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					               Graeme Ditri, Jason Ledger, Andrew Sorensen, Niel Wilson (group 4).


   HALOPHILE BACTERIA AS BIOMASS FOR AQUACULTURE


Abstract


In this experiment Halobacteria were grown with two different substrates, namely
glucose & acetate, a steady state for the former was not achieved as an acidic pH was
being recorded the latter showed a productivity (R) of ≈ 0.50 h-1, a yield coefficient (Y)
of ≈ 0.42 and a biomass concentration (X) of ≈ 8.5g/L.


Introduction


Aquaculture, although practiced by ancient civilizations, is still in commercial terms a
relatively fledgling industry with tremendous growth potential. A food source valued by
many aquaculture industries as the first exogenous feed of many invertebrates and larvae
fishes is Artemia sp1. Artemia are crustaceans of the family Artemiidae the most
common of which is Artemia fransiscana, which is found in large numbers in the salt
lakes of North America and made available commercially as an aquaculture feed2. In
addition A. fransiscana is the species found in the concentrator ponds of Dampier Salt in
Port Headland, Western Australia where it serves the dual purpose of being collectable as
an aquaculture feed and assisting in the water clarification of the salt concentrator ponds,
thus enhancing evaporation 1. Artemia will only survive in fresh water for about up to
five or six hours3 and when used as a feed for fresh water species this needs to be
considered as they can only be fed at a rate that will be consumed in this time, dead
artemia are both wasteful and will foul the water.


Artemia begin their life, depending upon the conditions of the parent, either as a free-
swimming nauplius or as a metabolically dormant cyst2. The cysts are able to withstand
severe environmental conditions including up to seven years of anoxia and evidence has
been published4 suggesting a unique metabolic pathway exists rather than complete
metabolic inactivity.
Whether life starts as a cyst or as a free swimming individual, the transition to sexually
mature artemia occurs over about 12 molts and after about 14 molts and eight days the
terminal size is reached2, resulting in approximately a 500 fold increase in biomass3. An
average length is 8mm but as long as 20mm have been recorded3. Under optimum
conditions a female may produce up to 300 nauplii every 4 days for a life span of
approximately 50 days. Cyst production is induced by high salinity, low feed and large
fluctuations in dissolved oxygen between night and day3. Optimum temperature is
between 25 and 30 degrees centigrade and optimum pH is 82.


Artemia are none-selective filter feeders and will eat whatever is in suspension, with that
in mind it is therefore possible to selectively enrich the artemia depending upon the
requirements of the final consumer, for example the essential fatty acids EPA and DHA
are of importance to marine aquaculture5 and it is beneficial to load artemia with these
fatty acids prior to use as an aquaculture feed.


Opportunity exists to identify or even develop food sources for artemia that enrich them
according to the requirements of the higher order aquaculturalist. In addition it may be
possible to identify a waste product that costs industry to dispose of in a biologically
responsible manner. From a commercial perspective it would be of obvious benefit if a
negative cost feed could be established, the benefit lies also in a potentially, ecological
responsible recycling of resources.


Artemia are relatively hardy organisms that remain viable in a range of environmental
conditions, they are however quite sensitive to pH levels2, which in turn can a function
of, feed type.


This procedure aims to achieve three principle objectives. Firstly a steady state chemostat
is to be arrived at; secondly the input and outputs are to be varied such that optimum
biomass production is achieved. Finally the bacterial biomass needs to be suitable to be
used as feed for artemia.




Materials & Methods




Figure 1: Chemostat setup.

Equipment
Mechanical stirrer
1L reactor
Aquarium
Airflow regulator
2x pumps
Aquarium air pump, airstone
2x thermostats
2x 5L Schott bottles.
Ice box
Retort stand
2x Timers
Silicon tubing
Clamps, connectors
Reagents
D-glucose
Sodium Acetate
Sodium chloride
Yeast extract
Salt-water aquarium mix
Anti foaming agent- polypropylene glycol
Ice
Artemia
Protocol
Week 1
The reactor was first run as a batch culture. Non-sterile culture of halobacteria was grown
in 20g/L glucose and 5 g/L yeast extract until an optical density of around 10 was reached
at 600nm. Airflow was kept high at 200 L/h to achieve optimal oxygen uptake rate.
Pumps were calibrated.


