Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Microbial populations and activities of mangrove, restinga and Atlantic forest soils from Cardoso Island, Brazil B. Pupin1 and E. Nahas2 1 Program of Agropecuary Microbiology, Universidade Estadual Paulista (UNESP), Jaboticabal, SP, Brazil 2 Department of Crop Production, Universidade Estadual Paulista (UNESP), Jaboticabal, SP, Brazil

Keywords bacteria, ecosystems, fungi, metabolic activity, season, soil depth. Correspondence Ely Nahas, Department of Crop Production, UNESP, 14884-900 Jaboticabal, SP, Brazil. E-mail: [email protected] 2013/1894: received 17 September 2013, revised 14 November 2013 and accepted 3 December 2013 doi:10.1111/jam.12413

Abstract Aim: Mangroves provide a distinctive ecological environment that differentiates them from other ecosystems. This study deal to evaluate the frequency of microbial groups and the metabolic activities of bacteria and fungi isolated from mangrove, restinga and Atlantic forest soils. Methods and Results: Soil samples were collected during the summer and winter at depths of 0–2, 2–5 and 5–10 cm. Except for fungi, the counts of the total, sporulating, Gram-negative, actinomycetes, nitrifying and denitrifying bacteria decreased significantly in the following order: Atlantic forest > mangrove > restinga. The counts of micro-organisms decreased by 11 and 21% from the surface to the 2–5 and 5–10 cm layers, but denitrifying bacteria increased by 44 and 166%, respectively. A larger growth of micro-organisms was verified in the summer compared with the winter, except for actinomycetes and fungi. The average frequency of bacteria isolated from mangrove, restinga and Atlantic forest soils was 95, 77 and 78%, and 93, 90 and 95% for fungi, respectively. Bacteria were amylolytic (33%), producers of acid phosphatase (79%) and solubilizers (18%) of inorganic phosphate. The proportions of fungi were 19, 90 and 27%. Conclusion: The mangrove soil studied had higher chemical characteristics than the Atlantic forest, but the high salinity may have restricted the growth of microbial populations. Significance and Impact of the Study: Estimates of the microbial counts and activities were important to elucidate the differences of mangrove ecosystem from restinga and Atlantic forest.

Introduction Brazil has the largest coastal extent in the world, with different ecosystems found in an excellent state of preservation, such as mangroves, restingas and Atlantic forest (EMBRAPA 2012). These systems are self-sustaining environments with a high deposition of plant material and biological productivity. Mangroves are a complex ecosystem characteristic of tropical and subtropical regions, forming a transition between terrestrial and marine environments, and are distributed worldwide. The mangrove area in Brazil is estimated at 963 km2, representing 7% of the world area (Giri et al. 2011), with vegetation composed mainly of Rhizophora mangle, Avicennia

schaueriana, Laguncularia racemosa and Conocarpus erectus. The soil is moist, salty, muddy, oxygen-poor and very rich in nutrients and organic matter (Kathiresan and Bingham 2001). Restingas comprise a number of herbaceous vegetation types situated on sandy and salty soil, under marine or fluvial–marine influence. The extension of restingas is about 5000 km, occurring on 79% of the Brazilian coast (Lacerda et al. 1993). The vegetation of the Brazilian restingas is characteristic of sandy coastal areas, with the prevailing species being Dalbergia ecastaphyllum, Sophora tomentosa and Tibouchina holosericea. In contrast to the restingas, the Atlantic forest is formed of large trees that reach 20–30 m tall and that are

Journal of Applied Microbiology 116, 851--864 © 2013 The Society for Applied Microbiology

851

Microbial populations of mangrove

B. Pupin and E. Nahas

found along the whole Brazilian coast. Although now discontinuous due to deforestation, it is globally one of the most biodiverse ecosystems, containing more than 60% of the plant species of the world (Faoro et al. 2010). Being self-sustaining systems, micro-organisms play an important role in the decomposition of organic matter, mineralization of organic compounds and making nutrients available to plants (McGuire et al. 2012). However, populations of micro-organisms and their activities can be influenced by changes in the physical and chemical characteristics of the soil (Oller Costa et al. 2012), and also by the seasons (Zhang et al. 2009). Additionally, it is possible that the composition of the microbial community is subject to maritime influence. Organic matter content and soil pH can be highlighted as the main characteristics influencing the number of micro-organisms (Pupin et al. 2009; Habibur et al. 2013). According to Fan et al. (2006), the total number of bacteria is positively correlated with the organic matter content. As the micro-organisms are directly involved in the cycling of nutrients in the soil, it is important to quantify them to indicate how these processes are occurring in ecosystems and are affecting soil quality (Hafich et al. 2012). The influence of the ecosystem on the number of bacteria (Silva et al. 2008), fungi (Oller Costa et al. 2012) and actinomycetes (Ravikumar et al. 2011) has shown great variability. Another important factor when considering mangroves and restingas is the availability of oxygen in the soil. Due to flooding, low levels of oxygen can occur, mainly in the deeper layers of the soil. Studies show that denitrifying bacteria are abundant in mangroves due to the anaerobic conditions associated with high levels of organic matter (Reef et al. 2010). A greater number of denitrifying bacteria were found at a 7 cm depth of the Tanshui River, northern Taiwan, but no correlation was found with the soil chemical characteristics (Fan et al. 2006).

Materials and methods Study area The Cardoso Island Park was created in 1962: it was the first protected island of Sao Paulo, Brazil, and has integrated the Biosphere Reserve of the Atlantic forest since 1991. It consists of several adjacent ecosystems, among which mangroves, restingas and Atlantic forest have high conservation value, and are preserved practically in their natural form. The park covers an area of 22
500 ha and is located at the coordinates 25°03′–25°18′S and 47º53′– 48º05′W (Fig. 1). The climate, according to the K€ oppen classification, is Af megathermic, superhumid, without a dry season and with excessive rain in summer. Annual rainfall is 2
216 mm and mean temperature is 22
3°C. In the summer, there was a rainfall of 328
3 mm and a temperature of 21
6°C, and in the winter 186
9 mm and 17
2°C, respectively (Fig. 2). 47 55’

48 00’ C an

an

ei

a

48 05’

Quantification of microbial populations in the soil as phosphate solubilizing (Ghosh et al. 2012), phosphatase (Kumar et al. 2012) and amylase producers (Grishko and Syshchikova 2010) can provide important information about organic matter decomposition and the solubilization and mineralization of the soil organic compounds. However, many of the relationships between microbial communities and biochemical activities are recognized, but many of the regulatory mechanisms and drivers are not known. Due to the scarcity of information about the functional role and participation of micro-organisms in coastal ecosystems, the objective of the present study was to analyse seasonal variation in microbial populations and the activities of the mangrove soil compared with the restinga and Atlantic Forest soils from Cardoso Island.

25 05’

Sao Paulo

MR F

Cardoso Island 25 10’

lan

Parana

At

25 15’

tic

Oc

ea

n

Brazil

Scale 0

852

2000n

Figure 1 Sampling sites of Cardoso Island, SP, Brazil. M, Mangrove, R, Restinga and F, Atlantic forest.

