Aquaculture Nutrition

2013 19; 895907

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doi: 10.1111/anu.12035

1,2 1,3 1,2

1
Institute of Biological Sciences, University of Malaya, Kuala Lumpur, Malaysia; 2 Institute of Ocean & Earth Sciences, Uni-
versity of Malaya, Kuala Lumpur, Malaysia; 3 Mushroom Research Centre, University of Malaya, Kuala Lumpur,
Malaysia

The production of high-quality live feed at the cheapest

Three species of purple non-sulphur bacteria (PB), Rhodo-
possible cost is crucial for the aquaculture industry. Com-
pseudomonas palustris, Rhodobacter sphaeroides and Rho-
mercial hatcheries worldwide require live feed of suitable
dovulum sulfidophilum, grown in palm oil mill effluent
sizes (2500 lm) (Sorgeloos & L
eger 1992) and nutritional
(POME) were successfully used for the first time as feed
value. For instances, most larval fishes require protein lev-
for rotifers (Brachionus rotundiformis). Rp. palustris cul-
els of 500600 g kg 1 (John & Paul 2012), and both doco-
tured in both POME and synthetic medium gave the high-
sahexaenoic acid (DHA) and eicosapentaenoic acid (EPA)
est rotifer density (332395 individuals mL 1) from 3 to
are essential fatty acids vital to the nutrition and survival
5 days at 10 g L 1 salinity. Other PB cultured in synthetic
of fish and invertebrate larvae (Brett 2009). Generally,
medium generally support higher rotifer density than PB
cultured fishes require an average DHA : EPA ratio of
cultured in POME. Rb. sphaeroides had the highest bio-
0.52.0, whereas cultured invertebrate species require a
mass (1.913.34 g L 1) and growth rate (0.641.11 g
ratio of 0.71.0 (Rodr
guez et al. 1997; Sargent et al. 1999;
day 1) in both types of culture medium. Nevertheless,
Brett 2009; Loo et al. 2012a). Live feeds commonly in use
only Rv. sulfidophilum grown in POME contained both ei-
are micro-autotrophs and heterotrophs such as microalgae
cosapentaenoic acid (EPA) and docosahexaenoic acid
and yeasts with costs of production that range from USD
(DHA), indicating its ability to biosynthesize them from
46 to USD 600 kg 1 dry biomass (Moon & Kim 1996;
POME nutrients. Rotifers fed Rv. sulfidophilum grown in
Spolaore et al. 2006; Brennan & Owende 2009; Pahl 2010)
POME had significantly higher amounts of protein, ara-
or 2070 per cent of hatchery operating costs (Coutteau &
chidonic acid, EPA and DHA than rotifers fed Rv. sulfi-
Sorgeloos 1992; Michael 1997). However, the costs of pro-
dophilum grown in synthetic medium. The nutritional
ducing photosynthetic microalgae in small-scale hatcheries
profile of lipid-deficient PB can be improved by growing
can be even higher from USD 770 to USD 1,000 kg 1 dry
them in POME, and these enriched PB produced at an
biomass (Pahl 2010). Little attention has however been
estimated cost of USD 8.7135.35 kg 1 dry biomass,
given to the use of phototrophic bacteria as a feed for roti-
depending on species, can support rotifer production in a
fers and other zooplankton, which has increasingly become
batch culture system.
important as live food in fish culture. Phototrophic bacteria
previously known as photosynthetic bacteria are prokary-
KEY WORDS: nutritional value, palm oil mill effluent, purple
otes that use light energy to metabolize useful chemical
non-sulphur bacteria, rotifers
energy via either chlorophyll- or bacteriochlorophyll-medi-
ated processes (Imhoff 1992). They derive energy from light
Received 5 July 2012; accepted 13 December 2012
by photophosphorylation to synthesis organic materials
Correspondence: Dr. Loo Poh Leong, Institute of Ocean & Earth
Sciences, University of Malaya, 50603 Kuala Lumpur, Malaysia. E-mail:
from inorganic components during photosynthesis (Imhoff
pohleong_loo@yahoo.com 1992). There are two groups of phototrophic bacteria,
namely oxygenic phototrophic bacteria (cyanobacteria) and

