Culture of Brachionus plicatilis (L-type) using anaerobically treated organic wastes

 

Received for publication, August 25, 2007

Accepted, October 10, 2007

 

G. IMMANUEL

Centre for Marine science and Technology, M.S. University

Rajakkamangalam – 629 502, Kanyakumari district, Tamilnadu, India

E. mail: gimmas@gmail.com  (or)  g_immas@yahoo.com

 

Abstract

Considering the high cost of production of Chlorella sp. as a source of food for live feed Branchionus plicatilis (L- type), an alternative cheap and waste organic substances such as cow dung (A), cabbage (B), poultry manure (C) and a mixture of the above three (D) at 1:1:1 ratio (anaerobically treated) have been tested as a food source to grow B. plicatilis at different salinity (10, 20, 30 and 40 ppt) conditions.  The population density of B. plicatilis was determined for every two days up to ten days. Among the tested dietary sources, B. plicatilis fed with mixed diet (D) showed a maximum population density (183±12 no/ml) on tenth day at 10 ppt salinity. But the other dietary sources exhibited relatively low density (P< 0.05).  B. plicatilis had highest specific growth rate (36%) when fed with mixed diet (D) at 10 ppt salinity than other tested dietary sources and salinities (P< 0.05). The result is compared with the result obtained with other food organisms like algae, yeast and inert food used for the culture of B. plicatilis.

 

Keywords:  Brachionus plicatilis; cowdung; cabbage; poultry manure; organic wastes

 

Introduction

 

The prime requirement of the aquaculture practice is the production of appropriate nutritionally balanced, non polluting, economically viable and acceptable feed in order to release optimum growth and survival of the cultured stock [1]. Live feed organisms are preferred by most of the cultured larvae compared to artificial feed: because in nature, they select one or the other live food organism as their principal food [2,3,4]. Live food organisms like Tubifex tubifex [5,6,7]. Daphnia [8] and Artemia [9,10,11,12] have been cultured using organic waste like cow dung, poultry manure, cabbage wastes, coconut mesocarp waste and pig dung in order to reduce the production cost as well as to utilize the waste in useful way for food production. The above mentioned live food organisms are the suitable candidate for raising them on the waste organic materials. But because of their relatively larger size, they are less/not preferred by newly born fish/crustacean larvae. In the mean time, the rotifer B. plicatilis and Artemia nauplii have been widely used as the food for newly born larvae due to its small size, nutrient condition as well as its tolerance to wide range of salinity. One of the main problems encountered with large scale production of the rotifer is the large scale requirement of unicellular algae like Chlorella sp. which requires substantial space, nutrient and time to produce sufficient quantities to feed the rotifer [13]. Hence, an alternative food like yeast has been used as a sole food [14,15] or in combination with several other nutrients such as algae [16,17] as well as inert food [18,19]. Marian et al. [11] found that anaerobically treated (12 to 20 days retention time) cow dung, poultry manure and cabbage (200-300g/l) supported growth of life food organisms. Hence I made a preliminary attempt to use these organic wastes as food source for the culture of the rotifer B. plicatilis.

 

Materials and Methods

 

A factorial design of experiment was undertaken for raising the rotifer B. plicatilis         (L-type)  at four salinities (10, 20, 30 and 40 ppt) and four food sources, viz cow dung (A), cabbage (B), poultry manure (C) and mixture of the these three  in 1:1:1 ratio (D). For this, 12-20 days anaerobically fermented cow dung, cabbage and poultry manure supplemented with vitamins B12 (10mg/l) (loading rate: 200-300g/l) were used as they supported more bacterial growth, more nutrient and less toxic substances [11].

 A stock B. plicatilis (L-type) with average lorica length of 220 ± 18 µm was selected for the present study. B. plicatilis was cultured in one liter capacity container with 0.5 l water. In all trials, triplicates were kept in each treatment and mild aeration was given to keep the oxygen concentration above 4mg/l. Initially, five B. plicatilis /ml were inoculated in to the culture medium. Water volume was maintained between 500-700 ml. The different salinity levels were prepared and maintained either by dilution of filtered sea water with filtered fresh water or by addition of solar salt dissolved in sea water. The feeding of respective fermented organic matter and their feeding regime adopted in the experiment is given in table 1. Population of B. plicatilis was recorded daily by taking triplicate samples (1 ml each) from the culture media after stirring gently. The whole experiment was conducted for a period of ten days. Specific growth rate (SGR - %) of  B. plicatilis was calculated using the formula

                                                         In Nt – In No   

                                   SGR (%) = -------------------

                                                                             t

Where,          No     = Initial number of rotifer

                      Nt     = Number of rotifer after t days

                        t      = Number of days (10 days)

Doubling time was calculated by dividing log e2 by Specific growth rate.

