|Marine single cell detritus; Ulva reticulate; Cellulase;
Fermentor; LAB; Yeast
|Species of the marine algal genus Ulva are known for their
various uses like feed, manure, folk medicines, dietary fiber contents
as well as for the rheological and gelling properties of their sulfated
polysaccharides. Uchida et al.  developed the protocol to perform
the lactic acid fermentation of seaweed. Single cell detritus (SCD) is
a product prepared by decomposing seaweed to a cellular level, and
is expected to be utilized as a fish-diet in place of unicellular algae.
The present Paper give details about the culture conditions standardized for the
effective preparation of SCD from Ulva reticulata based on a lactic acid
|Enzymatic hydrolysis of the seaweed cell wall is a procedure
used in many applications. It has been described for improving
protein digestibility by removing anti-nutritional factors such as
polysaccharides. Uchida and Murata  reported a successful
preparation of SCD by enzymatic and microbial fermentation. Uchida
et al.  combined the activity of both lactic acid bacteria (LAB) and
yeast for the preparation of the marine silage or MSCD.
|Uchida and Numaguchi  used the decomposing properties of the
marine bacteria Alteromonas espejiana to obtain SCD from Ulva pertusa
and showed that these particals are easily ingested by larvae of the clam
Ruditapes philippinarum. Uchida et al.  prepared a seaweed based
SCD successfully in large scale to feed young pearl oysters. MSCD was
prepared in this study to test its efficiency in shrimp larvae.
|Materials and Methods
|Collection and preparation of seaweed powder
|Seaweed Ulva reticulata (Chlorophyceae) was selected based on
their frond type, proximate composition and its abundant availability
and collected from the coastal waters of Gulf of Mannar, Thoothukudi.
Collected seaweeds were washed thoroughly, shade dried and oven
dried at 60°C for 4 hrs. Dried seaweed was ground to make a powder.
|The cell walls of seaweeds are made up of cellulose which needs
to be digested to convert them into individual cell components. The cellulase enzyme Deniwash 1000 L (Enzyme India Ltd) serves the
above purpose. Deniwash 1000 L is an acid cellulase liquid preparation
from Trichoderma viride. This enzyme has the activation temperature
of 50°C to 55°C. Seawater was filtered through the canister and UV
filters and the salinity was tested prior to use and during the process.
The hemocytometer (Neubauer counting chamber) is used for the
determination of enzyme digested particle numbers of seaweeds. A
thermostatic controlled incubator shaker was used to maintain the
activation temperature of respective enzymes. For in situ, fermentor of 10 litre capacity and an autoclavable 3 litre capacity were used to mass
|Standardization of enzyme digestion: To standardize the enzyme
type, concentration and time duration, three types of cellulase (Enzymes
India Pvt. Ltd) viz. Deniwash VT and Tropizyme-p, Deniwash-1000L
were added to the base material at different concentrations viz., 50,
100, 250, 500, 750 and 1000µl. The initial mean number of particles
was estimated to be 2.84 x 107 particles / ml and the increase in the
number of particles was estimated periodically i.e. after 20, 45, 60 and
120 minutes of enzyme inoculation.
|Standardization of microbial combination: To standardize the
microbial combination Lactobacillus plantarum (MTCC 1325) and
Saccharomyces ceriviceae (MTCC No.4780) were added in different
concentrations to the 100 ml base material treated with enzyme
Deniwash-1000L . The samples were drawn on the 4th, 6th and 8th day
and the parameters viz. odour, pH and lactic acid concentration 
|Standardization of substrates: To standardize the sugar substrate
concentration, potato powder at various concentrations viz. 1, 2.5, 5,
7.5 and 10% were added to 100 ml of the base material treated both
enzymatically and microbially. The samples were drawn from the
container periodically i.e., 2nd, 3rd, 4th, 5th and 6th day and the parameters
viz. odour, pH and lactic acid concentration were estimated / observed.
|To standardize the protein substrate concentration, soya powder
at various concentrations i.e., 1, 2.5, 5, 7.5 and 10 % were added to
100ml of the base material treated both enzymatically and microbially. The samples were drawn from the container periodically i.e., 2nd, 3rd, 4th, 5th and 6th day and the parameters viz. odour, pH and lactic acid
concentration were estimated / observed.
|Fermentation process: The 9:1 concentration of base material
(seawater and seaweed) was transferred to the fermentor sterilized at
121°C for 20 min after that the temperature was maintained at 55°C and
Deniwash 1000 L enzyme was injected through the inoculation port at
the rate of 7.5 ml per 100 ml of base material. The stirrer was set at 200
rpm .The whole process was carried out for 1 hr. The pH before and
after enzyme treatment were 5.75 and 5.86 respectively.
|L. plantarum strain (MTCC 1325) raised in MRS broth (Hi media)
and S. cerevisiae (MTCC No.4780) raised in YM broth (Hi media) for 24
hrs each were injected through the inoculation port. Prior to microbial
inoculation the temperature was dropped down to 35°C. L. plantarum
and S. cerevisiae were inoculated each at a final concentration of 107 cfu/ml of base material. The substrates potato powder and soya powder
were added at the rate of 1% w/v respectively of base material. The level
of lactic acid production, pH and odour were periodically estimated/observed. Fermentation was carried out till the pH reduced below 4.00.