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2013 John Wiley & Sons Ltd

anoxygenic phototrophic bacteria. The latter, in particular
the purple non-sulphur bacteria (PB), are evaluated in this
study.
The use of PB as a feed has several advantages over con-
ventional feed used in aquaculture. PB can be produced in
limited space on cheap substrate, have a digestible cell wall,
contain useful enzymes, may act as a probiotic and their
nutritional value can be improved by genetic manipulation
(Kobayashi & Kobayashi 2001). Furthermore, the protein
content of bacterial cells (600700 g kg 1 of the dry weight)
is superior to that of yeast (400600 g kg 1) (Sasaki et al.
1998) and is comparable to microalgae (500600 g kg 1),
egg and soybean meal (Kobayashi & Kurata 1978; Nop-
aratnaraporn et al. 1987). Bacteria can be a nutritious feed
for fish and shrimps (Hirotani et al. 1991; Cui et al. 1997).
Many PB, however, lack polyunsaturated fatty acids
(PUFA) and highly unsaturated fatty acids (HUFA), which
are essential to the survival of larval fish (Loo 2012). For
instance, koi (Cyprinus carpio) larvae were unable to sur-
vive long when given a sole diet of freeze-dried Rhodovulum
sulfidophilum grown in synthetic culture medium, but a diet
containing the bacteria supplemented with the yolk gran-
ules of hard-boiled Omega Plus hen egg significantly
improved survival and growth of koi larvae (Neik 2006).
Highly unsaturated fatty acids play a major role in sustain-
ing the structure and function of the cell membrane,
normal development and functioning of visual and neural
systems, as well as stress tolerance in young fish (Kanazawa
1997; Rainuzzo et al. 1997; Sargent et al. 1997). Poor
dietary essential fatty acids in fish gives rise to abnormal
pigmentation, fatty liver, poor swimming ability and raised
basal cortisol levels (Izquierdo 1996).
The culture of PB in wastewaters rich in lipids may
improve their content of PUFA and HUFA. At the same
time, PB can reduce the high organic loadings present in
agroindustrial wastewaters that pollute the environment if
left untreated (Kobayashi & Kobayashi 1995). Agroindus-
trial wastewaters such as palm oil mill effluent (POME)
which contain 46 g L 1 of oil and grease (Chow & Ho
2000) may, however, have applications in the aquaculture
industry because extracts of its oil droplets still contain
840 g kg 1 (wt/wt) neutral lipids and 160 g kg 1 complex
lipids (Chow & Ho 2000). POME also contains high con-
centrations of carbohydrate, protein, minerals and nitroge-
nous compounds (Phang 1990). Although the mass
production of PB by anaerobic fermentation has been esti-
mated at USD 79 kg 1 dry cells (Kim & Lee 2000), the pro-
duction cost of PB is expected to be reduced from the use
of agroindustrial wastewaters as substrate (e.g. POME).
It is however not known whether all PB can utilize oily
wastewaters and incorporate HUFA and other nutrients
into their biomass. It is also not well studied whether roti-
fers can be produced solely from a diet of nutritious bac-
teria, hence, making them useful microorganisms other
than microalgae in aquaculture. Rotifers are suitable small
zooplankton for rearing fish larvae, and their introduction
as larval food is often mandatory in the culture of certain
fish species. They act as suitable intermediaries or vehicles
of nutrients, for example, between microalgae and fish lar-
vae. In this study, three species of PB Rhodopseudomonas
palustris, Rhodobacter sphaeroides and Rhodovulum sulfido-
philum were investigated on their ability to utilize POME
as the sole source of carbon and nitrogen and to produce
HUFA. The three bacterial species were chosen for this
study based on some success in improving the survival of
larval fish and shrimps when used as a supplementary feed
(Getha et al. 1998; Azad et al. 2002; Neik 2006).
Hence, the objectives of the study were to determine (i)
the biomass production and nutritional profile of Rp. pa-
lustris, Rb. sphaeroides and Rv. sulfidophilum; (ii) whether
these PB could support the mass culture of rotifers and (iii)
the best species among the three PB for mass production
based on their HUFA level, protein level and cost-effective-
ness.
The stock cultures of previously isolated PB were obtained
from the Mycology and Plant Pathology Laboratory, Uni-
versity of Malaya. Two freshwater species, Rhodopseudo-
monas palustris strain PBUM001 and Rhodobacter
sphaeroides strain PBUM003, and one marine species Rho-
dovulum sulfidophilum strain PBUM002 were evaluated in
this study. The master stock cultures of the PB were main-
tained as stab cultures in 112 medium (Gest & Favinger
1983) solidified with 1.5% (w/v) agar. These stab cultures
were incubated at 30  2 C for 48 h and then topped with
sterile paraffin oil. The cultures were then kept at 4  2 C
(Azad et al. 2001). Prior to use, the stock cultures were pla-
ted out on agar plates and checked for purity.
Palm oil separator sludge henceforth referred to as
POME was collected from the Malaysian Palm Oil Boards
(MPOB) Experimental Palm Oil Mill, Labu, Negeri
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Aquaculture Nutrition 19; 895907 2013 John Wiley & Sons Ltd
 Sembilan, Malaysia. The fresh POME was kept in a cold
room for a day (to settle the solid particles) before it was
centrifuged (Beckman J2-M1) at 2300 g for 20 min at 4 C.
The supernatant was dispensed into two-litre plastic contain-
ers and then frozen at 20  2 C. The frozen supernatant
was thawed at room temperature when needed and filtered
through 25 lm pore size Whatman paper to further remove
fine solid particles. The pH of the supernatant was 4.55.
Preparation of bacterial stock inoculum The stock inocu-
lum of each bacterial species was produced in 112 medium.
For the marine species, 30 g of sodium chloride (NaCl)
was added to one litre of the medium. The mediums pH
was adjusted to 7.0 using acid buffer (hydrochloric acid) or
alkaline buffer (sodium hydroxide) before it was dispensed
into 20 mL McCartney bottles and autoclaved at 121 C
and 15 psi for 15 min.
A loopful of 96-h pure culture of each bacterial species
was separately inoculated into the autoclaved 112 medium
bottles. The bacterial cultures were then incubated under
anaerobic-light condition for 60 h at 30  2 C before they
were used as stock inocula in subsequent studies, unless
otherwise stated. Continuous illumination of 27.0 lmol
m 2 s 1 was provided to each bottle of bacterial culture by
a 100-watt tungsten bulb.
Comparing biomass production and growth of three species
of purple non-sulphur bacteria in POME and 112 medium
The pH of both 112 medium and 25% POME (v/v) in dis-
tilled water was adjusted to 7. As the 112 medium is a
defined nutrient formulation for optimal PB culture, it was
taken as a standard for comparison of bacterial growth in
POME. For the culture of Rv. sulfidophilum, 30 g of NaCl
was added to one litre of either 112 medium or diluted
POME. The medium was then dispensed into one-litre
Schott bottles and autoclaved at 121 C and 15 psi for
15 min. Each bottle containing 900 mL of medium was
inoculated with 10% (v/v) of the bacterial stock inoculum.
Triplicates were set up for each medium and bacteria
tested. The inoculated bottles were incubated anaerobically
under continuous light illumination of 27.0 lmol m 2 s 1
at 30  2 C. At intervals of 12 h over a period of three
days, 30 mL of the culture was collected from each inocu-
lated bottle for biomass analysis.
Biochemical analysis of biomass of purple non-sulphur bacte-
ria grown in POME and 112 medium Sufficient samples of
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Aquaculture Nutrition 19; 895907 2013 John Wiley & Sons Ltd
PB grown in either POME or 112 medium were harvested
at 60 h of cultivation. The bacterial samples were washed
several times with sterilized 0.9% (v/v) saline solution.
Samples of fresh POME, which had been centrifuged to
discard the solid particles, followed by filtration through
25 lm pore size Whatman paper, were also collected and
freeze-dried using a Christ Alpha 14 LD freeze dryer.
Protein, lipid, ash and moisture content of both freeze-
dried bacteria and POME were analysed using standard
methods of AOAC:981.10 (Kjeldahl method), AOAC:991.36
(Soxhlet extraction), AOAC923.03 and AOAC:950.46,
respectively (Association of Official Analytical Chemists
(AOAC) 1995), whereas carbohydrate was analysed using
the method as described by Pomeranz & Meloan (1987). A
muffle furnace and an oven were used to determine ash and
moisture content, respectively. The fatty acid methyl esters
(FAME) were prepared based on the International Union of
Pure and Applied Chemistry method 2.301 (Anonymous
1987). Fatty acid profiles of freeze-dried samples were deter-
mined using gas chromatography on a HP5890, while the
amino acid profiles of the samples were analysed using
Waters AccQTagTM method (Waters, Milford, MA, USA)
and run using high-performance liquid chromatography.
The biochemical analysis was carried out in triplicate
samples for each treatment.
Analytical techniques The dry weight of bacterial cell bio-
mass (g L 1) was determined according to Sawada & Rog-
ers (1977). A 10 mL of homogenous mixed bacterial
culture was transferred into a preweighed centrifuged tube
and centrifuged at 2300 g for 20 min. The cells were resus-
pended and recentrifuged twice at the same speed. The
packed cell mass in the tube was then oven-dried at 102 C
for 24 h. The tube after cooling in a desiccator was
reweighed. Triplicates were performed for each treatment.
The net dry weight of bacteria was calculated by taking the
difference between the final dry biomass and initial bacte-
rial dry biomass (0 h). The mean daily growth rate (g
day 1) was calculated by dividing the net dry weight of
bacteria by the number of days of culture.
Evaluation of PB biomass as a feed for rotifer culture All
three bacterial species grown in either 112 medium or
diluted POME medium were used as live microbial feed for
rotifer (Brachionus rotundiformis) culture. The freshwater
microalgae, Chlorella vulgaris Beijerinck commonly used as
a rotifer feed served as the control treatment for compari-
son. The rotifer feeding experiments were conducted in an
open-wall roofed hatchery.
 A stocking rate of 75 rotifers mL 1 was used and the roti-
fers were cultured in four-litre inverted amber bottles with
conical bottom (Helland et al. 1996), each with a working
volume of three litres of 10 g L 1 salinity water. This salinity
was previously evaluated to be the best among four tested
salinities (5 g L 1, 10 g L 1, 15 g L 1 and 20 g L 1) for
B. rotundiformis (Loo 2012), despite its ability to tolerate
salinities of 197 g L 1 (Treece & Davis 2000). Seawater was
diluted to 10 g L 1 salinity by mixing aged freshwater before
sterilization by UV-irradiation. No attempt was made to
manipulate or stabilize the physical conditions of the culture
water (e.g. temperature). Throughout the rearing period,
water quality parameters such as dissolved oxygen (DO) con-
centration (mg L 1), temperature (C) and pH were moni-
tored daily using a YSI 550A DO meter and YSI 100 pH
meter. Ammoniacal-nitrogen (mg L 1) level of the cultured
water was measured at two-day intervals using HACHs
8155-salicylate method (DR/2010 Spectrophotometer proce-
dure manual, HACH company, Loveland, CO, USA).
The stocked rotifers were fed thrice a day and each feed
treatment was executed in triplicates. Bacteria and microal-
gae were fed directly to the rotifers in aqueous form, pre-
pared by measuring the required wet weight of bacterial or
microalgal biomass (15 g) and suspending the cells in one
litre of autoclaved 10 g L 1 salinity water to give a feed
stock containing 1.68 g dry weight feed L 1. For each tank
of three-litre culture water, rotifers were fed a total daily
ration of 50 mL of the aqueous feed stock equivalent to
approximately 0.08 g dry weight of feed biomass. Through-
out the culture period, 50% water replacement was made
daily before feeding.
Rotifer density (individuals mL 1) was counted daily
under a microscope and three readings were taken from
each culture tank. Only rotifers fed with the biomass of
Rv. sulfidophilum were selected for biochemical analysis
because the PB contained both EPA and DHA, which are
beneficial to larval fish culture. These enriched rotifers were
harvested at 96 h, rinsed several times with filtered distilled
water to discard salt, immediately frozen for a day and
then freeze-dried before biochemical analysis. Biochemical
analysis of rotifer samples was carried out in triplicates.
The best PB for mass production was selected based sim-
ply on the computed total ranked score of each species
derived from the following criteria: HUFA (EPA+DHA)
content, protein content, rotifer density and cost-effective-
ness of their production. The latter took into consideration
the growth rate or biomass concentrations of the PB. A
ranked score of 1, 2 or 3 was assigned to each criterion, with
the highest score assigned to the best PB for a particular
criterion. The HUFA criterion was given a double weightage
for EPA+DHA given their importance in fish culture, but a
zero is assigned to either fatty acid in their absence.
Two-way ANOVA and post hoc Tukeys HSD were used to
test whether there was any difference in (i) bacterial biomass
production among the three PB species grown in POME or
112 medium by incubation time (0 h, 12 h,72 h), (ii) roti-
fer production among feed treatments (PB, microalgae) by
day of culture (Day 0, 1, 6) and (iii) ammoniacal-nitrogen,
pH, DO and temperature values of culture water among
feed treatments and day of culture (Day 0, 1, 6).
Each variable resulting from the proximate, fatty acid
and amino acid analyses of enriched bacteria were tested
using one-way ANOVA (six treatments) and rotifers (two
treatments) using Students t-test. All statistical analysis
was performed using the computer software Statistica, ver-
sion 10. Principal components analysis (PCA) as a multi-
variate procedure was used to interpret the amino acid and
fatty acid profiles of the PB. PCA was run on the software
CANOCO 4.5 (Ter Braak & Smilauer 2002).
The growth of the three tested species of PB was supported
by 25% POME (v/v) in distilled water (Fig. 1). The bacte-
rial biomass (0.381.91 g L 1) and daily growth rate
(0.150.64 g day 1) of the three species of PB grown in
POME were however significantly lower (P < 0.01) than
their biomass (1.443.34 g L 1) and daily growth rate
(0.581.11 g day 1) in 112 medium (Table 1). Overall,
Rb. sphaeroides had the highest dry biomass and growth
rate in both POME and 112 medium when compared to
Rp. palustris and Rv. sulfidophilum (Table 1). The initial
dry biomass of all PB grown in POME was higher than
those grown in 112 medium due to the presence of solid
particles in the diluted POME medium.
Within the 3-day culture period, all species of bacteria
showed a gradual increase in biomass without the exponen-
tial growth phase in POME and 112 medium, except
Rb. sphaeroides in 112 medium at 60 h (Fig. 1).
In all treatments, the bacterial biomass increased with
incubation time except Rp. palustris and Rv. sulfidophilum
grown in POME as their biomass production started to
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Aquaculture Nutrition 19; 895907 2013 John Wiley & Sons Ltd
 4.00 influenced (P < 0.01) by the type of feed. The highest roti-
B1-syn B1-POME
3.50
fer density (395 ind. mL 1) was obtained from Rp. palustris
Bacterial dry biomass (g l1)

