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 Composting of Wakame Using Halotolerant Bacteria

 Composting of Wakame Using Halotolerant Bacteria

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290



JING-CHUN TANG ET AL.

Table 3. Chemical composition of wakame used for the experiment.



Dried wakame

Washed wakame



Water (%)



N (%)



C (%)



C/N



NaCl (mg/g)a



8.22

13.72



3.64

4.14



29.82

36.95



8.19

8.92



89.0

46.2



a

NaCl concentration was determined for aqueous layer after mixing of water and

wakame with 10:1.



60

55



Temperature (°C)



50

45

40

35

30



15.2 mg/g NaCl

18.7 mg/g NaCl



25



23.6 mg/g NaCl

28.2 mg/g NaCl



20



0



8



16



24

32

Time (h)



40



48



Figure 2. Effect of salinity on the temperature changes during wakame composting by strain HR6.



53–55°C at a salinity level of 28.2 mg/g NaCl at 15.2°C, 18.7°C, and 23.6°C, and

48°C. The reduction of wet weight during 24–48 h of incubations was the highest in

the presence of 15.2 mg/g NaCl (Table 4). In the samples containing 15.2–28.2 mg/g

NaCl, there existed an insignificant difference in maximum temperature, which

proved that composting can be conducted in salinity as high as 28.2 mg/g of

NaCl with the inoculation of HR6, although a lower salinity is better for the

initialization time and activity of the composting process. This is important for

the field treatment of wakame when the salinity content is high. In addition, the

salinity of 15.2 mg/g of NaCl is also relatively high compared with that of the

common composting materials, suggesting that HR6 is superior to other bacteria for the disposal of waste with high salinity. The presence of Na+ sometimes

stimulates the decomposition of some components in wakame. For example,

alginate is degraded in the form of Na-alginate (Moen et al., 1997) and a Ca-crosslinked alginate matrix may present a limiting factor when microbes decompose



RECYCLING OF THE SEAWEED WAKAME THROUGH DEGRADATION



291



Table 4. Relative changes of wet weight during composting of wakame with strain HR6 under

different salinities.

Wet weight (%)

Incubation time(h)



15.2 mg/g NaCl



18.7 mg/g NaCl



23.6 mg/g NaCl



28.2 mg/g NaCl



0

24

48



100.0

98.7

97.2



100.0

99.2

97.4



100.0

99.2

97.6



100.0

99.6

98.5



brown algal tissue (Moen and Ostgaard, 1997). An appropriate level of salinity

may therefore be required for the composting and disposal of wakame.



2.2. ISOLATION AND CHARACTERIZATION OF HALOTOLERANT

BACTERIA FOR SEAWEED COMPOSTING

An attempt was made to examine the screening of marine bacteria for the degradation of wakame. As a result, an effective strain Halomonas sp. AW4 was isolated

and selected for further work. Strain AW4 initiated the wakame composting process

smoothly, showing a high decomposition rate within a short time. Dominant species in

the agar plate (5 g polypeptone, 3 g yeast extract, 30 g NaCl, 15 g agar in 1,000 mL

distilled water, pH 7.5) were further isolated and finally six predominant strains,

AW1–AW6, were selected for the present study. The characteristics of these strains

are shown together with those of HR6 for comparison (Table 5). The colonies

of these bacteria showed different colors: light green (AW2), light brown (AW1),

brown (AW5), light white (AW3, AW4), and white (AW5). All the strains grew

well in the presence of 0.5–1 M NaCl, but strains AW1 and AW2 could not grow

unless NaCl was supplemented. Morphologically, AW3 and AW6 were long rods

with low motility and cocci, respectively, while other strains were ellipse-shaped.

In small-scale composting, AW4 showed the highest decomposition rate when

compared with other species and HR6. In addition, AW4 revealed an optimal

growth rate in the presence of 0.5–1 M NaCl. Surprisingly, this strain still proliferated at a high salinity of 3 M NaCl. Inoculation of AW4 resulted in a more

rapid increase of the composting temperature than inoculation of HR6.