 

Table 1. Feeding regime adopted in the culture of B. plicatilis using the anaerobically      fermented organic wastes.

Culture period (Days)

Food Concentration (g/l)

Flow rate (ml/mt)

Duration of feeding

(hours of the day)

1-2

3

5

08-10

12-14

16-18

20-22

3-4

            3

10

08-10

12-14

16-18

20-22

5-7

6

15

08-10

14-18

20-24

-

 8-10

          6

                 20

08-10

14-18

20-24

  -

 

Salinity was measured daily by using a refractometer and oxygen by Winkler’s method. The experiments were conducted at 26±20C and the photoperiod was 16L: 8D. Salinity and water volume were maintained at the desired level one hour after feeding.

 

Statistical analysis

 

All experiments were performed in triplicate. The result obtained in the present study were analysed through Two way ANOVA test following Zar [20].

Results

 

Among all the fermented dietary sources, B. plicatilis cultured using the fermented cabbages (B) as food grew slowly in all the tested salinities and the population density reached to a maximum of 104±8 no/ml at the end of the experiment (10 th day) in 10 ppt salinity. Whereas B. plicatilis   fed on fermented mixture (D) dietary source exhibited a maximum density of 183±12 no/ml on 10th day at 10 ppt salinity. The other dietary source like fermented cow dung (A) and fermented poultry manure (C) showed the population density of 146±7.0 and 166±14.0 no/ml respectively in the same salinity at the end of the experiment. In the entire experimental dietary source, the growth of B. plicatilis   was slow up to fourth day, but from sixth day onwards, the multiplication was faster.

In all cases, the maximum density was observed at 10 ppt salinity level and the density become decreased with increasing salinity i.e. it was 96±8 to 26±2; 126±6 to 39±3; 161±11 to 39±3 and 143±10 to 48±4 no/ml. in the test salinities from 20- 40 ppt respectively in the test group of cabbage, cow dung, poultry manure and mixed group respectively (Fig. 1). A two way ANOVA test made on the effect of dietary source and salinity on population  density of B. plicatilis revealed that the variation between them was statistically more significant (F (2) =22.3 to 240.0; P<0.0001).

Likewise, among all the tested groups, the specific growth rate (%) of B. plicatilis was maximum (36%) in the mixed group (D) in the series containing 10 ppt salinity, whereas it was decreased in other salinities (33.5 to 22.6% in 20 to 40 ppt salinities). Similarly the specific growth rate of cabbage (B) was 30.3% at 10 ppt salinity, whereas it was reduced from 29.5 to 16.4% in the tested salinities of 20 to 40 ppt respectively. In cowdung (A) and poultry manure (C) also the specific growth rate was more ( 33.7 and 35.0%) at 10 ppt. Salinity, but it was  decreased in the order of 32.2 to 20.5% and 34.7 to 20.5% in  the  tested  salinities  of 20 to 40 ppt respectively  (Table 2).   The Two way

ANOVA test carried out for the specific growth rate of B. plicatilis revealed that the variation between the tested salinities and dietary sources were statistically more significant (F (2) 14.1371 to 89.7546; P< 0.001).

 

Table 2.  Specific growth rate (%) of B. plicatilis reared at different salinity

concentrations using the anaerobically fermented organic wastes.

 

Fermented Organic wastes

         SGR (%)  a t different   Salinity concentrations

 

 

10 ppt

 

20 ppt

 

30 ppt

 

40 ppt

 

Cow dung (A)

33.7

32.2

22.4

20.5

Cabbage (B)

30. 3

29.5

16. 8

16. 4

Poultry manure (C)

35. 0

34. 7

21. 2

20. 5

Mixture (D)

36. 0

33. 5

27. 7

22. 6

 

 

Density (no/ml)

          

           

   

 

Figure 1. Density (no/ml) of B. plicatilis cultured using fermented organic wastes as food at different salinity concentrations (10-40 ppt).

 

 

Discussion

 

The filter feeding habits of B. plicatilis led the possibilities of raising the organism on the variety of food types such as algae, yeast, bacteria and inert food. Algae of several species such as Chlorella sp., Synechococcus sp., Monochrysis lutheri, Dunaliella teriolecta, Cyclotella cryptica, Nitzschia clostelium, Tetraselmis terethe  [16,21] have been used as food for B. plicatilis. Others have used the green algae with blue green algae. For instance, Snell et al. [22] used the blue green algae, Schzothrix with Chlorella to increase of production.