Journal of Applied Microbiology 116, 851--864 © 2013 The Society for Applied Microbiology

400

15

300

10

200

5

100

0

0

Se

Au g

Ju l

Ju n

Ap

us pt t em be r O ct ob N ov er em be D r ec em be r

20

y

500

e

25

ril M ay

600

Ja nu a Fe ry br ua ry M ar ch

Figure 2 Monthly variation in precipitation (♦) and air temperature (●) during the year 2011. ( ) Months of soil samples collection.

30

Precipitation (mm)

Microbial populations of mangrove

Temperature (°C)

B. Pupin and E. Nahas

salinity was determined in aqueous extract, using the ratio 1 : 5 (v:v) (Table 1).

Soil sampling Soil samples were collected in two seasons, summer and winter, in mangrove (Sulfaquent), restinga (Spodosol) and Atlantic forest (Inceptisol) areas. In each area, five samples were taken randomly at depths of 0–2, 2–5 and 5–10 cm. Each sample consisted of three subsamples that were pooled to form a composite sample. Soil samples were taken using a Dutch auger in the restinga and Atlantic forest areas. In the mangrove area, trenches were opened and the samples were collected with a stainless steel spatula (sterilized). The samples were transported to the laboratory in plastic bags under refrigeration, sieved through a 4-mm sieve to remove the plant material and stored at 4°C until microbiological analysis. Part of the each sample was air-dried for chemical analysis. The

Microbiological analysis The number of colony-forming units (CFU) was counted by adding 10 g of wet soil in 95 ml 0
1% (w/v) sodium pyrophosphate. After serial dilutions, aliquots of soil suspensions were transferred to flasks containing culture media for micro-organism counts: Bunt and Rovira (1955), pH 7
4 for total and sporulating bacteria (dilutions of the suspensions were preheated in a water bath at a temperature of 80–85°C for 10 min before transfer to culture medium); TSA (Tryptic Soya Agar) supplemented with 5 lg ml 1 crystal violet for Gram-negative bacteria; starch casein (Kuster and Williams 1964) plus

Table 1 Chemical and physical characteristics of the mangrove, restinga and Atlantic forest soils Mangrove

Restinga

Atlantic forest

Depth

(cm)

0–2

2–5

5–10

0–2

2–5

5–10

0–2

2–5

5–10

pH OM Moisture Salinity P resin K+ Ca2+ Mg2+ H+ + Al3+ SB CEC V Texture

g kg 1 % mS cm mg kg (*) (*) (*) (*) (*) (*) %

4
6 27 45
0 11
62 9 7
6 45 66 52 118
6 170
6 70 Sandy loam

4
5 35 47
4 10
82 7 8
2 37 67 64 112
2 176
2 64 Sandy loam

4
5 36 45
8 10
83 7 7
9 39 67 72 113
9 185
9 61 Sandy loam

4
6 5 3
4 0
02 2 0
2 4 3 13 7
2 20
2 36 Sand

4
6 4 4
0 0
02 2 0
3 3 2 13 5
3 18
3 29 Sand

4
5 4 4
5 0
02 2 0
2 3 2 12 5
2 17
2 30 Sand

4
3 42 34
8 0
12 18 0
9 11 13 80 24
9 104
9 24 Sandy loam

4
4 47 35
5 0
08 21 1
4 17 16 88 34
4 122
4 28 Sandy loam

4
1 34 34
0 0
07 16 0
8 9 8 88 17
8 105
8 17 Sandy loam

1 1

OM, organic matter; SB, sum of bases; CEC, cation exchange capacity; V, base saturation; mS: millisiemens. (*) mmolc dm 3.

Journal of Applied Microbiology 116, 851--864 © 2013 The Society for Applied Microbiology

853

Microbial populations of mangrove

B. Pupin and E. Nahas

antibiotics (Williams and Davies 1965) for actinomycetes; and Martin (1950) medium, pH 5
6, plus 60 lg ml 1 penicillin, 40 lg ml 1 streptomycin and 70 lg ml 1 of rose Bengal for fungi. Most probable number (MPN) counts of nitrifying and denitrifying bacteria were made as proposed by Alexander (1965) and Schmidt and Belser (1982), respectively. Five tubes for each dilution series (10 2–10 8) were used. Denitrifying bacteria were grown under anaerobic conditions (Ulbrich et al. 2004). The media were incubated at 30°C for 24 h (bacteria), 72 h (fungi), 3 days (actinomycetes), 15 days (nitrifying bacteria) or 25 days (denitrifying bacteria). The results were expressed per g dry soil. The presence of nitrifying bacteria was indicated by the appearance of a pink colour resulting from NO2 production. For this, one drop of diazotizing reagent (50 mg of sulfanilamide dissolved in 100 ml 2
4 mol l 1 HCl) and one drop of coupling reagent (30 mg of N-(1-naphthyl)-ethylenediamine in 100 ml 0
12 mol l 1 HCl) were added to 0
1 ml of medium. The presence of denitrifying bacteria was indicated by the change in colour of the medium from green to blue and by active gasification. After incubation, the number of tubes showing growth was recorded and a five-tube table was used for the MPN calculation.

removed, and the plates were placed inverted on another plate containing NH4OH for 15 min. A yellow halo around the colony indicated the production of acid phosphatase. Micro-organisms solubilizing inorganic phosphate were detected using a solubilization medium containing calcium phosphate precipitate. A clear halo around the colony indicated a solubilizer micro-organism. The metabolic activity index of each isolate was determined by the ratio between the diameter of the activity halo and the diameter of the colony measured in mm (Fankem et al. 2006), and the results were grouped according to the interval activity (see Table 2). Statistical analysis The statistical analysis was carried out by variance analysis, following a factorial arrangement 3 9 3 9 2 the factors of which were as follows: three ecosystems (mangrove, restinga and Atlantic forest), three depths (0–2, 2–5 and 5–10 cm) and two periods (summer and winter). Statistical analysis was performed using SAS statistical software (1990). Means were compared using a Tukey test (P < 0
05). Microbial counts were converted to log (1 + x), with x being the number of CFU or MPN g 1 dry soil. A principal component analysis (PCA) was performed for all data using the Statistica program.