KS-syn KS-POME
3.00
PD1-syn PD1-POME
2.50
2.00
1.50
1.00
0.50
0.00
0 12 24
Hours
Figure 1 Biomass production of three species of PB grown in 112
medium or palm oil mill effluent (POME) (30  2 C; light illumi-
nation of 27.0 lmol m 2 s 1; pH 7) (data pointsmean of triplicate
values). Samples: B1-syn = Rp. palustris grown in 112 medium;
KS-syn = Rb. sphaeroides grown in 112 medium; PD1-
syn = Rv. sulfidophilum grown in 112 medium; B1-pome = Rp. pa-
lustris grown in POME; KS-pome = Rb. sphaeroides grown in
POME; and PD1-pome = Rv. sulfidophilum grown in POME.
level off after 60 h (Fig. 1). It was also observed that
increasing the incubation time to 72 h did not significantly
increase biomass production from 60 h (P > 0.05).
The rotifers grew well when PB grown in POME was used
as the sole feed. Rotifer peak production was significantly
grown in 112 medium, while the lowest rotifer density (102
ind. mL 1) was obtained from Rv. sulfidophilum grown in
POME. Peak rotifer densities for the different feeds were
highly variable, from 2 to 5 days (Table 2). In all feed
treatments, the rotifer populations fell or stagnated after
three days of culture, except those fed Rp. palustris grown
in POME and Chlorella vulgaris. Rotifers fed Rp. palustris
had significantly higher density and lower variability than
rotifers fed C. vulgaris. In general, significantly (P < 0.01)
36 48 60 72 higher densities of rotifers were obtained from PB grown in
112 medium as compared to PB grown in POME. Rp. pa-
lustris as feed gave the highest rotifer density, followed by
Rb. sphaeroides and Rv. sulfidophilum. However, Rv. sulfi-
dophilum gave the lowest variability in rotifer density com-
pared with Rb. sphaeroides and Rp. palustris (Table 2).
The recorded environmental parameters among treatments
were not significantly different (P > 0.05). In all treatments,
the water pH, DO and temperature fell within the ranges
of 6.718.34, 4.476.89 mg L 1 and 27.732.8 C, respec-
tively. However, the ammoniacal-nitrogen concentrations
varied among treatments. Tanks with rotifers fed PB grown
in 112 medium (Rp. palustris: 1.8011.0 mg L 1; Rv. sulfi-
dophilum: 1.603.60 mg L 1; Rb. sphaeroides: 0.412.40 mg
L 1) had significantly higher (P < 0.05) ammoniacal-nitro-
gen concentrations than tanks with rotifers fed PB grown
in POME (Rp. palustris: 0.030.51 mg L 1; Rv. sulfidophi-