For 16S rDNA analysis, strain AW4 was first incubated in modified LB

medium for 24 h. After extraction of DNA, we amplified 16S rDNA fragments

using the primers EUB27F (5¢-AGAGTTTGATCCTGGCTCAG-3¢) and EUB

533r (5¢-TTACCGCGGCKGCTGRC-AC-3). Partial 16S rDNA analysis suggested that AW4 belongs to the genus Halomonas. The phylogenetic allocation and

16S rRNA gene sequence similarities of AW4 and related bacterial strains are

presented in Fig. 3. AW4 was most closely related to the strain Halomonas venusta,

with 99.6% similarity in partial 16S rDNA analysis. Higher similarity with AW4

was also found in H. hydrothermalis, H. variabilis, H. magadiensis, H. boliviensis,



a



Awaji island

Awaji island

Awaji island

Awaji island

Awaji island

Awaji island

Soil



Light brown

Light green

Light white

White

Brown

Light white

White



Data by small scale composting.



AW1

AW2

AW3

AW4

AW5

AW6

HR6



Source



0

0.47

0

0

0.21

0.66

0.61



0.66

0.34

0.4

0.88

0.64

0.64

0.41



0.54

0.17

0.27

0.64

0.54

0.59

0.2



0.21

0

0.13

0.37

0

0.03

0.09







+

+









+

+

+

+



+

+



Ellipse

Ellipse

Rod

Ellipse

Ellipse

Coccus

Rod



<1

<1

<25

<1–2

<1

<1–2

<25



+



+

+

+

+

+



11.2

8.45

11

16.5

12.5

8.8

8.9



Growth at Growth at Microscopic observation

Degradation

30°C (3 M 50°C (0.5

of wakame

Color of colony 0 M NaCl 0.5 M NaCl 1 M NaCl 2 M NaCl NaCl)

M NaCl) Shape Size (m) Motility (%)a



30°C growth rate (max) (h−1)



Table 5. Characterization of six marine bacteria isolated from seaweeds for wakame composting.



292

JING-CHUN TANG ET AL.



RECYCLING OF THE SEAWEED WAKAME THROUGH DEGRADATION



293



Figure 3. Phylogenetic tree based on partial 16S rRNA gene of strain AW4. Evolutionary distances

were calculated using the “neighbor-joining” method. The scale bar represents 1% sequence divergence.



and H. alkantarctica with 98.7%, 98.5%, 97.4%, 97.2%, and 97.0%, respectively.

The genus Halomonas mainly consists of halotolerant, halophilic, and haloalkaliphilic species that were isolated from salterns, saline soil and lakes, and the Dead

Sea (Ventosa et al., 1998). Some of these species have already proven to be effective

bacteria for waste degradation at high salinity. H. campisalis showed a high capacity

for degradation of saline and alkaline wastes owing to denitrification, broad

carbon utilization, and pH as well as salinity tolerance (Mormile et al., 1999).

H. muralis was isolated from alkaline two-phase olive mill wastes (Ntougias et al.,

2006) and H. marina was found to be one of the main microorganisms that

degrade the brown alga Fucus evanescens (Ivanova et al., 2002).



2.3. THE COMPOSTING PROCESS OF WAKAME BY AW4

The initial moisture content of wakame, which was cut into pieces smaller than

1 cm, was adjusted to about 70%. About 1.5 kg of wakame was composted in

a 5-L container (Fig. 4) with an isolation film outside to keep the temperature

constant. The container was aerated with an air pump at a flow rate of 0.5 L/min.

Each bacterial strain was added as suspension. Changes of O2 and CO2 concentrations in exhaust gas as well as the temperature inside the container were measured.

Changes of the water content, pH, C, as well as N contents and viable cell numbers were also measured during composting. Figure 5 shows the changes of temperature as well as of O2 and CO2 in exhaust gas during composting with strains

HR6 or AW6. Temperature changes corresponded to changes of the O2 and CO2



294



JING-CHUN TANG ET AL.



Thermo recorder



Air out



Ice container



Wakame



O2, CO2 analyzer



Sawdust



Air pump

Air in



Figure 4. Composting system of wakame in a 5-L container.