Metabolic activity For each ecosystem and season, 40 fungal colonies (except that 60 fungal colonies were selected in summer) and 40 bacterial colonies were selected according to their phenotypic characteristics. The isolates were incubated in PDYA (potato, dextrose, yeast extract and agar) medium at pH 7
0 for fungi and NA (Nutrient Agar) medium at pH 5
5 for bacteria at a temperature of 30°C for 5 days. The isolates were inoculated in triplicate in culture medium and incubated at 30°C for 72 h (bacteria) and 120 h (fungi) to determine metabolic activity. The fungi were inoculated in the centre of three plates and the bacteria on the same plate. PDYA medium was used to detect amylase activity. After micro-organism growth, the medium was flooded with iodine reagent (0
5 g I, 1
0 g KI and distilled H2O to 100 ml) for 10 min. Amylase activity was detected by the formation of a colourless halo around the colony. Micro-organism producers of acid phosphatase were detected using the saline medium (Nahas 2002), correcting the pH to 7
4 for bacteria and 5
6 for fungi. The diameter of the colonies was measured, and the plates were then flooded with a substrate (4 mmol l 1 p-nitrophenylphosphate in 0
1 mol l 1 acetate buffer, pH 5
4) and incubated at 37°C for 90 min. Excess substrate was 854

Results In general, higher characteristics (moisture, salinity, K+, Ca2+, Mg2+, sum of bases, cation exchange capacity and base saturation) were found in the mangrove soil compared with the other soils studied, except for pH values and organic matter and P resin contents at all soil depth (Table 1). The texture ranged from sand (restinga soil) to sandy loam (Atlantic forest and mangrove). Figure 3 shows significant (Tukey, P < 0
05) differences in the counts of the total, sporulating and Gramnegative bacteria with regard to sampling depth and season. Total bacterial counts ranged from 6
6 to 15
2 9 106 g 1 in the Atlantic forest, 5
2 to 9
7 9 105 g 1 in the mangrove and 4
0 to 7
1 9 105 g 1 in the restinga soils (Fig. 3a). The average counts found in the Atlantic forest decreased drastically and significantly (P < 0
05), by 95% in the restinga and 93% in the mangrove soil. In general, a significant decrease was found in the counts between the two seasons (Fig. 3a), with a 79% reduction in the Atlantic forest, 27% in the mangrove and 15% in the restinga soils. The mean CFU number from the surface layer (0–2 cm) decreased (P < 0
05) by 7 and 16% in subsequent layers, 5–10 cm and 2–5 cm, respectively. However, only the counts

Journal of Applied Microbiology 116, 851--864 © 2013 The Society for Applied Microbiology

B. Pupin and E. Nahas

Microbial populations of mangrove

Table 2 Metabolic activity of the micro-organisms isolated in summer from mangroves, restinga and Atlantic forest soils Activity* (%) Ecosystems Amylase producers Mangrove Restinga Atlantic forest Average Mangrove Restinga Atlantic forest Average Acid phosphatase producers Mangrove Restinga Atlantic forest Average Mangrove Restinga Atlantic forest Average Phosphate solubilizing Mangrove Restinga Atlantic forest Average Mangrove Restinga Atlantic forest Average Overall average

Index of metabolic activity† (%)

With

Without

1
01–1
49

Fungi

88
3 86
7 83
3 86
1 50
0 51
7 56
7 52
8

11
7 13
3 16
7 13
9 16
7 15
0 10
0 13
9

11
7 13
3 16
7 13
9 13
3 8
3 8
3 10
0

0
0 0
0 0
0 0
0 3
3 5
0 1
7 3
3

0
0 0
0 0
0 0
0 0
0 1
7 0
0 0
6

5
0 11
7 10
0 8
9 11
7 21
7 30
0 21
1

95
0 88
3 90
0 91
1 55
0 43
3 36
7 45
0

76
7 76
7 66
7 73
3 26
7 38
3 28
3 31
1

13
3 10
0 21
7 15
0 21
7 1
7 6
7 10
0

5
0 6
7 1
7 4
4 6
7 1
7 1
7 3
3

73
3 66
7 83
3 74
4 56
7 58
3 60
0 58
3 50
3

20
0 33
3 15
0 22
8 10
0 8
3 6
7 8
3 32
5

20
0 33
3 13
3 22
2 6
7 6
7 5
0 6
1 26
1

5
0 0
0 3
3 2
8 1
7 1
7 0
0 1
1 5
4

1
7 0
0 0
0 0
6 1
7 0
0 1
7 1
1 1
7

Bacteria

Fungi

Bacteria

Fungi

Bacteria

1
50–3
00

>3
01

Isolates

*Percentage of isolates with or without metabolic activity. †Relationship between the halo diameter of activity and the colony diameter.

found in the winter in general decreased significantly (P < 0
05) from the 0–2 cm layer to other layers (Fig. 3a). The variation in sporulating bacteria counts followed the same tendency as total bacteria. The CFU number ranged from 3
6 to 5
1 9 105 g 1, 2
3 to 3
9 9 04 g 1 and 1
6 to 2
2 9 104 g 1 in the Atlantic forest, mangrove and restinga soils, respectively (Fig. 3b). The mean CFU found in the Atlantic forest soil also decreased (P < 0
05) by 95 and 93% in the restinga and mangrove soils, respectively. In general, counts decreased significantly from summer to winter (Fig. 3b), by 19% in average. Means for the sporulating bacterial counts decreased (P < 0
05) by 6 and 14% from the surface to the deeper layer, respectively. However, Fig. 3b shows that these decreases were significant only in the restinga soil during the winter, from the superficial to deeper layers. Gram-negative bacteria counts ranged from 3
5 to 9
1 9 105 g 1 in the Atlantic forest, 2
5 to 3
0 9 104 g 1 in the mangrove and 1
5 to 1
8 9 04 g 1 in the restinga

soils (Fig. 3c). Similarly, the restinga and mangrove soils showed a 97 and 96% reduction in Gram-negative bacteria (CFU) from the Atlantic forest soil. The mean value found in summer decreased (P < 0
05) by 40% compared with winter. Nevertheless, this reduction was only significant in the Atlantic forest soil (Fig. 3c). Mean Gramnegative bacterial counts were reduced (P < 0
05) by 10 and 28% from 0–2 to 2–5 and 5–10 cm soil depth, respectively. Significant (P < 0
05) differences were also found in the denitrifying and nitrifying bacterial counts relating to deep soil and seasons (Fig. 4). Denitrifying bacteria counts varied from 2
0 to 6
6 9 104 g 1 in the Atlantic forest, 1
2 to 10
1 9 104 g 1 in the mangrove and 1
1 to 75
7 9 102 g 1 in the restinga soils (Fig. 4a). Means found in the Atlantic forest and mangrove soils were similar (P < 0
05), being 12
8 and 11
6 times higher than found in the restinga soil. The MPN of denitrifying bacteria decreased (P < 0
05) in the winter compared with the summer (Fig. 4a) but, in contrast to the counts found

Journal of Applied Microbiology 116, 851--864 © 2013 The Society for Applied Microbiology

855

Microbial populations of mangrove

Total bacteria CFU x 106 g–1 dry soil

(a)

B. Pupin and E. Nahas

18 Ab

15

Ab

Bb

12 Aa 9

Aa Aa

6 3 Aa

Ab

Bb ABa

Ba

Cb

Ab Aa

Aa Bb

0 (b)

Aa Bb

6

Sporulating bacteria CFU x 105 g–1 dry soil

Aa

Aa

5

Aa

Ab

Ab

4

Bb

3 2 1

ABa Aa Ab Ab

Ba

Ab

Ab

Aa

Aa

Bb

Aa

Bb

(c)