Table 1 Maximum net dry biomass (mean  SD g L 1) and average daily growth rate of PB strain grown in 112 medium or palm oil mill
effluent (POME) (30  2 C; light illumination of 27.0 lmol m 2 s 1; pH 7) over 3 days of culture

Initial dry Final dry Net dry Daily growth Incubation

Treatments biomass1 (g L 1) at 0 h biomass1 (g L 1) biomass1 (g L 1) rate1 (g day 1) time (h)

112 medium
Rp. palustris 0.18  0.00 2.64  0.37 2.46  0.37 0.82 72
(PBUM001)
Rv. sulfidophilum 0.38  0.00 1.82  0.09 1.44  0.09 0.58 60
(PBUM002)
Rb. sphaeroides 0.33  0.00 3.67  0.07 3.34  0.07 1.11 72
(PBUM003)
POME
Rp. palustris 1.15  0.00 1.53  0.40 0.38  0.40 0.15 60
(PBUM001)
Rv. sulfidophilum 0.98  0.00 1.59  0.14 0.61  0.14 0.20 72
(PBUM002)
Rb. sphaeroides 0.66  0.00 2.57  0.07 1.91  0.07 0.64 72
(PBUM003)
1
Mean of triplicate values.
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Aquaculture Nutrition 19; 895907 2013 John Wiley & Sons Ltd
Table 2 Density of rotifers (mean  SD ind. mL 1) fed with bacterial or microalgal biomass in 10 g L 1
salinity water. Peak density is
bold faced

Treatments Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6

1
112 medium
Rp. palustris 75  0bc 18  3c 113  25bc 395  30a 248  41abc 91  14bc 39  3c
(PBUM001)
Rv. sulfidophilum 75  0bc 58  10c 127  12bc 147  12abc 125  14bc 92  18bc 13  14c
(PBUM002)
Rb. sphaeroides 75  0bc 38  11c 225  139abc 198  283abc 145  223abc 16  31c 0  0c
(PBUM003)
Palm oil mill effluent (POME)1
Rp. palustris 75  0bc 32  11c 129  87abc 198  45abc 246  24abc 332  19ab 225  88abc
(PBUM001)
Rv. sulfidophilum 75  0bc 50  3c 102  3bc 94  21bc 72  12c 15  13c 0  0c
(PBUM002)
Rb. sphaeroides 75  0bc 21  19c 96  89bc 113  178bc 107  173bc 27  43c 5  9c
(PBUM003)
Chlorella vulgaris 75  0bc 80  23bc 155  93abc 175  64abc 250  128abc 186  129abc 136  110abc
1
Mean of nine replicate values; mean rotifer density that does not share a common superscript letter differ significantly (P < 0.01).

lum: 0.801.04 mg L 1; Rb. sphaeroides: 0.372.20 mg L 1) POME contained significant amounts of DHA and LNA.
and rotifers fed microalgae (0.090.54 mg L 1). Rb. sphaeroides grown in 112 medium had significant
amounts of LA and EPA, while Rb. sphaeroides grown in
POME had higher amounts of LA and LNA (Fig. 2).