AW4



10



40



5



20



0



0



24



48



72



96



Time (h)



120



144



0

168



O2 and CO2 (%)



60



Temperature (ºC)



O2 and CO2 (%)



O2

CO2

Temperature



15



O2

CO2

Temperature



20



80



20



100



25



100



80



15



60



10



40



5



20



0



0



24



48



72



96



120



144



Temperature (ºC)



HR6



25



0

168



Time (h)



Figure 5. Changes of O2 and CO2 in exhaust gas and corresponding temperature during wakame

composting with strains HR6 and AW4.



concentration in exhaust gas, although a lag time of about 2 h was observed,

which suggests that the microbial activity was not always stable but variable during wakame composting. Higher temperatures and more O2 consumption and

CO2 production, however, were found during wakame composting with AW4

when compared with composting with HR6. The strain AW4 seems, therefore, to

be more adapted to the compost environment of wakame than strain HR6.

Table 6 shows the changes of dry weight, pH, and chemical composition

during the composting process of AW4 and HR6. Decomposition rates of dry



RECYCLING OF THE SEAWEED WAKAME THROUGH DEGRADATION



295



Table 6. Changes of dry weight, pH, and chemical composition during

wakame composting with strains AW4 and HR6.

Time (h)



Time (h)



Dry weight (%)



pH



N (%)



C (%)



C/N



AW4



0

72

168

0

72

168



100.0

87.8

73.6

100.0

93.4

87.8



7.16

8.22

9.00

6.98

8.24

8.48



3.09

3.01

3.15

2.28

2.10

1.91



31.34

30.19

28.95

32.84

31.16

32.37



10.1

10.1

9.2

14.4

14.9

16.9



HR6



Values of C and N are average from two independent experiments.

Table 7. Changes of cell viability in wakame composting with strains

AW4 and HR6 (×108 CFU/g).

Time (h)



0



8



24



48



55



72



120



168



AW4



6

6

1.1

1.1



25.3

25.3

2.8

2.8



23.3

23.3

1.2

1.2



1.5

12.7

0.4

18.5









4.3

26.3

0.0

6.5



1.5

22.5

0.0

9.8



1.8

35.0

0.0

10.8



HR6



AW4

Total

HR6

Total



0.1

183.0



weight wakame were 12.2% and 26.4% after 3 and 7 days in the experiment of

AW4, respectively, which were much higher than that of HR6 under the same

condition, 6.6% and 12.2%. pH values during the decomposition with AW4

increased to 8.2 and 9.0 after 72 and 168 h of incubations, respectively, and high

NH3 content, about 500 ppm, was detected in exhaust gas showing that an

alkaline environment was caused by the production of NH3 in the exhaust gas. C

and N contents decreased and increased slightly after 168 h, respectively, leading

to a reduction of the C/N ratio, 9.2. Since the N content was relatively high compared with other composts (Tang et al., 2003), the C/N ratio was lower than that

of general compost, >15 (Bernal et al., 1998).

Changes of viable cell numbers of strain HR6, AW4, and total bacteria were

observed during the incubation process (Table 7). The viability of strain HR6

increased within 24 h of composting. Then it decreased gradually, although a sharp

increase of total bacteria to 1.83 × 1010 CFU/g was observed at 55 h of incubation.

After 3 days of composting, total viable cell numbers decreased to the level of 6.5

× 108 CFU/g. On the other hand, the cell viability of AW4 increased during 24–72

h of composting, which corresponded with temperature and respiratory changes.

The cell viability remained at a high level until the end of composting, although the

temperature and respiratory rates gradually decreased. The appearance of the

wakame biomass showed great change after composting, that is, its color changed

from brown-green to grey-black and the original shape of the wakame was gradually destroyed. It became fine granule as that of matured compost, which suggests

that a diverse microbial community was induced by inoculating AW4, which was

effective in decomposing wakame during composting.



296



JING-CHUN TANG ET AL.



3. Role of Alginate-Degrading Bacteria in the Recycling of Wakame

Alginate is a linear copolymer of b-1,4-d-mannuronic acid and a-1,4-l-guluronic

acid with the residues organized in blocks of not only polymannuronate and

polyguluronate but of heteropolymeric sequences of both uronic acids (Moen

and Ostgaard, 1997). Alginate can be degraded by radiation or thermal treatment

(Nagasawa et al., 2000). Biological degradation, on the other hand, has been generally conducted by alginate lyase that acts on the 4-O-linked glycosidic linkage

of alginate (Iwamoto et al., 2001). Alginate lyases have been isolated from a wide

range of organisms such as algae, marine invertebrates, marine microorganisms,

etc. (Wong et al., 2000). Since the degradation of alginate is not easily preceded

by most microorganisms, which is mainly because of its complicated molecular

structure, there has been no report on the isolation of alginate-degrading bacteria

and their application in the decomposition of seaweed wastes. However, isolation

of alginate-degrading bacteria and their use is interesting and indispensable for

the effective degradation of these wastes.