10

Gram-negative bacteria CFU x 105 g–1 dry soil

0

8

Aa Aa Ba Ab

6

Ab 4

Bb

2 Aa 0

Aa

0-2 cm

Aa Aa

Ab Aa

2 - 5 5 - 10 cm cm Mangrove

Aa Aa 0-2 cm

Aa Ba

ABa Aa

2 - 5 5 - 10 cm cm Restinga

0-2 2 - 5 5 - 10 cm cm cm Atlantic forest

in other soils, increased on average by 17
5 times in the restinga soil. The NMP denitrifying bacteria increased (P < 0
05) on average by 44 and 166% from the 0–2 cm layer to the 2–5 and 5–10 cm layers, respectively. Counts found in the summer decreased in the following order: 5–10 cm > 2–5 cm > 0–2 cm, but in the winter, this decrease varied between the different soils (Fig. 4a). Populations of nitrifying bacteria ranged from 1
8 to 20
1 9 105 g 1 in the Atlantic forest, 2
9 to 9
2 9 104 g 1 in the mangrove and 2
7 to 5
8 9 103 g 1 in the restinga soils (Fig. 4b). On average, a 99 and 90% decrease (P < 0
05) from the Atlantic forest to the restinga and mangrove soils, respectively, and by 62% from summer to winter were found. The reduction in counts from summer 856

Figure 3 Total (a), sporulating (b) and Gramnegative (c) bacteria populations of mangrove, restinga and Atlantic forest soils found in summer (■) and winter (□). Means followed by the same letter, uppercase (depth) and lower (season), do not differ by Tukey test at 5%.

to winter was only significant in the 0–2 and 5–10 cm layers in the Atlantic forest soil and in the 0–2 cm layer of the mangrove soil (Fig. 4b). A significant reduction in the means by 56 and 70% was found in the NMP of the surface layer to the lower layers of the soil. While this reduction was found in the Atlantic forest soil, MPN counts increased in the deeper layer in the mangrove and restinga soils (Fig. 4b). The effect of deep soil and season on actinomycetes and fungi counts is shown in Fig. 5. Actinomycetes counts ranged from 5
1 to 8
9 9 104 g 1, 1
4 to 1
9 9 103 g 1 and 1
2 to 1
6 9 103 g 1 in the Atlantic forest, mangrove and restinga soils, respectively (Fig. 5a). Drastic reductions (98%) in the mean counts were found

Journal of Applied Microbiology 116, 851--864 © 2013 The Society for Applied Microbiology

B. Pupin and E. Nahas

Microbial populations of mangrove

Denitrifying bacteria CFU x 104 g–1 dry soil

(a)

12 Aa

10 8

Ab Ab

6

ABa Ba

Ca 2

Bb

Ab

ABb

Ba

4

Bb

Bb Cb

Ba

Bb

Aa

Ab

Aa

0

Figure 4 Denitrifying (a) and nitrifying (b) bacteria populations of mangrove, restinga and Atlantic forest soils found in summer (■) and winter (□). Means followed by the same letter, uppercase (depth) and lower (season), do not differ by Tukey test at 5%.

Nitrifying bacteria CFU x 105 g–1 dry soil

(b)

21

Aa

18 15 12 9 Ab

Ba

6 3 0

Ca Aa Aa

Ba Bb 0-2 cm

Aa Aa

2 - 5 5 - 10 cm cm Mangrove

from the Atlantic forest to the mangrove and restinga soils. No significant variations between counts were found between summer and winter or between soil layers, except from 0 to 2 cm to deeper layers in the Atlantic forest (Fig. 5a). However, mean counts decreased (P < 0
05) by 28 and 40% with increasing soil depth. The number of fungi CFUs varied from 4
2 to 7
1 9 104 g 1, 1
1 to 2
3 9 103 g 1 and 6
1 to 13
4 9 102 g 1 in the Atlantic forest, restinga and mangrove soils, respectively (Fig. 5b). Population means decreased (P < 0
05) by 97–98% from forest to restinga and mangrove soils, and by 28% from winter to summer. Counts found in winter were higher (P < 0
05) than in summer, except in the 2–5 cm layer of the mangrove and 5–10 cm layer of Atlantic forest soils (Fig. 5b). Means of the fungi numbers from the topsoil layer decreased by 17 and 24% in the deeper layers, respectively. However, count variations were only found in the 2–5 and 5–10 cm layers (Fig. 5b). The principal component analysis plot (PCA) explained 91
1% of microbiological behaviour (11 variables): the first principal component, PC1, explained 67
1%, and the second PC2 explained 24
0% of the variation. Three distinct groups were found: Atlantic forest, mangrove and restinga (Fig. 6). The main variables

Bb ABa Ba Aa Bb Ab ABa 0-2 2 - 5 5 - 10 cm cm cm Restinga

Cb

0-2 2 - 5 5 - 10 cm cm cm Atlantic forest

selected by PCA for the Atlantic forest soil were total, sporulating, Gram-negative, nitrifying bacteria, actinomycetes and fungi. pH was influenced by the resting soil, but denitrifying bacteria and the organic matter content, moisture and salinity were influenced by the mangrove soil. PCA for winter was similar to summer, explaining 90% of the accumulated variance: 68% was explained by component 1 and 22% by component 2 (Fig. 7). Moreover, the same trend was observed in winter, whereby the PCA clustered the variables into Atlantic forest, mangrove and restinga groups. Of the total bacteria isolates sampled in summer, metabolic activity was shown in 93% from mangrove, 78% from restinga and 58% from Atlantic Forest soils (data not included). The proportions found in winter were 98, 95 and 98%, respectively. From the fungi isolates, the percentages found were 97, 93 and 95% in summer and 90, 88 and 95% in winter, respectively (data not included). Of the fungi isolates sampled in summer, 14% on average had amylase activity, 91% acid phosphatase and 26% solubilized inorganic phosphate (Table 2). The proportions found for bacteria were 21, 68 and 13%, respectively. The isolates had more than one metabolic activity.

Journal of Applied Microbiology 116, 851--864 © 2013 The Society for Applied Microbiology

857

Microbial populations of mangrove

B. Pupin and E. Nahas

10

(a) Actinomycetes CFU x 104 g–1 dry soil

A A 8 AB AB 6

B B

4 2 A A

AA

AA

AA

AA

AA

0 8

(b) Fungi CFU x 104 g–1 dry soil

Aa 6

ABa Aa

Ab

Ba Aa

4

2

0

Aa Ab 0-2 cm

Ba

Ba

Aa Ab 2 - 5 5 - 10 cm cm Mangrove

Ba ABa Ab Ab Ab 0-2 2-5 5 - 10 cm cm cm Restinga

Figure 5 Actinomycetes (a) and fungi (b) populations of mangrove, restinga and Atlantic forest soils found in summer (■) and winter (□). Means followed by the same letter, uppercase (depth) and lower (season), do not differ by Tukey test at 5%.

Aa

0-2 2-5 5 - 10 cm cm cm Atlantic forest

2

CP 2: 23,97%

1

R_2 R_3 R_1

ACT FT F_3 F_1 BN BGN BE BT F_2

pH 0

M_1 M_2

–1

OM BD

M_3 Salinity –2 –2

–1

Moisture 0

1

CP 1: 67,13%

Thus, data included in Table 2 show that a higher proportion of fungi and bacteria had a metabolic activity index in the range of 1
01–1
49 (mean 30%), followed by 1
50–3
00 (7%) and >3
01 (1%). 858

2

Figure 6 Graph biplot of principal component analysis (PC1 9 PC2) during the summer season. M, Mangrove, R, Restinga, and F, Atlantic forest. M_1, R_1 and F_1 correspond to the 0–2 cm depth; M_2, R_2 and F_2 correspond to the 2–5 cm depth; M_3, R_3 and F_3 correspond to the 5–10 cm; BT–total bacteria, BE–sporulating bacteria, BGN–Gram-negative bacteria, BN–nitrifying bacteria, BD–denitrifying bacteria, ACT–actinomycetes, FT–total fungi and OM–Organic matter.