PB grown in 112 medium had significantly higher

(P < 0.05) amounts of protein and moisture content than
PB grown in POME (Table 3). Rb. sphaeroides grown in
POME recorded the lowest amount of PUFA (16.9% of
total fatty acids), while Rp. palustris grown in POME had
the highest PUFA (54.3%) as compared to other treat-
ments (Table 3). Only Rv. sulfidophilum grown in POME,
which contained 42.8% PUFA, had both DHA and EPA
(Table 3).
The rotifers fed PB grown in POME had significantly
higher (P < 0.05) contents of protein, moisture, oleic acid
(OA, C18:1n-9c), linoleic acid (LA, C18:2n-6), arachidonic
acid (ARA, C20:4n-6), EPA, DHA, total saturated fatty
acids (SFA), total monounsaturated fatty acids (MUFA)
and n-3 PUFA, as compared to rotifers fed PB grown in
112 medium, which instead had significantly higher
(P < 0.05) contents of lipid, carbohydrate, total PUFA and
n-6 PUFA (Table 3).
Principal components analysis biplots of the bacterial
samples and the types of fatty acids and amino acids they
contained are shown in Fig. 2. The first (x-axis) and second
PCA axis explained 43.2% and 31.7% of the total varia-
tion, respectively. Rp. palustris grown in POME contained
higher amounts of a-linolenic acid (LNA, C18:3n-3), ARA
and EPA. Rv. sulfidophilum grown in 112 medium or
The present study has shown that PB species, Rp. palustris,
Rb. sphaeroides or Rv. sulfidophilum grown in POME had
significantly lower (P < 0.01) mean daily growth rates as
compared to PB species grown in 112 medium. As POME
is not nutrient deficient, this is likely due to the culture
medium itself. POME is naturally dark brown and contains
a high amount of suspended particles (Ma 2000). Under
anaerobic-light condition, these properties would reduce
the transmission of light that is required by PB, whereas
the yellowish-tinged 112 medium is clear and thus pro-
motes better cell growth. Nevertheless, the bacteria could
grow in POME despite its high organic loading. Rb. sph-
aeroides grew well in POME as compared to the other
tested bacterial species (Table 1). Basically, the differences
in mass culture production among bacterial species are due
to limitation in light transfer, type and concentration of
substrate and the species of PB (Kim & Lee 2000).
The selection of the right PB species and strain for aqua-
culture purpose is however not only based on their capabil-
ity of rapid growth but also their nutritional profile. PB
generally lack PUFA and HUFA due to their low lipid
compositions and fatty acids (Imhoff & Imhoff 1995).
Their fatty acid profile is basically influenced by the
cultivation medium (Imhoff & Imhoff 1995). Thus, it is
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Aquaculture Nutrition 19; 895907 2013 John Wiley & Sons Ltd
 Table 3 Proximate composition (g kg 1), fatty acids (% total fatty acids) and amino acids (g 100 g 1 amino acids) of palm oil mill effluent (POME), biomass of three species of
freeze-dried purple non-sulphur bacteria (Rp. palustris, Rb. sphaeroides, Rv. sulfidophilum) and biomass of enriched rotifers. Bacteria were cultured in either synthetic 112 medium or
POME

Rotifers
Biomass of Biomass of Biomass of Biomass of Biomass of fed with biomass Rotifers fed with
Rp. palustris Rp. palustris Rb. sphaeroides Rb. sphaeroides Rv. sulfidophilum Biomass of of Rv. sulfidophilum biomass of
Nutritional grown in grown in grown in grown in grown in 112 Rv. sulfidophilum grown in 112 Rv. sulfidophilum
profiles1 POME 112 medium POME 112 medium POME medium grown in POME medium grown in POME

Proximate
composition
Protein, gkg 1 107 616 360 599 511 637 443 422 476
Lipid, gkg 1 56 86 58 71 61 44 74 123 67
1
Carbohydrate, gkg 471 54 10.2 43 84 81 74 69 55
Ash, gkg 1 241 157 420 193 268 132 334 110 106 ns
Moisture, gkg 1 125 87 60 94 76 106 75 276 296
Total,% 100 100 100 100 100 100 100 100 100
Energy, kJ 1184 1449 995 1348 1231 1373 1147 1294 1142
Fatty acids
(% total
fatty acids)
Stearic acid 4.0 0.0 0.0 2.2 3.5 0.8 0.9 9.3 7.5

Aquaculture Nutrition 19; 895907 2013 John Wiley & Sons Ltd
(C18:0)

..............................................................................................
OA 35.8 0.0 0.0 1.4 0.0 2.5 1.5 17.5 25.8
LA 10.7 0.0 0.0 5.6 4.7 0.3 0.9 3.8 4.9
ns
LNA 0.9 0.0 4.5 0.0 2.3 2.9 2.2 0.5 0.6
ARA 0.0 0.0 10.3 0.0 0.0 0.0 0.0 0.0 0.7
EPA 0.0 0.0 18.0 29.1 1.0 0.0 3.9 0.9 2.6
DHA 0.0 0.0 0.0 0.0 0.0 0.8 2.6 0.8 1.2
Total SFA 52.0 16.9 33.0 29.1 47.0 53.6 45.4 36.5 41.2
Total MUFA 36.4 53.6 12.8 27.6 36.1 18.9 11.8 33.3 40.9
Total PUFA 11.6 28.3 54.3 43.3 16.9 27.4 42.8 30.2 17.9
ns
Percentage 100.0 98.8 100.0 100.0 100.0 100.0 100.0 100.0 100.0
total fatty acids
n-3 PUFA 0.9 0.0 22.5 30.3 3.4 3.7 10.7 2.8 5.4
n-6 PUFA 10.7 15.4 18.2 11.9 7.3 9.9 18.2 23.1 12.0
ARA/EPA 0.0 0.6 0.0 0.0 0.0 0.0 0.3
DHA/EPA 0.0 0.0 0.0 0.0 0.7 0.9 0.5
DHA/ARA 0.0 0.0 1.8
Amino acids
(g 100 g 1
amino acids)
ns
Total essential 43.6 51.8 51.4 52.8 51.2 51.0 51.2 46.5 47.0
amino acids
ns
Total non-essential 56.4 48.1 48.6 47.1 48.9 49.1 48.8 53.3 53.0
amino acids

