg)
CK
3.79 ± 0.11a
46.89 ± 0.94a
24.85 ± 0.99a
0.37 ± 0.00a
9.859 ± 0.112a
UV-B
3.89 ± 0.12a
49.00 ± 0.98a
25.97 ± 1.04a
0.32 ± 0.01b
7.977 ± 0.195b
TN total nitrogen, TC total carbon, SOC soil organic carbon, TP total phosphorus, AP available phosphorus. We obtained the data by amalgamating the samples of two depths of three replicates for each treatment. So the value is mean ±SD (n = 6). Values within the same column not followed by the same letter differ significantly (P \ 0.05)
was very different due to their multiplex distribution in different depths with different treatments. The total OTU number for CK and UV-B of the upper layer was 20 and 13, and 16 and 13 for CK and UV-B foe the lower layer, respectively (Fig. 2). Obviously, there was a decrease due to enhanced UV-B, compared with CK,and samples of the upper layer showed a greater change than that of the lower layer. However, some OUT (e.g., QT4, QT6) appeared while others (e.g., QT14, QT19, QT20) disappeared, and some OTUs (e.g., QT7, QT8, QT22, QT25 and QT27) were only detected in the lower layer with treatment of enhanced UV-B radiation. Fig. 1 The effect of enhanced UV-B on culturable bacterial abundance in soil of Qinghai–Tibet Plateau alpine meadow ecosystem. The values are the average of three replicates for each sample. Different letters over the bars show significant differences
bacteria obviously decreased with the increase of soil depth. As a result of enhanced UV-B, the abundance was slightly reduced, from 2.94 9 106 to 2.46 9 106 CFU/g for the upper layer and from 1.65 9 106 to 1.44 9 106 CFU/g for the lower layer, though the differences were not significant statistically between different treatments for the same soil layer (P [ 0.05). RFLP and phylogenetic analyses Through restriction fragment length polymorphism (RFLP) analysis, all 154 isolates distinguished by the morphological characteristics were clustered into 41 groups. After comparing 16S rDNA sequence of the representative strains of each group, they formed 30 groups. The recovered populations fell into four categories: Firmicutes, Actinobacteria, Proteobacteria and Bacteroidetes (Fig. 2). All samples were dominated by Firmicutes and Proteobacteria (Fig. 3), especially the Bacillus of Firmicutes and the Pseudomonas of Proteobacteria. Bacteroidetes was subordinate. Actinobacteria was the least, and reduced nearly by half (from 15 to 7.69 % for the upper layer and from 12.5 to 7.69 % for the lower layer) due to enhanced UV-B radiation (Fig. 3). The diversity in each soil sample
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Microbial activity measured by microcalorimetric method The results obtained from calorimetric determination for all soil samples are shown in Fig. 4 and Table 2. The recorded power–time curves (Fig. 4) showed evolution changes of microbial activity. The mean values of the maximum peakheat output power, Pmax (lw), the time to reach the maximum of the peak, tmax (min), and the microbial growth rate constant, k (min-1), were obtained through integration of each curve. The total heat evolution, QT (J/g), was also calculated for these samples (Table 2). The two curves of the lower layer samples displayed a longer lag phase than that of the upper layer which showed a rapid microbial growth and the curves of UV-B were lower than that of CK for the two layers (Fig. 4). The Pmax of the former were lower than the latter. Between enhanced UV-B and CK of either upper or lower layer, there were no significant differences (P [ 0.05) (Table 2). The values of k, representing microbial growth rate constant during the log phase or the exponential phase of microbial activity curves, reduced with the increase of soil depth as well as with the decrease of numbers of bacteria (Fig. 5a). Furthermore, the k of upper layer samples showed a significant difference between enhanced UV-B and CK (P \ 0.05) (Table 2). Compared to the bacterial population, the time to reach the peak, tmax, presented a reverse trend in each soil layer
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Fig. 2 Phylogenetic dendrogram based on a comparison of the 16S rDNA sequences of the 30 representative isolates from the soil samples of alpine meadow ecosystem of Qinghai–Tibet Plateau, with the treatment of enhanced UV-B, and some of their closest phylogenetic relatives. The tree created by the neighbor-joining
method. aC(a-CK) and aU(a-UV-B), the soil samples above 10 cm with the treatments of control and enhanced UV-B, respectively; bC(b-CK) and bU(b-UV-B), the soil samples below 10 cm with the two treatments, respectively. ‘‘?’’, stand for the presence of corresponding bacteria
(Fig. 5b), although there were also no significant differences between different treatments (P [ 0.05) (Table 2). The QT, the value of total heat released by soil microorganisms, negatively but implicitly correlated with the value of CFU (Fig. 5c).