During winter, the proportions of the fungi having amylase, acid phosphatase and inorganic phosphate-solubilizing activities were 27, 88 and 29% and 45, 89 and 23% of bacteria, respectively (Table 3). The mean index

Journal of Applied Microbiology 116, 851--864 © 2013 The Society for Applied Microbiology

B. Pupin and E. Nahas

Microbial populations of mangrove

2

CP 2: 22,02%

1

R_1 R_3 R_2 F_1 ACT BT FT F_2 BE F_3 BGN BN

pH 0

M_1 M_2

–1

BD OM

M_3 Salinity Figure 7 Graph biplot of principal component analysis (PC1 9 PC2) during the winter season. Abbreviations see Figure 6.

–2 –2

given in Table 3 shows that the proportion of fungi and bacteria with metabolic activity was 31, 19 and 2%, respectively. Discussion Micro-organism populations This research showed that populations of micro-organisms varied according to the influence of the ecosystems studied, predominating in the Atlantic forest in relation to mangrove and restinga soils. This variation in the counts can be attributed both to the ecosystem characteristics, such as soil (Table 1) and vegetation (see Introduction), and to the environment in which they are found, including the maritime influence on the mangroves. Variation in the microbial community in the Atlantic forest soil as a result of seasonal, physical and physicochemical factors is also reported by Jung et al. (2012). Therefore, physicochemical soil characteristics (Table 1) may have contributed greatly to the differences in the counts observed in this study. Except for organic matter and P resin, the chemical characteristics of the mangrove soil studied were higher than that from Atlantic forest soil, but the high salinity (Table 1) might have restricted the growth of microbial populations. The soils may be considered strongly acidic, given the pH varied on average from 4
1 to 4
6 (Table 1). These results were similar to others reported, from 3
1 to 5
5 in mangrove (Effiong and Ayolagha 2010), 3
7 to 4
7 in restinga (Gomes et al. 2007) and 4
5 to 4
6 in the

–1

Moisture 0

1

2

CP 1: 67,67%

Atlantic forest soils (Valpassos et al. 2007). The organic matter content of restinga, mangrove and Atlantic forest soils was 4
3, 32
7 and 41
0 g kg 1 (Table 1), considered arbitrarily very low, medium and high, respectively. Studies have shown that the organic matter content of these ecosystems varies widely with location, but generally is medium to high. Organic carbon contents of 23
8– 102
8 g kg 1 were reported in China (Zhang et al. 2009), 2
9–25
6 g kg 1 in India (Saravanakumar et al. 2009) and 82
0–185
6 g kg 1 in Brazil mangrove soils (PradaGamero et al. 2004). Variations from 2
0 to 13
6 g kg 1 in restinga (Oliveira et al. 2010) and 30
3 g kg 1 in the Atlantic Forest soils (Valpassos et al. 2007), both from Brazil, have been found. These characteristics, together with the biological activity of plant species, can control the composition and activity of microbial communities, determining their conditions of survival and growth (Agnelli et al. 2004). In addition to soil characteristics, variations in the amount of litter in the ecosystems studied can differ, influencing the microbial counts between soils (Leff et al. 2011). The forests are recognized as being the most productive ecosystem, providing large amounts of organic matter derived from the higher litter amount, and responsible for the retention of large numbers of compounds and important sources of nutrients returned to the soil (Staelens et al. 2011; Wood et al. 2012). Mangroves, despite harbouring only a few tree species, daily produce large amounts of litter (Mukherjee et al. 2012); however, much of this is carried away by the tides, which also influence the high moisture content of the soil.

Journal of Applied Microbiology 116, 851--864 © 2013 The Society for Applied Microbiology

859

Microbial populations of mangrove

B. Pupin and E. Nahas

Table 3 Metabolic activity of microorganisms isolated in winter from mangroves, restinga and Atlantic forest soils Activity* (%) Ecosystems Amylase producers Mangrove Restinga Atlantic forest Average Mangrove Restinga Atlantic forest Average Acid phosphatase producers Mangrove Restinga Atlantic forest Average Mangrove Restinga Atlantic forest Average Phosphate solubilizing Mangrove Restinga Atlantic forest Average Mangrove Restinga Atlantic forest Average Overall average

Index of metabolic activity† (%)

Isolates

With

Without

1
01–1
49

1
50–3
00

>3
01

Fungi

75
0 67
5 77
5 73
3 57
5 57
5 50
0 55
0

25
0 32
5 22
5 26
7 42
5 42
5 50
0 45
0

22
5 17
5 15
0 18
3 12
5 15
0 20
0 15
8

2
5 12
5 7
5 7
5 30
0 22
5 25
0 25
8

0
0 2
5 0
0 0
8 0
0 5
0 5
0 3
3

10
0 17
5 7
5 11
7 5
0 20
0 7
5 10
8

90
0 82
5 92
5 88
3 95
0 80
0 92
5 89
2

80
0 67
5 85
0 77
5 35
0 27
5 30
0 30
8

10
0 15
0 7
5 10
8 52
5 45
0 57
5 51
7

0
0 0
0 0
0 0
0 7
5 7
5 5
0 6
7

82
5 55
0 75
0 70
8 82
5 72
5 75
0 76
7 49
7

17
5 45
0 25
0 29
2 17
5 27
5 25
0 23
3 50
3

15
0 45
0 22
5 27
5 10
0 20
0 22
5 17
5 31
3

2
5 0
0 2
5 1
7 5
0 7
5 2
5 5
0 17
1

0
0 0
0 0
0 0
0 2
5 0
0 0
0 0
8 1
9

Bacteria

Fungi

Bacteria

Fungi

Bacteria

*Percentage of isolates with or without metabolic activity. †Relationship between the halo diameter of activity and the colony diameter.