Mean pair of enriched rotifers showed significant difference (P  0.05) except those labelled ns (not significant).
1
Mean of triplicate values.
1.5

C 12:0

C 17:0 Arg
Glu C 18:2n6
C 4:0
PD1-syn C 18:3n6
C 22:6n3 (DHA)
C 18:1n9 (OA)

C 22:2
PD1-pome
C 22:1n9
Iso Val
Asp
Leu
C 21:0

C 20:3n3
C 18:3n3 (LNA)
C 20:0
Pro Figure 2 PCA of the fatty acid compo-
C 20:2
sitions of freeze-dried bacterial bio-
C 15:1 mass. Samples (filled circles): B1-
Ala
B1-syn syn = biomass of Rp. palustris grown
C 14:0
C 18:1n9
in 112 medium; PD1-syn = biomass of
C 16:0 C 14:1
Met C 6:0 C 18:0
KS-pome Rv. sulfidophilum grown in 112 med-
C 13:0
C 23:0
C 22:0
C 24:1 C 16:1 ium; KS-syn = biomass of Rb. sphaero-
C 20:1n9 C 20:4n6 (ARA)
B1-pome C 17:1 Thr
C 20:3n6 ides grown in 112 medium; B1-
C 24:0 C 8:0
C 11:0 C 15:0 C 18:2n6 (LA)
C 10:0
pome = biomass of Rp. palustris grown
KS-syn
in POME; PD1-pome = biomass of
C 20:5n3 (EPA)
Rv. sulfidophilum grown in POME; KS-
Gly
pome = biomass of Rb. sphaeroides
Ser Phe
1.0

Tyr
Lys grown in POME; Variables (arrows):
His
37 fatty acids and 16 amino acids in
1.0 1.5 conventional notations.

important that bacteria destined for use in aquaculture are All the four important fatty acids, namely stearic acid,
cultured on an appropriate substrate. For example, OA, LA and LNA, which are the basic precursors of
Rv. sulfidophilum grown in sardine processing wastewater PUFA and HUFA (Milan & Sakayu 1999) were found in
had low amounts of PUFA and no HUFA despite having only Rv. sulfidophilum, whereas Rp. palustris and Rb. sph-
more than 95% C-14, C-16 and C-18 straight chain SFA aeroides lacked one of the four important fatty acids
and MUFA; this low-quality feed did not promote good (Table 3; Fig. 2). Although POME-cultured Rp. palustris
survival of penaeid shrimp larvae (Azad et al. 2002). and Rb. sphaeroides contained LNA, they appear to have
Cultured in POME, both Rp. palustris and Rb. sphaeroides limited ability to biosynthesis PUFA and HUFA as DHA
lack DHA, while Rv. sulfidophilum contains significant was absent. However, both species contained a significantly
amounts of EPA and DHA (Table 3; Fig. 2). Nevertheless, higher amount of EPA (Table 3; Fig. 2) as compared to
the fatty acid profiles of these PB are still superior to their cul- their substrate (POME) (Table 3). The higher amount of
ture in 112 medium. Cultured in 112 medium, Rb. sphaeroides EPA in POME-cultured Rp. palustris, as compared to
contains only EPA, Rv. sulfidophilum only DHA and Rp. pa- Rb. sphaeroides and Rv. sulfidophilum cultured in POME, is
lustris none of the essential fatty acids mentioned above likely the result of conversion of ARA to EPA since ARA
(Table 3; Fig. 2). The present study shows that all the PB are was significantly higher in Rp. palustris. On the other hand,
capable of incorporating the nutrients from POME into their EPA in both POME-cultured Rb. sphaeroides and Rv. sulfi-
cells, and perhaps even producing HUFA. For instance, Rho- dophilum seems directly synthesized from LNA because
dopseudomonas spp. are reportedly able to produce DHA ARA was not found in both bacteria (Table 3; Fig. 2).
(Singh & Ward 1997; Ratledge 2001). The presence of EPA Methionine has been reported to be the limiting amino
and DHA in POME-cultured Rv. sulfidophilum indicates their acid in PB (Shipman et al. 1975). The present study reveals
biosynthesis as POME does not contain these two fatty acids that all PB tested contained fair amounts of methionine.
(Table 3). Thus, this study is the first to report on the ability The amount of methionine in all three species of PB grown
of Rv. sulfidophilum to biosynthesis HUFA. in POME ranged from 2.56 to 3.54 g 100 g 1 amino acids,
..............................................................................................