Week 2
A feed vessel (5L) and product vessel (5L) were attached to the reactor. The feed vessel
contained 20g/L glucose, 5g/L yeast extract, 30g/L NaCl and was kept on ice to prevent
microbial growth. The medium flow was set to 60mL/h. Polypropylene glycol was added
as necessary to avoid foam overflow of the reactor. The reactor was kept at a level of
approximately 1 litre and a temperature of 28ºC.


A standard curve was constructed by preparing dilutions of the product suspension. Five
product samples were prepared in 100mL each with the following percent culture; 20, 40,
60, 80 and 100. The 100% sample was then diluted 1 in 5 to obtain accurate optical
density readings at 600nm. The product samples were then placed in separate foil
containers and oven dried to determine dry weight. Absorbance versus dry weight curves
was then plotted.


Approximately 5 litres of salt water mix and 40 artemia were added to the aquarium.
The aquarium was fitted with a pump and air stone to maintain adequate levels of oxygen
in the water. A temperature of ≈ 28ºC was maintained with a thermostat. At this stage it
was not being fed from the reactor (as in fig. 1). The artemia were fed with aliquots from
the product bottle, which was brought to the appropriate pH by the addition of 5M
NaOH.


Aliquots from the reactor were read at 600nm and read of the standard curve to determine
the biomass concentration. pH readings were taken each day.


The product was refrigerated to be used as artemia feed. Feed that was not used was
autoclaved.


Week 3
The feed was changed to 20g/L acetate, 5g/L yeast extract and 3g/L NaCl.
The same protocol was carried out as in week 2.
Results


                                      Biomass Production
                    9

                    8

                    7
      Biomass (g)




                    6

                    5

                    4

                    3

                    2

                    1

                    0
                        15   16        17          18           19           20            21           22

                                                    Time (days)


Figure 2: Biomass production reaching a steady state of ≈ 8.5 g/L using acetate and yeast extract as the
substrate, at a pH of 8.5 (suitable as a feed source for Artemia).


                                                pH range

                    10
                     9
                     8
                     7
                     6
    pH




                     5
                     4
                     3
                     2
                     1
                     0
                         0        5             10                   15              20                25
                                                        Day

Figure 3: pH range from the initial batch culture fed on glucose through to the final steady state with an
acetate substrate.
Table 1: Biomass productivity and the relative yield coefficients using acetate & yeast extract as the
substrate.
                                                                      -1
 Time (days)      Biomass X (g)         Y (X/S)         R (D x X) (h )
        16        5.429109072          0.271455         0.325746544
        18        8.392783875          0.419639         0.503567032
        20          8.4273793          0.421369         0.505642758
        21        8.456208822           0.42281         0.507372529


At steady state the productivity (R) was ≈ 0.50 h-1, the yield coefficient (Y) was ≈ 0.42
and the biomass concentration (X) was ≈ 8.5g/L.


Discussion


The major problem encountered through running a continuous flow system is that the
aeration rate was not sufficient as the maximum airflow rate that was allowed was
250L/h. The airflow rate for the chemostat was therefore kept high at 200L/h to ensure
that sufficient oxygen was being supplied to culture. Initially there was an continual
increase in biomass density with glucose as a substrate and there was a substantial
decrease in the pH of the reaction vessel and the product collected, indicating that the
halobacteria which were present in the reactor were strictly fermentative bacteria or
perhaps facultative anaerobes.