Restingas are characterized by the formation of a thin layer of litter (Martins et al. 2008). Subject to the influence of sea currents, such as in the mangroves, the presence of bacteria in the environment can ensure nutrient cycling and therefore greater sustainability in extreme conditions. Therefore, quantification of these groups of bacteria can give an insight into the transformations in the studied soils. Gram-positive bacteria, and among these the genus Bacillus, were the predominant group in mangroves from Suva, Fiji Islands, due to its characteristic ability to form endospores (Kumar et al. 2007) and therefore to its greater sustainability in the ecosystem in extreme conditions. Soil Gram-negative bacteria have functions both in the decomposition of organic substances and the transformation of N-chemical compounds, as nitrifying, denitrifying and N2-fixing bacteria (Mishra et al. 2012). Populations of total, Gram-negative and sporulating bacteria from mangrove soils were lower than those found in the Atlantic forest and higher than in the restinga soil. The 860

number of total bacteria from the mangrove soils studied was similar to that reported in the mangrove of Marambaia, Brazil, at 2
0 9 107 g 1 of dry soil (Maciel-Souza et al. 2006), and larger than that found in the mangrove from Suva, Fiji, at 1
07–1
35 9 104 g 1 dry soil (Kumar et al. 2007). However, unlike total bacteria counts, the frequency of sporulating bacteria and Gram-negative bacteria was less than that found in the mangrove forest of and 44– Odisha, India, at 11–21
7 9 105 5 1 175 9 10 CFU g dry soil, respectively (Mishra et al. 2012). The results from the mangrove in southern China showed that the frequency of actinomycetes and fungi was smaller than that for total bacteria (Lu et al. 2008). In our study, we found a similar response, with a drastic decrease in the actinomycetes and fungi populations, corresponding to less than 0
2% of the total bacteria. Similarly, in the restinga and Atlantic forest soils, the counts were less than 0
6% for each. Large variations in the actinomycetes counts have been observed, depending on

Journal of Applied Microbiology 116, 851--864 © 2013 The Society for Applied Microbiology

B. Pupin and E. Nahas

the ecosystem studied. So, in the mangroves of Tamil Nadu, India, the number of CFUs ranged from 2 to 19 9 106 g 1 dry soil (Ravikumar and Suganthi 2011). These results can be considered fairly high when compared to those for mangrove and restinga soils, but the results found in the Atlantic forest soil were similar to those from the mangrove of Manakkudi, India, where a value of 1
2–32
4 9 104 g 1 soil was found (Ravikumar et al. 2011). The fungi frequency was very restricted in the mangrove soil when compared to the Atlantic forest or to the results found for the mangrove from Suva, Fiji (Kumar et al. 2007), of 0
3–6 9 103 g 1 and the restinga at 4
6– 90 9 103 g 1 dry soil (Fraga et al. 2010). This result is intriguing, because the chemical composition of mangrove soil and the moisture content were higher than those found for other soils (Table 1). This probably resulted from flooding by saline sea water (Oliveira et al. 2010), restricting the growth of fungi. Nitrification occurs in the soil through various chemical reactions performed by ammonifiers and nitrifying bacteria, which catalyse the production of NH4+ from N-organic compounds and oxidation producing nitrate, respectively (Sher et al. 2012). Our results showed that the abundance of nitrifying bacteria in mangrove soil was lower than for the Atlantic forest, but was 17
5 times that for the restinga, showing the importance of these bacteria in mangrove soil (Das et al. 2012). In general, higher populations of micro-organisms were found in the surface layer compared with the deeper layers of the soil, with the exception of denitrifying bacteria that increased with soil depth. According to Krishna et al. (2012), it is to be expected that the number of CFUs will vary with sampling depth due to reduced exposure to sunlight and the decreased organic matter content in the lower layers of the soil. Denitrifying bacteria catalyse the conversion of nitrate via nitrite in NO, N2O and N2, which are lost to the atmosphere under anaerobic conditions and in the presence of organic matter as an energy source (Barta et al. 2010). The frequency of denitrifying bacteria in the order of 104 g 1 dry soil was similar between mangrove and Atlantic forest soils, and greater than under restinga soil. Our results are consistent with previous results found in an estuarine setting from northern Taiwan (Fan et al. 2006). The mangrove and Atlantic forest soils have high organic matter content (Table 1) and increased populations of denitrifying bacteria in the deeper layers, which suggests a gradual limitation on the amount of oxygen. Season variations have been considered to be a determining factor in the frequency of soil microbial populations (Oller Costa et al. 2012). The largest population growth of bacteria was observed in summer compared

Microbial populations of mangrove

with winter (Fig. 2) in all ecosystems studied, with the exception that the fungi were inexplicably contrary to this result. The different climatic conditions caused by the seasons, with higher air temperature and rainfall during summer, alter the relationships that occur between the soil and vegetation, especially in the subtropics, and consequently influence the growth of soil micro-organisms. Metabolic activity of isolates Micro-organisms synthesize and secrete enzymes and metabolites in the environment to obtain nutrients for their growth. They thus provide a function in the decomposition of organic matter and soil nutrient recycling. Using solid culture media, such as those used in this study, allows the detection of specific enzymes and metabolites and the differentiation and rapid screening of large populations of micro-organisms. Few studies have been conducted to determine the metabolic activity of soil micro-organisms from the mangrove compared with other ecosystems. Knowledge of the activity of micro-organisms in an environment that is subject to maritime influence, such as the mangrove, is relevant. The microbial cultures exhibited microbial activity of the selected metabolic tests based on the assays of this study. Of the total bacterial isolates, 58– 93% showed metabolic activity in the summer and 95–98% in the winter, and the values were 93–97% and 90–95% for fungi isolates, respectively (data not included). These results show that a significant number of micro-organisms perform transformations that enable the sustainability of ecosystems studied. Moreover, bacterial and fungal isolates had one, two or even three metabolic activities. Few micro-organisms seem to be able to degrade starch, the most important organic compound reserve in green plants. The amylolytic enzymes hydrolyse a-glucosidic bonds from amylose and amylopectin (RondanSanabria et al. 2012). The proportions of amylolytic isolates were 15–25% for the bacteria and 12–17% for fungi. Little difference was found between the numbers of isolates for the different soils studied and seasons. Phosphorus has been well studied because it is one of the most limiting elements in the mangrove (Reef et al. 2010), and only 0
1% of total soil P is available to plants. The mechanisms used by micro-organisms to obtain P involve the solubilization of inorganic phosphates and the synthesis and secretion of phosphatases. Most inorganic phosphates in acid soils, such as in our study, are aluminium or iron salts. Therefore, the presence of inorganic phosphate-solubilizing micro-organisms is important for providing soluble P to plants. Our results show that the solubilizing activity of inorganic P was detected in 10–15% of bacteria and 17–33% of fungi.

Journal of Applied Microbiology 116, 851--864 © 2013 The Society for Applied Microbiology