Aquaculture Nutrition 19; 895907 2013 John Wiley & Sons Ltd
 whereas those grown in 112 medium ranged from 2.71 to
6.98 g 100 g 1 amino acids. These values are higher than
those (2.20 g 100 g 1 amino acids) reported in the Food
and Agriculture Organization (FAO) guidelines for amino
acid composition in single-cell protein (FAO 1980). There-
fore, POME is a rich substrate for enriching both the
amino acid and fatty acid profiles of PB.
As a feed, PB grown in POME improves the nutritional
profile of rotifers. For instance, rotifers can biosynthesize
ARA when cultured on Rv. sulfidophilum grown in POME
but not on the bacteria grown in 112 medium. The reason
is not clear, but LA, the precursor of ARA, as present in
the former (0.9% of total fatty acids) was three times more
than in the latter (0.3%). Rotifers are also able to biosyn-
thesize HUFA from LNA although at a very low rate
(Lubzens et al. 1985). This ability is exemplified in the pres-
ent study where EPA is present in rotifers if fed with
Rv. sulfidophilum grown in 112 medium despite the bacteria
having no EPA but LNA (Table 3).
An issue with rotifers in aquaculture is that its high lipid
content often results in a lower content of protein that is
necessary to promote high growth rate in larval fish
(Rnnestad et al. 2003a). The protein and amino acid
requirements of many species of exogenously feeding fish
larvae are quite high due to muscle protein deposition, turn-
over and catabolism of amino acids, which serve as an
important energy source (Rnnestad et al. 2003b). Conse-
quently, high lipid rotifers with lower protein content may
not fulfil the protein and amino acid requirements of larval
fish. This is observed in rotifers fed Rv. sulfidophilum grown
in synthetic medium, with the result that the rotifers had a
decreased protein level (from 637 g kg 1 to 422 g kg 1) but
increased lipids (Table 3). On the other hand, rotifers cul-
tured from Rv. sulfidophilum grown in POME were quite
lean with higher protein content than their bacterial diet
(from 443 to 476 g kg 1) and decreased lipid content. This
consistent response by rotifers to Rv. sulfidophilum grown in
POME is advantageous in terms of larval fish nutrition and
cost-effectiveness. For instance, rotifers cultured from
Rv. sulfidophilum grown in POME need not be further
enriched prior to feeding the larvae of the marble goby,
Oxyeleotris marmorata (Loo et al. 2012a).
None of the three species of enriched PB given as live
feed to rotifers were adverse to their production. It has
been reported that PB as a feed supplement favours the
growth of zooplankton such as brine shrimp and is supe-
rior to green algae (Kobayashi 1995). However, it is not
obvious why in this study rotifers fed with PB cultured in
112 medium grew denser than those fed with POME-cul-
tured PB, or why Rp. palustris regardless of the growth
medium was superior to others in term of stimulating roti-
fer growth. There were however differences between the PB
feed; those in 112 medium were 100% pure bacteria,
whereas those in POME were a mixture of 79% bacteria
and 21% fine plant particulates. The latter presents a prob-
lem that cannot be entirely removed in practical terms

Table 4 Comparisons on the advantages, limitations and costs of production among the three PB species grown in palm oil mill effluent
(POME), with selection criteria for best PB to mass culture

Criteria Parameter Rp. palustris Rb. sphaeroides Rv. sulfidophilum

Bacterial Bacterial biomass (g L 1) 0.38  0.40 1.91  0.07 0.61  0.14

performance Daily growth rate (g day 1) 0.15 0.64 0.20
Nutritional profiles Contained highest Contained highest amounts Contained highest
amounts of carbohydrate, of protein, LA, total amounts of lipid,
LNA, ARA, EPA, total PUFA, SFA and MUFA; no DHA DHA and n-6 PUFA
n-3 and n-6 PUFA; no DHA
The ability to biosynthesis HUFA No No Yes
Rotifer Rotifer density (ind. mL 1) 332  19 113  178 102  3
production (in batch culture)
Stability of rotifer culture Moderate variability Highest variability Lowest variability

Peak density reached 5 days 3 days 2 days

Cost of bacterial Estimated cost USD 35.35 USD 8.71 USD 23.29
production (kg 1 dry weight)
Selection criteria1 EPA+DHA 3+0 1+0 2+3
Protein 1 3 2
Production costs 1 3 2
Rotifer density 3 2 1
Total rank score 8 9 10
1
Based on rank scores of 1, 2 or 3, with highest score assigned to highest lipid content, protein content, rotifer density or lowest pro-
duction cost; absence of DHA is assigned zero. Production costs consider biomass concentration of bacteria.
..............................................................................................

Aquaculture Nutrition 19; 895907 2013 John Wiley & Sons Ltd
Table 5 Estimated cost to produce 1 kg dry weight of Rhodovulum sulfidophilum grown in palm oil mill effluent (POME) based on 0.61 g
bacterial dry biomass harvested from 1 L of Rv. sulfidophilum after 60 h of cultivation

Phototrophic bacterium Chemicals/Materials Unit cost (MYR) Amount Cost (MYR) Cost (USD)a
1
(a) To produce 163.9 L of Dipotassium phosphate 3.94 kg 163.9 g 0.65
inoculum (bacteria grown (K2HPO4)
1
in 112 medium) needed Magnesium sulphate 0.32 kg 82.0 g 0.03
for (b) below (MgSO4.7H2O)
Yeast extract 1.51 kg 1 1639.3 g 2.48
NaClb 0.32 kg 1 4917.9 g 1.57
Freshwater Commercial rate: 163.9 L 0.34
1 m3 or 1000 L = MYR 2.07
Electricity (8 bulbs) Commercial rate: MYR 2.34 9 8 18.72
1kWh = MYR 0.39 x a
100-watt tungsten
bulb = 0.1 kWh
x culture period
(60 h) = MYR 2.34
20 L culture container 20 unit 1 8 units 1.10
(reusable for 1 year)
1
100-watt tungsten 3.00 bulb 8 bulbs 4.29
bulbs (bulb life = 14 d)
Non-skilled labourc 3.33 h 1 or 900 2h 6.66
month 1, working
9 h day 1
Total 35.82 11.37
(b) To produce 1 kg dry Inoculum from (a) 163.9 L 35.82
weight of bacteria grown Freshwater Commercial rate: 1 m3 1106.6 L 2.29
in POME and sold in or 1000 L = MYR 2.07
ziplock bagg POME 368.9 L 0.00d
Transportation cost 100 for 4000 L of POME 368.9 L of POME 9.22
to collect POME
20 L culture container 20 unit 1 82 units 11.23
1 L sealed ziplock bag 0.06 bag 1 1 bage 0.06
NaCl 0.32 kg 1 4426.5 g 1.42
Non-skilled labour 3.33 h 1 or 900 month 1, 4 hf 13.32
working 9 h day 1
Total 73.36 23.29h
a
1 USD = 3.15 MYR; b NaCl is not needed for freshwater strain; c Preparation of bacterial inoculum; d POME is not costed because it is an
effluent discharge; e A 1 L ziplock bag is required to pack 1 kg dry weight of bacteria grown in POME; f include preparation of POME
medium and inoculation (1.5 h) on first day and harvesting of bacteria grown in POME and cleaning of culture containers (2.5 h) on last
day; g bacteria are cultured under anaerobic condition, in open area under direct sunlight. The condensed cells are air-dried in the open
area; h The total cost of production does not include the cost of land and building.

