Bacteria biomass and carbonic anhydrase activity in some

karst areas of Southwest China

W. Lia,*, L.J. Yua, D.X. Yuanb, H.B. Xua, Y. Yanga

aSchool of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, ChinabKarst Dynamics Laboratory, Ministry of Land and Resources, Guilin 541004, China

Received 24 June 2002; revised 12 September 2003; accepted 13 October 2003

Abstract

To determine the role of microbes in karst processes, it is necessary to investigate the ecological distribution and characteristics of soil

microorganisms in karst areas. In this paper, a preliminary study was carried out in two different karst areas of Southwest China: Nongla and

the Guilin Yaji Karst Experimental Site (Guangxi). Soil samples from 10–20 cm in depth were analyzed for the number of bacteria, and the

predominant bacteria were identified. Analysis showed that the amount of soil bacteria correlated highly with characteristics of the karst

ecosystems, including their different geochemical environments and vegetation. The predominance of Azotobacteraceae colonies showed

that the soil fertility of both types of karst areas may be improving. Also, the origin of carbonic anhydrase (CA), which could accelerate karst

processes, was explored. The CA-producing bacteria were screened, and activities of extracellular and intracellular CA were measured.

Obvious differences existed in intracellular and extracellular CA activities of soil bacteria between the two karst ecosystems with different

vegetation conditions. This suggests that the activity of CA from soil bacteria in the two different karst areas was also correlated with karst

ecosystem characteristics, including their different geochemical environments and vegetation features.

q 2003 Elsevier Ltd. All rights reserved.

Keywords: Karst ecosystem; Soil bacteria; Carbonic anhydrase; Vegetation

1. Introduction

Based on the results of research under the auspices

of UNESCO’s International Geological Correlation

Programme (IGCP) Project 379, living organisms and

their specific enzymes may play an important role in the

operation of karst dynamic systems (Yuan and Jiang,

2000). The evolution of epikarst (the near-surface

component of a karst system) has been shown to result

from a complex interaction of lithologic, biologic, soil,

and ecological components (Pan and Cao, 1999). Biomass

growth and soil formation clearly impact the evolution of

karst ecosystems, with the processes of biological activity

and soil medium as the major functions. Therefore,

biological processes plays an important role in the karst

processes, and thus it is critical that karst scientists turn

their attention to the biological role in studies of karst

evolution.

Soil often contains a variety of habitats occupied by a

great diversity of organisms that perform a wide variety of

functions (Waid, 1999). As the major category of soil

microbes, bacteria are very small, propagate quickly, and

exist in large quantities. They can even be found in many

extreme environments, such as those with high or low

temperatures, high salinity, high acidity, high alkalinity,

high osmotic pressure, and low nutrition, that were

previously believed to be unfavorable for life. Bacteria,

along with other microorganisms, are essential components

of an ecosystem, as they play key roles in material and

energy cycling. They also form important symbiotic

associations with plants, in many cases increasing soil

fertility and plant growth. Therefore, bacteria have become

a major biological focus of karst research since the 1950s,

and their important role in the precipitation of carbonate

rock had been recognized even earlier (Greenfield, 1963;

Krumbein, 1979; Kellerman and Smith, 1914). Previous

researchers have focused their attention on describing the

properties of erosional action on karst produced by bacteria.

The study of the biochemical mechanisms of carbonate

1367-9120/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.

doi:10.1016/j.jseaes.2003.10.008

Journal of Asian Earth Sciences 24 (2004) 145–152

www.elsevier.com/locate/jaes

* Corresponding author. Tel.: þ86-27-87543633; fax: þ86-27-

87540184.

E-mail address: huliwei@hotmail.com (W. Li).

corrosion by bacteria involves analyzing their metabolic

products, especially CO2 and organic acids (Zhang et al.,

1997). It has been shown that CO2 correlates with soil

microorganisms and that the main producer of CO2 in soil

may be bacteria (Jakucs, 1977). However, few studies of

soil bacterial diversity in karst areas have been reported.