The primary objective of a bacterial cell is to utilize substrate as fast as possible and if a
cell is respiring, as a result of NADH oxidation being slower than required, then there
will be an accumulation of intermediates in the glycolytic pathway, in particular fructose
bis-phosphate and pyruvate. These intermediates are of crucial importance as they then
act as allosteric activators for vital enzymes required for fermentation pathways and
inhibitors of enzymes of the pyruvate dehydrogenase complex required for the
conversion of pyruvate to acetyl-CoA and entry into the TCA cycle6. Basically in the
presence of high glucose concentrations the crabtree effect, as this above phenomenon is
known, will result in an increase in fermentation which can best be described with
reference to the following diagram6.
Crabtree effect 6 [Accessed 11/10/02]
Therefore the subsequent decrease in pH with glucose as a substrate can be explained by
the crabtree effect. The high levels of glucose in the initial feed resulted in a chain of
events, which led to an increase in the amount fructose bis-phosphate present, and
ultimately the activation of various fermentation enzymes. High levels of glucose in the
feed also resulted in the inhibition in pyruvate carboxylase, which is part of the pyruvate
dehydrogenase complex involved in the conversion of pyruvate to acetyl-CoA, so the
cells switched to fermentation rather than respiration6.


Looking at Figure 3 further emphasizes this point, as there is a substantial decrease in pH
with the initial chemostat feed between day 8 and day 16 indicating an accumulation of
acetate. Further analysis with the glucose analyzer indicated that there was still
significant glucose present when measured with product collections on day 11 and day 15
with 0.035g/l of residual glucose present on day 11 and 0.111g/l present on day 15.
Therefore the substrate feed was switched to sodium acetate to prevent a marked decrease
in pH as artemia are sensitive to changes in the pH range and require an optimal pH of 8
for proliferation. Sodium acetate was chosen as a substrate feed as it contains the
necessary ‘building blocks’ for growth of bacteria in a continuous flow culture, and
looking at Figure 3 the pH remained fairly constant between a pH of 8-9 which is
comparable with the optimum pH for growth and proliferation of artemia.
In this experiment a non-sterile chemostat was used and this resulted in a few problems
whilst the chemostat was being run with blocked lines and blocked needles, which
resulted in a loss of reactor volume. For future reference it may be necessary to alter the
chemostat set-up or perhaps even use a sterile batch culture and feed vessel although this
is not necessary to achieve the objectives of the experiment. In addition the dilution rate
could have been varied in order to obtain an accurate determination of Ymax, Umax and
Rmax for the chemostat.


Also for future reference the salt concentration of the culture should be comparable with
seawater, which is 3.6% not 30%, which was the initial salt concentration of the
chemostat. Although some halobacteria can tolerate these high salt concentrations
artemia will grow significantly slower at higher salt concentrations and optimum growth
conditions for artemia occur between 30-35 ppt (1.022-1.0260 density) saline and low
light levels. A minimal amount of light is necessary for hatching as the greater the light
intensity the greater the kinetic energy of the artemia so they exhaust there energy
reserves and cannot multiply. At low light levels the artemia will spread throughout the
water column, swimming slowly so there will be good food conservation. Of particular
importance is the feed rate of product to the artemia, as artemia will be adversely
affected in the water quality is poor and possesses to much organic matter. Therefore
there must be some significant washout of volume from the aquarium to maintain a high
water quality, which is required for, continued survival and proliferation of artemia.
Approximately 20% washout per week is sufficient to maintain good water quality3.


The most important factor with reference to artemia is the oxygen level in the water, as
they will determine what the artemia will consume. If there is a good oxygen supply
present in the aquarium the artemia appear to be pale pink and yellow and in this ideal
condition growth and reproduction is rapid. However, if there is a low oxygen level in the
water with a significant quantity of organic matter or high salinity, the artemia will feed
on bacteria and detritus and under these conditions they will produce haemoglobin and
look red or orange in colour3.
Basically, if low oxygen supply is maintained the colony size will drop so a good air
supply must be maintained so food will remain in suspension were it can be filtered out
and it will promote a good oxygen supply3.