861

Microbial populations of mangrove

B. Pupin and E. Nahas

Phosphatases are a group of hydrolases that catalyse the release of phosphate from esters and anhydrides of phosphoric acid (Olsson et al. 2011). Bacterial producers of acid phosphatase ranged from 55 to 83% and 88 to 95% for fungi. Under different systems of soil fertilization, it has been found that 58% of bacteria and 85% of fungi show acid phosphatase activity (Nahas 2002). Therefore, the high percentage of micro-organisms that produce acid phosphatase, as determined in the present study, suggests that the P content was fairly limiting in the studied soils in stimulating the secretion of this enzyme. In conclusion, we showed that the higher chemical characteristics, except P resin and organic matter, were found in the mangrove soil, but that microbial counts were lower than those found in Atlantic forest soil, probably due to the influence of high soil salinity. The low moisture and organic matter content of restinga soil led to the lowest microbial counts. Shifts in the soil microbial populations found in this study were directly attributed to the ecosystem characteristics. The PCA biplot features three clusters that provided a clear picture of the influence of ecosystems on microbial counts and chemical properties. We conclude that the trends identified by multivariate analysis were similar to the univariate analysis and that the microbiological and chemical variables were more susceptible to the effect of soil characteristics than to seasonal variations. The topsoil tends to accumulate organic matter and nutrients that stimulate the increase in microbial populations. Consequently, decreased fertility with increasing depth of soil causes a reduction in microbial populations. In contrast, there is an increase in the denitrifying bacteria counts, probably favoured by the absence of O2. Except for the populations of actinomycetes and fungi, the hot and humid climate in summer led to an increase in the microbial counts in comparison with winter. Little variation between soils and station was found for the number of isolates showing metabolic activity, ranging from 88 to 98%, except for the restinga and Atlantic Forest soils during the summer. The production of acid phosphatase by the bacteria and fungi isolates was more frequent than that of amylase or inorganic phosphate solubilization. The results of the present study also suggest the need of further study to understand the biogeochemical activities related to C, N and P cycles in the mangrove under consideration in relation to other ecosystems. Acknowledgements We thank CAPES (B. Pupin) and CNPq (E. Nahas) for fellowships and Forestry Institute (Department of Environment of the Sao Paulo State) for research support in the Cardoso Island Park. 862

Conflict of interest The authors declare no conflict of interest. References Agnelli, A., Ascher, J., Corti, G., Ceccherini, M., Nannipieri, P. and Pietramellara, G. (2004) Distribution of microbial communities in a Forest soil profile investigated by microbial biomass, soil respiration and DGGE of a total and extracellular DNA. Soil Biol Biochem 36, 859–868. Alexander, M. (1965) Denitrifying bacteria. In Methods of Soil Analysis, Part 2. Chemical and Microbiological Properties. eds Page Miller, A.L. and Keeney, R.H. pp. 1485–1489. Madison, WI: American Society Agronomy. Barta, J., Melichova, T., Vanek, D., Picek, T. and Santruckova, H. (2010) Effect of pH and dissolved organic matter on the abundance of nirK and nirS denitrifiers in spruce forest soil. Biogeochem 101, 123–132. Bunt, J.S. and Rovira, A.D. (1955) Microbiological studies of some subantartic soils. J Soil Sci 6, 119–128. Das, S., De, M., Ray, R., Chowdhury, C., Jana, T.K. and De, T.K. (2012) Microbial Ecosystem in Sunderban Mangrove Forest Sediment, North-East Coast of Bay of Bengal, India. Geomicrobiol J 29, 656–666. Effiong, G.S. and Ayolagha, G.A. (2010) Characteristics, constraints and management of mangrove soils for sustainable crop production. Electron J Environ Agric Food Chem 9, 977–990. EMBRAPA (Empresa Brasileira de Pesquisa Agropecuaria) (2012) A aquicultura e a atividade pesqueira. Available at: http:// www.cnpma.embrapa.br (accessed: 25 December 2012). Fan, L.F., Shieh, W.Y., Wu, W.F. and Chen, C.P. (2006) Distribution of nitrogenous nutrients and denitrifiers strains in estuarine sediment profiles of the Tanshui River, northern Taiwan. Estuar Coast Shelf Sci 69, 543–553. Fankem, H., Nwaga, D., Deubel, A., Dieng, L., Merbach, W. and Etoa, F.X. (2006) Occurrence and functioning of phosphate solubilizing microorganisms from oil palm tree (Elaeis guineensis) rhizosphere in Cameroon. Afr J Biotechnol 5, 2450–2460. Faoro, H., Alves, A.C., Souza, E.M., Rigo, L.U., Cruz, L.M., Al-Janabi, S.M., Monteiro, R.A., Baura, V.A. et al. (2010) Influence of soil characteristics on the diversity of bacteria in the Southern Brazilian Atlantic Forest. App Environ Microbiol 76, 4744–4749. Fraga, M.E., Pereira, M.G. and Souza, F.A. (2010) Soil mycobiota in a dune area in the municipality of Restinga da Marambaia, State of Rio de Janeiro, Brazil. Floresta e Ambient 17, 30–36. Ghosh, U., Subhashini, P., Dilipan, E., Raja, S., Thangaradou, T. and Kannan, L. (2012) Isolation and characterization of phosphate-solubilizing bacteria from sea grass rhizosphere soil. J Ocean Univ China 11, 86–92.

Journal of Applied Microbiology 116, 851--864 © 2013 The Society for Applied Microbiology

B. Pupin and E. Nahas

Giri, C., Ochieng, E., Tieszen, L.L., Zhu, Z., Singh, A., Loveland, T., Masek, J. and Duke, N. (2011) Status and distribution of mangrove forests of the world using earth observation satellite data. Global Ecol Biogeogr 20, 154–159. Gomes, F.H., Vidal-Torrado, P., Macıas, F., Gherardi, B. and Otero, X.L. (2007) Soils under restinga vegetation on the Cardoso Island (SP). 1 - Characterization and classification. Rev Bras Ci Solo 31, 1563–1580. Grishko, V.N. and Syshchikova, O.V. (2010) Structural and functional properties of actinomycetal communities in chernozems and saline soils of Ukraine. Eurasian Soil Sci 43, 202–209. Habibur, R.M., Rafiqul, I.M., Jahiruddin, M., Puteh, A.B. and Monjurul, A.M.M. (2013) Influence of organic matter on nitrogen mineralization pattern in soils under different moisture regimes. Int J Agr Biol 15, 55–61. Hafich, K., Perkins, E.J., Hauge, J.B., Barry, D. and Eaton, W.D. (2012) Implications of land management on soil microbial communities and nutrient cycle dynamics in the lowland tropical forest of northern Costa Rica. Trop Ecol 53, 215–224. Jung, J., Yeom, J., Han, J., Kim, J. and Park, W. (2012) Seasonal changes in nitrogen-cycle gene abundances and in bacterial communities in acidic forest soils. J Microbiol 50, 365–373. Kathiresan, K. and Bingham, B.L. (2001) Biology of mangroves and mangrove ecosystems. Adv Mar Biol 40, 81–251. Krishna, M.P., Rinoy, V. and Mohamed, H.A.A. (2012) Depth wise variation of microbial load in the soils of midland region of Kerala: a function of important soil physicochemical characteristics and nutrients. Indian J Edu Inform Manage 1, 126–129. Kumar, S., Hatha, A.A.M. and Christi, K.S. (2007) Diversity and effectiveness of tropical mangrove soil microflora on the degradation of polythene carry bags. Int J Trop Biol 55, 777–786. Kumar, G., Kanaujia, N. and Bafana, A. (2012) Functional and phylogenetic diversity of root-associated bacteria of Ajugabracteosa in Kangra valley. Microbiol Res 167, 220–225. Kuster, E. and Williams, S.T. (1964) Selection of media for isolation of Streptomycetes. Nature 202, 928–929. Lacerda, L.D., Araujo, D.S.D. and Maciel, N.C. (1993) Dry coastal ecosystems of the tropical Brazilian coast. In Dry Coastal Ecosystems: Africa, America, Asia, Oceania ed. Van der Maarel, E., pp 477–493. Amsterdam: Elsevier. Leff, J.W., Nemergut, D.R., Grandy, A.S., O’Neil, S.P., Wickings, K., Townsend, A.R. and Cleveland, C.C. (2011) The effects of soil bacterial community structure on decomposition in a tropical rain forest. Ecosystems 15, 284–298. Lu, J.K., Kang, L.H., Chen, J., Lu, C.O., Huang, B.L. and Jiang, Y.G. (2008) Research on soil microbe and the relativity between microbe and soil chemical factor of mangrove forest in south China. Forest Res 21, 523–527.