Understanding soil bacterial diversity and their ecological

distributions in karst areas will be helpful in discovering

which specific bacteria play an important role in the karst

process. This paper offers a comparative study on the

amount of soil bacteria and species of predominant bacteria

in the two different karst areas. The experimental results

could provide a scientific basis for further study of the

ecological characteristics of soil bacteria in karst areas.

Moreover, some specific enzymes produced by soil

bacteria that may be important in karst evolution have not

been thoroughly studied. Carbonic anhydrase (CA), for

example, is a Zn-containing enzyme critical to catalyzing

the CO2 conversion reaction (Fridlyand and Kaler, 1987;

Lindskog et al., 1971). It was found in one study that after

adding CA to a karst system, the erosion rate of limestone

increased by a factor of about 10 (Liu and Dreybrodt, 1997);

therefore, the karst process was significantly intensified.

However, these experiments were carried out in the

laboratory and the CA used in the experiments came from

bovine sources, so a detailed understanding of the driving

action of biological CA on karst dynamic systems in nature

remains to be fully revealed. In order to understand the

actual role of biological CA in the karst processes, it is

necessary to examine the distribution and activity of

biological CA in karst field areas (Liu, 2001). In the current

study, CA-producing bacteria were screened from the two

different types of karst areas, and the activities of

intracellular and extracellular CA were measured. The

experimental results could provide a scientific basis for

further study on the role of CA in biokarst.

2. Materials and methods

2.1. Study areas and sampling

The study areas (Fig. 1) include two types of karst in

Southwest China: (1) the Nongla karst of Mashan, Guangxi,

and (2) the Yaji Karst Experimental Site near Guilin,

Guangxi. These sites were selected as representative of

different types of karst and vegetation distribution. Nongla

is located in the southern subtropical region with an annual

average rainfall of 1700 mm and annual mean air tempera-

ture of 19.8 8C. Approximately 85% of the rainfall occurs

from April to October. Its elevation is 450–500 m above sea

level (ASL). Karst landforms typical of peak-cluster

depressions occur there, and the site is covered with an

abundant plant community in Nongla. The limestone and

dolomite within which the karst has developed belong to the

Dongganling group of Devonian age. The Yaji Karst

Experimental Site near Guilin City is also in the subtropical

zone; however, bushes provide the most common vegetation

cover. The karst formed within the Rongxian limestone of

Devonian age. The highest peak at the Yaji site is 650 m

ASL, and the elevation of the adjacent plain in Guilin is

150 m ASL. The annual mean rainfall is 1915 mm and

annual mean air temperature is 18.8 8C. Approximately 70%

of the rainfall occurs from April to August.

A total of nine surface soil samples (five from Nongla

and four from Guilin) were collected at a depth of

10–20 cm in July 2001. The soil samples were placed into

sterilized sacks, taken to the laboratory, stored at 4 8C, and

used for experiments within one week. The geographic

distribution of various soil sampling sites is shown in Fig. 1.

The vegetation and geological background of the sample

sites as well as some chemical properties of the surface soils

at the collection sites are presented in Table 1. These brown

limestone soils are categorized as Cab-udic Luvisols

according to Soil Taxonomy (Gong, 1999).

2.2. Determination of moisture of the soil samples

A quantity of each soil sample was weighed and then

dried at 105 8C to a constant weight. Then the dry weight of

the sample was determined gravimetrically, and the

moisture content was calculated.

2.3. Enumeration of the soil bacteria

The soil bacterial numbers were determined following the

Research Methods for Soil Microorganisms recommended

by Nanjing Institute of Soil (1985). Each soil sample was

diluted in a series of 10-fold dilutions, and 0.1 ml of three

kinds of dilutions were spread on broth-peptone agar plates.

The plates were then incubated at 34–37 8C for 24–48 h,

and visible colonies were counted and recorded.