For future reference the use of airstones needs to be considered carefully as airstones can
produce fine bubbles that can lodge in the swimming appendages and some artemia may
ingest the fine bubbles, which will all, result in considerable death in the colony.
Moderate aeration with very coarse or no airstones and good water quality are all of
significant importance in maintaining a population of adult artemia. Finally, artemia feed
constantly, so faster and more sustainable growth rates will be achieved if the artemia are
fed constantly over a 24-hour period. If all of these requirements are met then the
objectives of the chemostat experiment may then be more easily met.
References

1
    Molony B. & Parry G. (2002). Preliminary assessment and seasonal fluctuations in the
fish biota inhabiting the concentrator ponds of Dampier Salt, Port Headland with options
for the potential applications of results. Department of Fisheries, WA Marine Research
Division.


2
     Fox R, 1994, Invertebrate Anatomy [online], Department of Biology, Lander
University Greenwood SC, Available : http://www.lander.edu/rsfox/artemia.html (Accessed
3/10/02)


3
    Schumann Kai, 1995, Artemia (Brime Shrimp) FAQ 1.1 [online], Portland State
University, USA, Available: http://www.ee.pdx.edu/~davidr/discus/articles/artemia.html (Accessed
3/10/02)


4
    Warner A H., & Clegg J S. (2001).Diguanosine nucleotide metabolism and the survival
of artemia embryos during years of continuous anoxia. European Journal of
Biochemistry. 268:1568-1576.



5
    Narciso L. Pousao-Ferreira P., Passos A. & Luis O. (1999). HUFA content and
DHA/EPA improvement of Artemia sp.with commercial oils during different enrichment
periods. Aquaculture Research. 30:21-24.

6
    NeegeeAnn Polytechnic, [Last updated] 1998, Metabolic control of the Crabtree effect,
School of Life Sciences and Chemical Technology, Available: http://www.np.edu.sg/~dept-
bio/biochemical_engineering/lectures/ferm1/bioferm16c.htm (Accessed 11/10/02)
Appendix


                                          Glucose Calibration Curve
                          9

                          8
                          7
  Glucose (g/L)




                          6
                          5                                                       y = 1.9731x - 0.0459
                                                                                      R2 = 0.9996
                          4

                          3

                          2

                          1

                          0
                              0       1            2                3                 4                    5
                                                    Dextrose (g/L)
Figure 4: Glucose calibration curve for the determination of residual D-glucose (dextrose), in the product
vessel.




                                      O.D vs Dry Weight of Product 1

                          2.5

                              2
            O.D (600nm)




                          1.5

                              1
                                                                             y = 0.5911x + 0.3249
                                                                                  R2 = 0.9919
                          0.5

                              0
                                  0   0.5          1              1.5             2                  2.5
                                                       Mass (g)

Figure 5: Calibration curve for the determination of biomass concentration (g/L, X), with a glucose
(20g/L) and yeast extract (5g/L) substrate medium.
                                     O.D vs Dry Weight Product 2

                    0.5
                   0.45
                    0.4
    O.D (600 nm)



                   0.35
                    0.3
                   0.25                                                      y = 0.5203x + 0.0485
                    0.2                                                           R2 = 0.9977

                   0.15
                    0.1
                   0.05
                      0
                          0         0.2            0.4              0.6            0.8                 1
                                                         Mass (g)

Figure 6: Calibration curve for the determination of biomass concentration (g/L, X), with an acetate
(20g/L) and yeast extract (5g/L) substrate medium.

Table 2: Biomass production during week 3, determining the yield coefficient and productivity.
(calculations viewable in the attached spreadsheet (chemostat project calcs)).

 Time                O.D        Biomass X    [S]                    D (flow rate (1mL/min) /reactor
 (days)              (600)nm        (g)      g/L     Y (X/S)                    vol (1L))                  R (D x X)
   16                   2.85   5.429109072   20      0.27146                      0.06                     0.325747
   18                  4.392   8.392783875   20      0.41964                      0.06                     0.503567
   20                   4.41    8.4273793    20      0.42137                      0.06                     0.505643
   21                  4.425   8.456208822   20      0.42281                      0.06                     0.507373