Microbial populations of mangrove

Maciel-Souza, M.D., Macrae, A., Volpon, A.G.T., Ferreira, P.S. and Mendonca-Hagler, L.C. (2006) Chemical and microbiological characterization of mangrove sediments after a large oil-spill in Guanabara Bay, RJ, Brazil. Braz J Microbiol 37, 262–266. Martin, J.P. (1950) Use of acid, rose bengal, and streptomycin in the plate method for estimating soil fungi. Soil Sci 69, 215–232. Martins, S.E., Rossi, L., Sampaio, P.S.P. and Magenta, M.A.G. (2008) Floristic characterization of “restinga” plant communities at Bertioga, S~ao Paulo State, Brazil. Acta Bot Bras 22, 249–274. McGuire, K.L., Fierer, N., Bateman, C., Treseder, K.K. and Turner, B.L.(2012) Fungal community composition in neotropical rain forests: the influence of tree diversity and precipitation. Microb Ecol 63, 804–812. Mishra, R.R., Swain, M.R., Dangar, T.K. and Thatoi, H. (2012) Diversity and seasonal fluctuation of predominant microbial communities in Bhitarkanika, a tropical mangrove ecosystem in India. Rev Biol Trop 60, 909–924. Mukherjee, J., Ray, S. and Ghosh, P.B. (2012) A system dynamic modeling of carbon cycle from mangrove litter to the adjacent Hooghly estuary, India. A modelling study. Procedia Environ Sci 13, 391–413. Nahas, E. (2002) Soil microorganisms phosphatase producers in different agricultural systems. Bragantia 61, 267–275. Oliveira, A.P., Ker, J.C., Silva, I.R., Fontes, M.P.F., Oliveira, A.P. and Neves, A.T.G. (2010) Spodosols pedogenesis under barriers formation and sandbank environments in the south of Bahia. Rev Bras Ci Solo 34, 847–860. Oller Costa, P.M., Souza-Motta, C.M. and Malosso, E. (2012) Diversity of filamentous fungi in different systems of land use. Agroforestry Sys 85, 195–203. Olsson, R., Giesler, R., Loring, J.S. and Persson, O. (2011) Enzymatic hydrolysis of organic phosphates adsorbed on mineral surfaces. Environ Sci Technol 46, 285–291. Prada-Gamero, R.M., Vidal-Torrado, P. and Ferreira, T.O. (2004) Mineralogy and physical chemistry of mangrove soils from Iriri River at the Bertioga Channel (Santos, S~ao Paulo State, Brazil). Rev Bras Ci Solo 28, 233–243. Pupin, B., Freddi, O.S. and Nahas, E. (2009) Microbial alterations of the soil influenced by induced compaction. Rev Bras Ci Solo 33, 1207–1213. Ravikumar, S. and Suganthi, P. (2011) Biodiversity of actinomycetes along the south East Coast of Tamilnadu, India. World Appl Sci J 13, 119–124. Ravikumar, S., Fredimoses, M. and Gokulakrishnan, R. (2011) Biodiversity of actinomycetes in Manakkudi mangrove ecosystem, South west coast of India. Ann Biol Res 2, 76–82. Reef, R., Feller, I.C. and Lovelock, C.E. (2010) Nutrition of mangroves. Tree Physiol 30, 1148–1160. Rondan-Sanabria, G.G., Valcarcel-Yamani, B. and FinardiFilho, F. (2012) Effects on starch and amylolytic enzymes during Lepidium meyenii Walpers root storage. Food Chem 134, 1461–1467.

Journal of Applied Microbiology 116, 851--864 © 2013 The Society for Applied Microbiology

863

Microbial populations of mangrove

B. Pupin and E. Nahas

Saravanakumar, A., Rajkumar, M., Sun, J., Serebiah, J.S. and Thivakaran, G.A. (2009) Forest structure of arid zone mangroves in relation to their physical and chemical environment in the western Gulf of Kachchh, Gujarat, Northwest coast of India. J Coast Conservat 13, 217– 234. SAS Institute. (1990) Statistical Analysis System, SAS/STAT User’s Guide (Version 6). 3rd edn. Cary, NC: SAS Institute. Schmidt, E.L. and Belser, L.W. (1982) Nitrifying bacteria. In Methods of Soil Analysis, Part 2. Chemical and Microbiological Properties. eds Page Miller, A.L. and Keenney, R.H. pp. 1027–1042. Madison, WI: American Society Agronomy. Sher, Y., Baram, S., Dahan, O., Ronen, Z. and Nejidat, A. (2012) Ammonia transformations and abundance of ammonia. FEMS Microbiol Ecol 81, 145–155. Silva, F.S., Pereira, D.C., Nu~ nez, L.S., Krepsky, N., Fontana, L.F. and Baptista Neto, J.A. (2008) Bacteriological study of the superficial sediments of Guanabara Bay, RJ, Brazil. Braz J Oceanogr 56, 13–22. Staelens, J., Ameloot, N., Almonacid, L., Padilla, E., Boeckx, P., Huygens, D., Verheyen, K., Oyarz un, C. et al. (2011) Litterfall, litter decomposition and nitrogen mineralization

864

in old-growth evergreen and secondary deciduous Nothofagus forests in south-central Chile. Rev Chilen Hist Nat 84, 125–141. Ulbrich, A.V., Leite, C.R.F., Souza, J.R.P. and Andrade, D.S. (2004) Action of imazapic + imazapyr on purple nutsedge (Cyperus rotundus L.) and denitrifying bacteria in corn. Planta Daninha 22, 577–582. Valpassos, M.A.R., Maltoni, K.L., Cassiolato, A.M.R. and Nahas, E. (2007) Recovery of soil microbiological properties in a degraded area planted with Corymbia citriodora and Leucaena lecocephala. Sci Agri 64, 68–72. Williams, S.T. and Davies, F.L. (1965) Use of antibiotics for selective isolation and enumeration of actinomycetes in soil. J Gen Microbiol 38, 251–261. Wood, T.E., Cavaleri, M.A. and Reed, S.C. (2012) Tropical forest carbon balance in a warmer world: a critical review spanning microbial- to ecosystem- scale processes. Biol Rev 87, 912–927. Zhang, Y.Y., Dong, J.D., Yang, B., Ling, J., Wang, Y.S. and Zhang, S. (2009) Bacterial community structure of mangrove sediments in relation to environmental variables accessed by 16S rRNA gene-denaturing gradient gel electrophoresis fingerprinting. Sci Mar 73, 487–498.

Journal of Applied Microbiology 116, 851--864 © 2013 The Society for Applied Microbiology