 Normally, to maintain rotifer culture, one has to subsequently maintain 5-10 times volume of algal cultures [23]. In order to alleviate/ reduce the problem of using expensive algae- culture system, an alternative, inexpensive, commercially available food like yeast and other inert foods have been tried by scientists. Hirata and Mory [23] first used the yeast Saccharomyces cereviciae to raise the rotifer, B. plicatilis. Later on, several species of marine yeast have been used [15,24]. Though Mastuda et al. [24] could raise B. plicatilis on isolated specific marine yeast more than 2-3 times higher than those achieved with Chlorella, the relatively expensive production cost due to specific techniques led a limited applicability as foods. The bacteria Pseudomonas (P-1 and P-7) have been found to improve the growth of B. plicatilis by Ushiro et al. [25] and Yamasaki and Hirata [26].

The inexpensive supplies of bacteria produced in waste water treatment plants or from alcohol fermentation industries have been used successfully by Hino et al. [27] and Fukuhara et al. [14] for B. plicatilis. Groeneweg and Schluter [28] raised B. plicatilis in the effluent of the algal ponds used for the treatment of piggery waste. Another way adopted to reduce the cost was using inert food, eg. Spray dried Chlorella or Platymonas succica [29]; commercial Spirulina, Chlorella and methanol grown yeast [18]. Utilization of microencapsulated diet, some time has the limitation of constant and continuous support of growth to the rotifers [19]; however it opens the chance for permitting the enrichment of rotifers with micronutrients required by the fish and prawn larvae. Comparison of the growth characteristics of B. plicatilis reared by giving different foods like algae, yeast, inert food with that of the waste organics used in the present study revealed that the growth rate and doubling time were considerably equal to the values reported by James et al. [13] for yeast food, and for Chlorella  by Matunog [30] at > 20ppt. The high growth rate obtained in the lowest salinity may be the reason for the comparable growth rate obtained with other works; however, it is important to note that James et al. [13] obtained the growth rate at 30 ppt (Table 3). If they have raised the rotifer in low salinity, they might have raised the rotifer ay faster rate. Hence, further research is required before stepping into a large scale production of B. plicatilis using the organic wastes by changing the feeding regime, supplementation with other algae /yeast in order to obtain a very good growth rate and continuous production.

 

Table 3. Comparison of growth characteristics of B. plicatilis fed with different

kinds of food

Kinds of food

Feeding regime frequency

Food quantity

Temp (0 C)

Salinity (ppt)

Culture periods (days)

SGR (%)

Doubling Time (Days)

Source

Algae

Tetraselmis

Tetrathele

Chlorella sp

,,

,,

,,

,,

,,

,,

 

Once / day

Once / day

--

--

--

--

--

--

--

 

5x10 cells/ml

15x10 cells/ml

25x10 cells/ml

--

--

--

--

--

--

 

20-25

20-25

25-31

--

--

--

--

--

--

 

35

35

35

10

15

20

25

30

40

 

10

10

6-7

--

--

--

--

--

--

 

24-49

16-47

20.0

59.0

52.0

37.0

28.0

16.0

13.0

 

2.9-1.4

4.3-1.5

3.4

1.2

1.3

2.0

2.3

5.0

5.3

 

Okauchi  and  Fukusho (1985) Matunog (1977)

 

Yeast

Marine yeast

Baker’s yeast

 

20 h/day

 

20 h/day

 

38±8 mg/l

 

23±1 mg/l

 

25-27

 

25-27

 

30

 

30

 

10

 

10

 

46.0

 

31.0

 

 

1.7

 

2.3

 

 

James

et al., (1986)

Inert Food

Artificial diet & algae (4:1)

 

Continuous

 

-

 

22-23

 

18

35

 

15

27

 

37.0

--

 

1.9

--

Gatesoupe and   Luquet (1981)

Organic wastes

Cow dung (A)

3-4 times/day

3-6 g/l

26±2

10

20

30

40

10

10

10

10

 

33.7

32.2

22.4

20.5

 

2.1

2.3

3.3

3.8

 

Present study

Cabbage

(B)

3-4 times/day

3-6 g/l

26±2

10

20

30

40

10

10

10

10

 

30.3

29.5

16.8

16.4

2.3

2.4

4.0

4.3

 

Present study

Poultry manure

(C)

 

3-4 times/day

3-6 g/l

26±2

10

20

30

40

10

10

10

10

 

35.0

34.7

21.2

20.5

2.0

2.1

2.6

3.3

 

Present study

Mixture  diet

(D)

 

 

3-4 times/day

3-6 g/l

26±2

10

20

30

40

10

10

10

10

 

36.0

33.5

27.7

22.6

 

2.1

2.1

3.6

3.6

 

Present study

 

 

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