Discussion The effects of enhanced UV-B on soil bacteria from alpine meadow ecosystem of Qinghai–Tibet Plateau were investigated. The abundance of soil culturable bacteria was
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Fig. 3 The effect of enhanced UV-B on the distribution of Firmicutes, Actinobacteria, Proteobacteria and Bacteroidetes in different soil samples of alpine meadow ecosystem of Qinghai–Tibet Plateau
Fig. 4 Power-time curves recorded microcalorimetrically from different soil samples of Qinghai–Tibet Plateau alpine meadow ecosystem with two treatments of enhanced UV-B and CK, amended with glucose and ammonium sulphate. In these curves Pmax (lw) is plotted against tmax (min)
slightly reduced due to enhanced UV-B radiation, although there were no significant statistically differences between different treatments in the same soil layer (Fig. 1). As we known, fungi and bacteria are generally more sensitive to damage by UV-B radiation in direct sunlight than are plants (Braga et al. 2001; Mu¨hlenberg and Stadler 2005; Yan et al. 2012), however, sunlight scarcely penetrates the soil surface (Aucamp 2007; Avery et al. 2004), less than 0.3 % of light is transmitted through to 2 mm soil depth (Jeffery et al. 2009). So the bacteria may receive the direct effects of UV-B radiation scarcely. Moreover, Avery et al. reported there were no significant effects of UV-B treatment on culturable counts of bacteria in undisturbed plot similar to ours. Thus we speculate that the slight changes may be mediated by the vegetation (Avery et al. 2004). Bacterial community composition, however, were altered due to the enhanced UV-B. In Greenland and Antarctica, altered solar UV-B radiation did result in qualitative changes in the soil microbial communities, this was thought to be due to altered quantity and/or quality of root exudates (Johnson et al. 2002; Rinnan et al. 2005). However, the root production was apparently unaffected by the UV-B radiation between 5 and 10 cm (Zaller et al. 2002). Hence, the number of soil bacteria species in the lower layer (below 10 cm) showed insignificant changes as well as the small changes of culturable bacterial abundance in this layer. Nevertheless, the distribution of these detected species displayed great differences in all of soil samples (Fig. 2). Some bacteria (e.g., QT4, QT6) presented in the enhanced UV-B treatment, not in CK. This may because species diversity was affected by solar UV-B radiation because of species-specific sensitivity to the radiation and another studies reported that UV-B radiation could alter the plant and its root exudates, so some of the soil bacteria were stimulated or inhibited (Kashimada et al. 1996; Piccini et al. 2009; Ulevicˇius et al. 2004). In addition, the Bacillus of Firmicutes and the Pseudomonas of Proteobacteria are dominant. As is well known,
Table 2 The effect of enhanced UV-B on microbial activity of samples in different soil depth of Qinghai–Tibet Plateau alpine meadow ecosystem Depth Above 10 cm Below 10 cm
Treatment
Pmax (lw)
tmax (min)
k (min-1)
QT (J/g)
CK
180.04 ± 18.04a
1,015.5 ± 87.60a
0.0054 ± 0.0007a
5.18 ± 0.39a
UV-B
169.31 ± 27.38ab
993.83 ± 83.93a
0.0040 ± 0.0008b
5.52 ± 0.36a
CK
136.19 ± 23.79b
1,184.83 ± 22.55ab
0.0039 ± 0.0004b
5.66 ± 0.84a
UV-B
147.26 ± 17.90ab
1,246.33 ± 207.13b
0.0030 ± 0.0003b
6.25 ± 1.46a
The data obtained from power–time curves with 1.0 g soil samples supplemented with 0.5 ml solution containing 5.0 mg of glucose and 5.0 mg of ammonium sulphate. Pmax (lw), the maximum peak-heat output power; tmax (min), the time to reach the maximum of the peak; k (min-1), the microbial growth rate constant; QT (J/g), the total heat evolution. The value is mean ±SD (n = 3). Values within the same column not followed by the same letter differ significantly (P \ 0.05)
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other groups also presented with special pigment, e.g., some of QT24 and QT28 groups were lemon yellow, creamy yellow or orange, even some species of Firmicutes produce pigments, e.g., some of QT3 group were bright mauve, and some of QT11 were pastel pink. Pigment plays an important role in resisting UV for bacteria (Sandmann et al. 1998; Sundin and Murillo 1999), UV-B radiation sensitivity was inversely correlated with pigmentation and well pigmented species were considered to be tolerant of UV-B radiation corresponding to solar UV-B radiation with substantially depleted ozone (Aucamp 2007). Thus, the result that Bacillus and Pseudomonas are better to grow in the conditions like Qinghai–Tibet Plateau is creditable. The fact that Bacteroidetes was subordinate in these detected species may also because of its ability of producing pigments, e.g., some OUT of QT1 and QT2 produce yellow or orange, and some of QT16 produce purple. Soil microbial activity
Fig. 5 Log of CFU against growth rate, k (a), peak time, tmax (b), and total heat, QT(c), respectively, for soil samples treated with enhanced UV-B. With the decrease of bacterial quantity (Log of CFU), the value of k (a) decreased, the value of tmax increased (b) and QT (c) showed an increasing tendency
Bacillus are easily to mass culture with its lower nutritional requirements and tenacious viability, and some species of Pseudomonas produce pigments, e.g., some species of QT17 group which belong to Pseudomonas in this work produce beige by morphology observing, some of QT5 and QT23 groups produce grayish blue. Besides Pseudomonas,