3.1. ISOLATION AND IDENTIFICATION OF ALGINATE-DEGRADING

BACTERIA

In the primary screening, four strains from a total of 56 isolated microorganisms

were selected based on the ability of alginate degradation. Four bacteria designated as A7, N7, N10, and N14 were Gram positive, long rods in shape, and of

white color colony on agar plates. All these bacteria were unable to grow in the

medium without alginate supplemented. Quantitative changes of reducing and

unsaturated polysaccharides that were produced by each bacterium in a medium

containing initially 5 g/L alginate are shown in Fig. 6. The reducing sugar was

detected by 3,5-dinitrosalicylic acid (DNS) method (Miller, 1959). The supernant

of culture medium was mixed with DNS solution and boiled for 5 min. Absorbance of the reaction solution was analyzed at 540 nm after dilution by distilled

water at the rate of 1:10, and the result was expressed as mg glucose/mL culture.

The production of unsaturated sugar through b-elimination reaction by alginate

lyase was determined by measuring the absorbance at 235 nm (Iwamoto et al.,

2001). Both saccharides were lower than in the control after inoculation of the

marine bacterium AW4, but alginate-degrading bacteria showed an increase of

both saccharides after 48–96 h of incubations. An increase of OD235 at 48 h of

incubation compared with that of reducing sugars might be caused by the existence of endo-alginate hydrolase, which was reported in fungi (Schaumann and

Weide, 1990). Strain A7 showed the most rapid degradation of alginate among the

strains that were examined.

A7 was long rod-shaped, 5–10 mm in size, and filamentous, and its colonies

were of circular morphology and creamy white (data not shown). Partial 16S



Reducing sugars (mg-Glucose/ml)



a



8



6



4



2



0



b



50



Unsaturated sugars (OD 235)



RECYCLING OF THE SEAWEED WAKAME THROUGH DEGRADATION



40



297



30



20

10

0



0



24



48



72



96 120 144 168



Incubation time (h)



0



24



48



72



96 120 144 168



Incubation time (h)



Figure 6. Changes of reducing and unsaturated sugars by four alginate-degrading bacteria and marine

bacterium AW4 as reference. Changes of reducing sugars (a) and unsaturated sugars (b) were followed for

A7 (open diamonds), N7 (open squares), N10 (open triangles), N14 (open circles), and AW4 (closed circles).



rDNA analysis showed that A7 was most closely related to Gracilibacillus halotolerans, at a similarity of 99% (Waino et al., 1999). In the phylogenetic tree based

on partial 16S rRNA genes (Fig. 7), some alginate-degrading bacteria such as

Vibrio sp. O2 (Kawamoto et al., 2006) or Bacillus algicola (Ivanova et al., 2002)

have been described, but this is the first time to report alginate-degrading bacteria

within the genus Gracilibacillus.



3.2. CHARACTERIZATION OF ALGINATE-DEGRADING

BACTERIUM A7

The growth of strain A7 in the presence of low concentrations of nutrients, 0.1 g

polypeptone and 0.06 g yeast extract in 1 L, was characterized under different pH,

NaCl concentrations, temperature, and nutrient contents (Fig. 8). Rapid growth

was observed at pH 8.5–9.5, suggesting that an alkaline environment is favorable

for growth. The bacterium grew well in the presence of 0.5–2 M NaCl, whereas

no growth was found in the absence of NaCl. At a temperature of 30°C, the

highest growth rate was observed after 48 h of incubation, although a sharp

increase of growth within 24 h was observed at 45°C and a much slower rate was

observed at 20°C. The optimal condition for growth of A7 was observed when the

strain was incubated in the presence of 5 g/L of sodium alginate. Thus, the best

overall condition for growth was as follows; pH 8.5–9.5, NaCl 0.5 M, temperature

30°C, and nutrient content of 2–5 g/L of polypeptone.



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