2.4. Analysis and identification of the predominant bacteria

The methods of isolation and incubation were identical to

those described above. The predominant bacteria were

analyzed by colony morphologies and cell morphologies.

Then the predominant bacteria were separated and inocu-

lated onto broth-peptone agar slants. The slants were

incubated at 34–37 8C for 24–48 h, and then stored at

4 8C for further use. The predominant bacteria were

identified according to Bergey’s Manual of Determinative

Bacteriology (Holt, 1994).

2.5. Screening for carbonic anhydrase-producing bacteria

A number of different dilutions of each soil sample were

spread onto broth-peptone agar plates that contained

60 g l21 calcium carbonate. The plates were then incubated

at 34–37 8C for 24 h, and visible colonies were collected

and purified using the conventional streaking method.

W. Li et al. / Journal of Asian Earth Sciences 24 (2004) 145–152146

Fig. 1. (a) Location map of the study areas in Southwest China. (b) Schematic map of sampling sites at the Nongla karst area of Guangxi, China. The

geomorphologic structure and vegetation distributions are based on Jiang (2001). (c) Schematic map of sampling sites in the Guilin Yaji Karst Experimental

Site, China. The geomorphologic structure and distributions are based on Yuan et al. (1996).

W. Li et al. / Journal of Asian Earth Sciences 24 (2004) 145–152 147

The activity of CA for each pure bacterial culture was

measured, and the bacteria that can produce CA were

isolated.

2.6. Determination of carbonic anhydrase activity

CA catalyses the following reaction:

H2O þ CO2 , HCO23 þ Hþ

The activity of the enzyme was determined by measuring

the net rate of proton production during the reaction, as

carried out under controlled conditions. Pure bacterial

cultures were centrifuged under 5000g for 5 min and the

supernatant retained for extracellular enzyme assay. The

harvested biomass was washed twice with distilled water.

To prepare cell extracts, 0.2–0.6 g of fresh cells were placed

into a chilled mortar and ground in 7 ml 100 mM Tris–

H2SO4 buffer (pH 8.3). These operations were conducted at

0–5 8C. The ground material, after being centrifuged at

5000g for 5 min, was then subjected to intracellular enzyme

assay.

CA activity was determined from the rate of CO2

hydration at 2 8C by following the change of pH traced on a

chart recorder according to a method modified from

Brownell et al. (1991). The assay was carried out in a

2 8C coldroom. Calibration was carried out by monitoring

the drop in pH from 8.3–7.3 units during sequential

additions of 100 ml of 0.1 M H2SO4 to the assay medium

which contained 5 ml 20 mM barbitone buffer (pH8.3). It

was found that there was a drop of 0.2 pH units per 100 ml of

0.1 M H2SO4. Therefore, 1 pH unit was equivalent to

0.1 mmol Hþor 0.1 mmol CO2 hydrated. The CA activity

was determined by the difference in the rates of decrease in

pH in the assay medium containing 4.5 ml ice-cold CO2-

saturated water in the presence of 0.5 ml boiled and

unboiled bacterial extracts or culture supernatant. The

enzyme Units were calculated according to the formula U ¼

10 ðTo=Te 2 1Þ; where To and Te represent the time for pH

change with boiled and unboiled bacterial extracts or culture

supernatant respectively. The values presented are the

means of three replicates. The specific activities of CA are

expressed as U mg21 protein. Protein concentrations were

determined by the method of Lowry et al. (1951).

3. Results

3.1. Effects of different karst ecosystems on the numbers

of soil bacteria and the predominant bacterial species

Table 2 shows the differences in the numbers of soil

bacteria and the predominant bacterial species between the

two different karst ecosystems. From Table 2, it is clear that

site NL#1 yielded the largest number of bacterial colonies.

The humus substrate and the presence of a large quantity of

plant root material may be probable reasons. The color of

NL#1 soil sample was black indicating that more abundant

organic materials were in the NL#1 soil sample (Table 1).