Microcalorimetric technique is sensitive enough to detect very low heat rates and can provide qualitative and quantitative indicators of soil microbial activity(Barros et al. 1997; Nu´n˜ez-Regueira et al. 2006b). This method had been applied in soil environments subjected to various factors such as land-use, heavy metal and pesticides (Barros et al. 2006; Nu´n˜ez-Regueira et al. 2006a; Prado and Airoldi 2002; Zheng et al. 2009). In this research, it is the first time that microcalorimetric technique was applied to soil microbial activity subjected to enhanced UV-B radiation and the results demonstrated its importance. It is clear from the foregoing results that k (min-1) showed a visible lower growth rate of microbe in UV-B treatment, while the number of culturable bacteria was also less than in control (Fig. 5a). This result indicated that the number of soil bacteria reduced slightly due to the enhanced UV-B radiation, and the growth of soil microbe become slowly, accordingly. Similar results also can be found in Zheng’s study(Zheng et al. 2009). In upper layer, Pmax (lw) of samples treated by elevated UV-B which had lower bacterial numbers displayed a lower value and tmax (min) showed a reverse trend on the whole (Fig. 5b). These results were consistent with previous studies which suggested that shorter lag phases, lower peak times (tmax), higher Pmax and greater growth rate constant (k) were all good indicators of the high microbial metabolic activity in a microcalorimetric analysis (Barros et al. 1997; Nu´n˜ez-Regueira et al. 2006b; Zheng et al. 2009; Zheng et al. 2007). This indicated that enhanced UVB radiation would change the quantity and quality of soil microbes to a certain extent and prefer to inhibition. In addition, Zheng et al. indicated that the deficiency of available P can limit microbial activity (Zheng et al. 2009).
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In this work, available P reduced due to enhanced UV-B (Table 1) and microbial activity for samples treating with enhanced UV-B was relatively lower than control (Fig. 4). It showed that enhanced UV-B can change the soil chemical properties and then change microbial activity. The QT (J/g) negatively correlated with culturable bacterial abundance in this work (Fig. 5c). The highest bacterial numbers corresponded to the lowest QT. In fact, besides UV-B manipulation, soil microbial metabolism is mainly associated with many factors such as soil pH, nutrient, temperature, community structure and so on. So QT is probably associated with microbial diversity in soils since enhanced UV-B had little effects on microbial abundance but altered microbial community composition obviously (Rinnan et al. 2005). Additionally, in contrast to other microorganisms, bacteria are numerically dominant, but the other microorganisms in soils, including actinomycetes, fungi, protozoa and algae, developed multiple cooperative and competitive interactions as well as their relations with heat evolution of microbial activity (Zheng et al. 2007). Still less the majority of bacteria are uncultivable organisms. What’s more, compared to the calorimetric method which measured the total interaction of all microorganism growth in the system, plate count operation which reflected a specific growth of bacteria did not distinguish between active and dormant bacteria. If an inactive bacterial was spread on a culture medium, it will germinate and produce a colony that will be counted, but the inactive unit in the calorimetric system is not measured by this method (Critter et al. 2002). Therefore, the particularly change of QT, whose change was opposite from the CFU of culturable bacteria and the growth rate constant (k) of microbes in soil, may be due to these facts. On the other hand, our result that samples with the greatest value of QT showed the smallest value of k (Table 2), was consistent with previous research (Barros et al. 1997), which suggested that samples with the greatest percentages of organic matter showed the greatest values of QT and the smallest values of k. This may be due to a bigger availability of substrate sensible to microbial attack and organic matter suffers a slower decomposition, or may be due to the different vegetation distribution of sampling sites (Zheng et al. 2007). Therefore, there was a possible correlation between QT and organic matter or vegetation quantity. In conclusion, the results illustrated that the enhancement in 14 % of UV-B irradiation led to the change of soil microbial communities in alpine meadow ecosystem of Qinghai–Tibet Plateau subjected to nearly consecutive 10 years. Despite detrimental effects of enhanced UV-B on some species and activating effects on some others, an evidently inhibition of the diversity and activity was observed, especially in the upper soil layer. Since sunlight
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scarcely penetrated the soil surface, changes in microbial metabolic potential were likely to be plant-mediated. Thus, the changes that soil microbe manifested possibly due to the alterations in the quality and/or quantity of root exudation induced by UV-B. Although responses of soil culturable bacteria abundance to ambient even enhanced UVB were relatively slight in present, the bacteria community composition and the total activity of soil microbe have been changed considerably and there is no doubt that a possible future increase of UV-B levels will be occur more seriously due to the aggravating circumstances. Therefore, we cannot rule out the possibility that these increases would change the below-ground function of alpine meadow ecosystem. Acknowledgments This research was supported by National Basic Research Program (2012CB026105), National Natural Science Foundation (31170482, 31370450), PhD Programs Foundation of Ministry of Education (2010021111002, 20110211110021), The Fundamental Research Funds for the Central Universities (LZUJBKY-2013-92) in China, and State Key Laboratory of Frozen Soil Engineering, Chinese Academy of Sciences (SKLFSE200901). We are grateful to Dr Yantian Ma for his help. Conflict of interest of interest.
The authors declare that they have no conflict
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