(Loo 2012). Nevertheless, this difference in bacterial bio-
mass had been shown not to affect rotifer production (Loo
2012). On the other hand, the 112 medium may contain
unknown growth stimulants (e.g. vitamins) even for the
rotifers or Rp. palustris may contain the unknown factor
for rapid rotifer growth.
One important factor that affects prey selection by roti-
fers is particle or prey size (Rothhaupt 1990). Despite their
ability to capture, ingest and process particles of various
sizes, food clearance must be sufficiently fast to sustain a
constant ingestion rate in rotifers. This depends on particle
size where a range of 115 lm seems suitable for most
Brachionus species (Rothhaupt 1990; Hansen et al. 1997;
Baer et al. 2008). The cellular sizes of Rp. palustris
(1.8 lm x 0.5 lm) (Maheswari 1997), Rb. sphaeroides
(1.2 lm x 0.8 lm) (Khanom 2001) and Rv. sulfidophilum
(0.9 lm x 0.4 lm) (Azad 2004), all fell within the range of
suitable prey size. Thus, it is possible that B. rotundiformis
may prefer the largest (i.e. Rp. palustris) of the three bacte-
rial species or the larger green alga C. vulgaris (510 lm),
in spite of the fact that PB can self-flocculate into larger
cell masses. However, apart from prey size, rotifers appar-
ently graze non-selectively on foods (Rothhaupt 1990).
The best density for B. rotundiformis in the batch culture
system based on microalgae as feed was 1,500 rotifers
mL 1 (Zhang et al. 2005) and for B. plicatilis was 800 roti-
fers mL 1 (Penglase et al. 2011). The present study based
on PB shows much lower rotifer density (Table 2) than in
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Aquaculture Nutrition 19; 895907 2013 John Wiley & Sons Ltd
 these studies. However, the culture conditions were differ-
ent among these studies. For example, 32 g L 1 salinity
and a controlled temperature of 29 C in Zhang et al.
(2005) and 1822 g L 1 salinity and 25 C in Penglase
et al. (2011), as compared to 10 g L 1 salinity and 27.7
32.8 C in the present study. The lower salinity used in our
study (for the purpose of culturing marble goby) may be
the reason why our rotifer densities were lower as salinity
has a greater effect on rotifer availability than temperature
(Fielder et al. 2000). Nonetheless, the early decline of our
rotifer population at 10 g L 1 is likely attributed to the
large variability in water temperature observed throughout
the rearing period in the open-air hatchery as all other
culture water parameters including pH, DO and ammonia-
cal-nitrogen were within safe limits. Assavaaree et al.
(2003) reported that B. rotundiformis fed with microalgae
at 15 g L 1 salinity exhibited reduced growth rate and
higher resting egg production when water temperature was
brought down from 30 C to 25 C. Thus, a stable water
temperature sustained in an indoor hatchery appears neces-
sary to maintain a higher rotifer density.
The batch culture system based on unfiltered or unsettled
POME-grown Rv. sulfidophilum as sole feed can sustain a
much higher mean rotifer density of 900 mL 1 and a mean
egg ratio of 0.270.56 egg rotifer 1 (Loo et al. 2012b).
These rotifers fed to larval marble goby gave much higher
larval fish survival of 71.481.9% at 5 g L 1 salinity and
25.131.7 C (Loo 2012), as compared to the best survival
of 20% at 10 g L 1 salinity and 25.330.5 C based on
microalgae fed rotifers tested at 0, 5, 10, 15, 20 and 30 g
L 1 salinity (Senoo et al. 2008). Importantly, the batch cul-
ture method of producing rotifers for fish larvae is viewed
as suitable for many small-scale hatcheries in the tropics
where the technological know-how for producing microal-
gae is low and production cost is high. The commercial
production of PB grown in POME is viable given that one
kilogram of dry weight POME-cultured PB can be pro-
duced at a cost of USD 8.7135.35 (Table 4), compared
with the lowest production cost of USD 46.44 for microal-
gae (Spirulina and Chlorella) (Brennan & Owende 2009). In
particular, the cost of producing one kilogram of dry bio-
mass of Rv. sulfidophilum grown in POME at USD 23.29
includes 31.10% for raw materials, 29.10% for utilities and
39.80% for labour and others (Table 5).
In conclusion, the advantages of using the PB as an
aquaculture feed include (1) rich in protein and PUFA, (2)
give reasonable yield of rotifers without the need for fur-
ther enrichment, (3) economical and (4) simplicity in PB
culture which is amenable to low technology small-scale
..............................................................................................
Aquaculture Nutrition 19; 895907 2013 John Wiley & Sons Ltd
farms. PB can grow in POME although their growth rates
are slower than in synthetic 112 medium. However, PB
produced from POME has a better nutritional quality par-
ticularly in essential fatty acid compositions. Therefore,
they are expected to have a wider application in larval fish
culture, as for instance in the larviculture of the problem-
atic marble goby (Loo et al. 2012a). Further investigation
on their wider use in the hatchery production of other fish
species is thus warranted if POME-grown bacteria are to
be mass produced on a commercial scale. Although Rp. pa-
lustris is the fastest grower in both substrates tested and
Rb. sphaeroides gives the highest rotifer density, only
Rv. sulfidophilum grown in POME contains both EPA and
DHA, gives the lowest variability in rotifer density and
incurs moderate production cost. Finally, the total ranked
score based on the selection criteria is highest for Rv. sulfi-
dophilum (Table 4). Hence, of the three species investigated
in this study, Rv. sulfidophilum grown in POME would be
the best live feed for rotifer culture.
The authors wish to thank University of Malaya for pro-
viding research grants (FS271/2007C, PS166/2008A, PS192/
2009A and PS310/2010A) and research facilities. We also
thank the staff at the University of Malaya Marine Culture
Unit and Mycology and Plant Pathology Laboratory for
all their valuable assistance.
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