The average numbers of soil bacteria (3.47 £ 107

ind. g21 dry soil) in Nongla were higher than those

(1.15 £ 107 ind. g21 dry soil) at the Yaji Karst Experimental

Site but the difference was not significant ðP ¼ 0:287Þ: This

difference may be related to the karst ecosystems’

Table 1

The conditions of sampling sites and some chemical properties of surface soils

Site No. Textures and description Plant species Limestone

denudation

rate (mg cm22 a21)

Organic

matter

(g kg21)

Total N

(g kg21)

C/N ratio pH (H2O)

NL#1 Forest of Shangnongla; black humus soil

with lots of plant roots

M.repandus, Bambusa

blumeana

Sample lost 95.27 5.13 10.77 6.72

NL#2 The edge of the Jidanbao forest at Landiantang;

yellow brown loose soil

Evergreen broadleaf,

Nephrolepis auriculata

Sample lost 71.31 3.75 11.03 6.54

NL#3 Bush cluster, Jidanbao hillside; yellow brown,

less loose soil

Vitex negundo, Nephrolepis

auriculata

1.99 74.84 3.46 12.55 7.02

NL#4 Under a tree of Zenia insignis Chun, Jidanbao;

yellow soil with little stone and more herbage

Zenia insignis Chun,

Nephrolepis auriculata

Sample lost 88.18 4.16 12.30 7.00

NL#5 Under a tree of Ilex Kudingcha, Jidanbao; grey

brown loose soil with more herbage

Ilex Kudingcha, loose

Nephrolepis auriculata

Sample lost 81.65 4.07 11.63 6.50

YJ#1 Depression of No.1, bush cluster; grey black,

loose and soft soil

Pterolobium punctatum,

Broussonetia papyrifera

7.73 63.65 3.58 10.31 6.38

YJ#2 Depression of No.1, under a small tree;

yellow brown soil

Vitex negundo, Loropetalum

chinensis

Sample lost 54.36 2.84 11.10 6.87

YJ#3 Slope of depression of No.1; yellow brown,

granule and lump soil

Vitex negundo,

Phyllostachys sulphurea,

Loropetalum chinensis

1.85 45.47 2.15 12.27 6.55

YJ#4 Puerto; red brown soft soil Vitex negundo 7.87 61.46 3.02 11.80 6.00

W. Li et al. / Journal of Asian Earth Sciences 24 (2004) 145–152148

characteristics, including their geochemical environments

and vegetation conditions.

On the other hand, it is evident that different species of

predominant bacteria existed in soil samples collected from

the different sites (Table 2). Nonetheless, Azotobacteraceae

colonies were predominant in both karst areas.

3.2. Comparison of intracellular or extracellular CA

activity in soil bacteria between the two different karst

ecosystems

In the 10 bacterial isolates screened for their capability of

producing CA, nine strains produced detectable activity of

intracellular or extracellular CA. NLCa602, isolated from

the NL#5 soil sample collected at Nongla, had the highest

intracellular CA activity of 13.522 U mg21 protein, and

GLCa102, isolated from the YJ#1 soil sample of the Karst

Experimental Site, had the highest extracellular CA activity

of 1.116 U mg21 protein. The results of the CA activities

are shown in Fig. 2. In addition, the CA activities of

predominant bacteria from the two karst areas were also

measured. The results showed that most of the predominant

bacteria had intracellular or extracellular CA activity. It is

evident that the strains with the higher activity of CA can be

screened from special plates containing calcium carbonate.

The intracellular activities of this enzyme are significantly

higher than the extracellular ones. On the other hand, the

strains isolated from the soil samples of Nongla displayed

obvious intracellular CA activities, while the strains isolated

from the soil samples of the Yaji Karst Experimental Site

displayed mostly both intracellular and extracellular CA

activities.