The particle size of the single cell unit was measured as 2.5-7.5 µm.
MSCD product was aseptically transferred to a jar and closed air tight
and stored at room temperature with proper labeling.
|Deniwash 1000 L enzyme at a concentration of 7.50 ml per 100 ml
of base material for duration of 60 min that showed the highest particle
numbers (Figure 1) was chosen for the enzymatic digestion. The
treatments with the L. plantarum strain (MTCC 1325) and S. cerevisiae (MTCC No.4780) at a final concentration of 105 cfu/ml was effective
in lactic acid production (Table 1). The substrate (sugar and protein)
concentration at the minimum i.e., 1% was found effective for MSCD
preparation. The progress of fermentation in the fermentor vessel was
monitored by observing the lactic acid production and the reduction
in pH level (Figure 2 and Figure 3). The microbial propagation study
undertaken in an interval of 24 hours revealed that an increment of
L. plantarum from 1.23 x 105 cfu/ml to 3.00 x 108 cfu/ml . Similarly
S. cerevisiae recorded an increment from 4.30 x 105 to 5.00 x 108 in
duration of 72 hrs. The viability of the microbes i.e., L. plantarum and
S. cerevisiae in MSCD was studied periodically for duration of 50 days.
Initially L. plantarum was at 3.00 x 108 cfu/ml which reduced to 1.86
x 106 cfu/ml and S. cerevisiae was at 5.00 x 108 cfu/ml that reduced to
8.00 x 106 cfu/ml at the end of 50 days.
|In this study, Ulva reticulata with frond type, flat, foliaceous and
thallus with two cell thicknesses  and with crude protein level of
19.5% was selected. Further, Ulva sp. also produce high amount of lactic
acid during fermentation (0.76g/100ml of fermentation products),
which help in the long term preservation of the MSCD . Cellulase
enzymes are advantageous because of their better agar recovery rate
and their potential application in the degradation of cellulosic wastes
into glucose which could be utilized as the carbon source for the
|Based on this concept, cellulase enzyme was assigned for the studies
on seaweed. Deniwash 1000 L was found to produce large number of
particles i.e. 2.84 x 107 particles / ml in 60 minutes at a concentration
of 750 µl/10ml of base material at 55°C. Stability and high cell
concentration of the MSCD are the advantages of the present method
compared to microalgae culture . A microbial mixture of one type
of 'LAB', Lactobacillus brevis and two types of yeast, Debaryomyces
hansenii and Candida sp. were used to prepare the marine silage of
Undaria pinnatifida . It showed that yeast and 'LAB' can coexist in
a same medium. Lactobacillus plantarum produce lactic acid with high
efficiency and retard the growth of contaminating bacteria . Yeast,
S. cerevisiae was selected, as it helps in fermentation apart from its
unique bioremediatory effect in aquaculture system . Uchida et al. 
recorded that L. plantarum in combination with S. cerevisiae produced
lactic acid at a rate of 1.64 g/l and pH also was observed to be reducing
to 3.7 . In the present study, the concentration of 'LAB' and yeast
to be inoculated was standardized as 107 cfu/ml each. To promote
the lactic acid fermentation a sugar substrate, namely potato powder
and protein substrate soya powder, was added to the base material at a
concentration of 1% w/v of base material. The ester odour in the MSCD
was presumably contributed by S. cerevisiae in the culture medium .
|The duration of MSCD preparation took a relatively less duration
than that of Uchida et al.  who took 8 days for fermentation [11-13]. The difference in the duration could be due to the efficiency of the
fermentor as well as the right combination of microbes incorporated in
the present study. The microbial propagation during MSCD preparation
in this study reached a maximum of 108 (both LAB and yeast) at the 60th
hour of microbial inoculation. The yeast viability tested during a period
of 50 days, at an interval of 15 days, showed that it was declining as the storage duration increases, which could be due to the lack of salt
tolerance capability of S. cerevisiae as reported by Uchida et al. .
|The project investigator acknowledges the grand provided by the Department
of Biotechnology, Government of India, New Delhi for carrying out works on this
area of research.
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