4. Discussion

The growth of soil microorganisms is closely correlated

with the content of organic matter (Zak et al., 1994),

along with the content and proportion of available elements

in soil. Table 1 and Table 2 clearly show that the differences

in the number of soil bacteria between the two different

karst ecosystems are caused by a combination of several

factors. The first involves interactions between the karst

geochemical environment, the intensity of karst processes,

and the stages of karst development at the different sites.

Plants in karst areas can grow not only in soil but also

directly on bare rock, indicating a petrophilic character for

the vegetation (Yuan and Cai, 1988). Inorganic ions in karst

waters that result from dissolution of carbonate bedrock by

rain water, soil water, and ground water provide an

important source of available elements for microorganisms.

The rock type in Nongla is mainly dolomite, which contains

more magnesium and trace elements such as zinc,

manganese, and iron relative to limestone (Yuan et al.,

1996; Jiang, 1997), and may be more suitable for the growth

of microorganisms (Black, 1996). Moreover, karst pro-

cesses in Nongla have been shown to be intensive (Jiang,

2000). As a result, not only do K, Na, Ca and Mg occur in

high concentrations, but some relatively less soluble

elements such as Si, Al, Fe and Mn are also found in

these karst waters. These elements provide source materials

for microorganisms, which may explain the higher amount

of soil bacteria at Nongla than at the Yaji Karst

Experimental Site. Secondly, differences in soil bacterial

numbers result from plant growth conditions, plant species

diversity, and the amount of leaf litter at different sites. A

mountain pass was sealed to allow reforestation in the early

1960s in Nongla of Mashan, so at present the vegetation has

been restored and Nongla lies in a forest environment. Plant

species diversity is far more abundant in Nongla than in the

Yaji Karst Experimental Site, and so may be more suitable

for the growth of microorganisms. Thirdly, during the

process of plant growth, plant root systems secrete some

metabolites, such as carbohydrates, amino acids, organic

acids, fatty acids, auxins, ribonucleotides, and enzymes to

the rhizosphere. These metabolites not only provide bacteria

with available C and N, but also increase the number of soil

bacteria in the vicinity of the plant root because of the

presence of stimulants for bacterial growth. This is the so

called ‘rhizosphere effect’, which results in the largest

bacterial numbers at site NL#1 (Table 2). Finally, different

species, ages, and growth phases of plants at the different

sites result in different varieties, amount and characteristics

of their secretions, which leads to differences in bacterial

numbers.

There was a certain difference in demands on the soil

environment for growth of different bacterial species. The

predominant bacterial species can reflect soil character-

istics, and are closely related to the karst geochemical

environments, karst landforms, and corresponding veg-

etation. Table 2 shows that Azotobacteraceae colonies were

predominant in both karst areas, indicating that the soil

fertility of both karst areas may be improving because

Table 2

Numbers of soil bacteria and species of predominant bacteria in two types

of karst sites (Nongla and the Guilin Yaji Karst Experimental Site)

Site No. Bacterial numbers

(107ind. g21 dry soil)

Predominant bacteria

NL#1 9.98 ^ 0.50 Bacillaceae

NL#2 1.26 ^ 0.12 Bacteroidaceae

NL#3 4.26 ^ 0.30 Azotobacteraceae

NL#4 0.79 ^ 0.08 Brevibacteriaceae

NL#5 1.08 ^ 0.15 Brevibacteriaceae

YJ#1 2.27 ^ 0.20 Corynebacteriaceae,

Propionibacteriaceae

YJ#2 0.67 ^ 0.06 Azotobacteraceae

YJ#3 0.27 ^ 0.02 Azotobacteraceae

YJ#4 1.41 ^ 0.10 Corynebacteriaceae

Values are the means of six replicates ^ SD. The difference in mean

values between the two types of karst sites is not significant ðP ¼ 0:287Þ

W. Li et al. / Journal of Asian Earth Sciences 24 (2004) 145–152 149

Azotobacteraceae bacteria are related to soil fertility

(Kahindi et al., 1997).

CA has been found in virtually all mammals, as well as

plants and algae, and is fundamental to many eukaryotic

biological processes such as photosynthesis, respiration,

CO2 and ion transport, and calcification and acid–base

balance (Smith and Ferry, 2000). While previous reports

have suggested that this enzyme is not prevalent in the

Bacteria and Archaea domains, recent research has

demonstrated that CAs are far more prevalent in

prokaryotes and distributed among far more metabolically

diverse species than previously recognized (Smith et al.,

1999). This study has provided further evidence in support

of this observation. The experimental results showed that

CA activity could be detected in most of the strains

isolated from soil samples in the two types of karst areas.

There were obvious differences in intracellular and

extracellular CA activities of soil bacteria between the

different karst ecosystems. Different karst ecosystems

have different karst geochemical environments, karst

landforms, and vegetation, which may lead to differences

in CA activities. Moreover, the highest intracellular CA

activity was detected in a strain isolated from Nongla,

where there is more abundant vegetation, and the highest

extracellular CA activity was detected in a strain isolated

from depression No.1 at the Yaji Karst Experimental Site.

These suggest that the activity of CA from soil bacteria

between the two different karst areas is correlated with

karst ecosystem characteristics, including their different

geochemical environments and vegetation features.

Fig. 2. Comparison of carbonic anhydrase activity of soil bacteria between the two karst areas: (a) Nongla study area of Guangxi, China; (b) Guilin Yaji Karst

Experimental Site. Values are the means of three replicates, and error bars represent standard errors.

W. Li et al. / Journal of Asian Earth Sciences 24 (2004) 145–152150

5. Conclusions

There were differences in the numbers of soil bacteria

and the predominant bacterial species between the two

different karst ecosystems. It was shown that the average

numbers of soil bacteria in Nongla (3.47 £ 107 ind. g21 dry

soil) were higher than those at the Yaji Karst Experimental

Site (1.15 £ 107 ind. g21 dry soil). The comparative

analysis showed that the amount of soil bacteria was

strongly correlated with characteristics of the karst ecosys-

tems, including their different geochemical environments

and karst developmental status, as well as plant species and

growth conditions. The large numbers of Azotobacteraceae

colonies in both Nongla and the Guilin Yaji Karst

Experimental Site showed that the soil fertility of both

areas may be improving.

On the other hand, the origin of CA, which could

accelerate karst processes, was also explored. The CA-

producing bacteria were screened and the activities of

extracellular and intracellular CA were measured. The

results showed that there were obvious differences in

intracellular and extracellular CA activities of soil bacteria

between the two karst ecosystems with different vegetation

conditions. This suggests that the activity of CA from soil

bacteria in the two karst areas was also correlated with karst

ecosystem characteristics, including geochemical environ-

ments and vegetation features. Further work is needed to

monitor the stabilization of CA in karst environments and to

explore the role of CA from soil bacteria in karst dynamic

systems.

Acknowledgements

This work was jointly supported by the Major Research

Plan of the National Natural Science Foundation of China

(Grant No. 90202016), the General Programs of the

National Natural Science Foundation of China (Grant No.

40152002, 40302034), and the Karst Dynamics Laboratory,

Ministry of Land and Resources, China. The authors are

grateful to Zaihua Liu, Zhongcheng Jiang, Jianhua Cao,

Yunqiu Xie, Shiyi He, Guanghui Jiang and Fang Guo for

their cooperation with sampling and for providing some

information about karst geology and climate. The authors

thank Zhongcheng Jiang for providing the data on limestone

denudation in the Yaji Karst Experimental Site and in

Nongla, and the authors also thank Yun Wu for experimen-

tal assistance. Many thanks to Prof. Zaihua Liu and an

anonymous reviewer, their very useful comments and

suggestions improved this manuscript profoundly. The

authors appreciate Dr Chris Groves and Dr John Mylroie,

and the journal Editor-in- Chief, Dr Kevin Burke, who

checked and improved the English.

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