The Cultivation of Single Cell Protein as Feed to Brine Shrimps
N301 Industrial and Environmental Microbiology
Group 7 – Cynthia Wong, Isabelle Lean, Jean Tan and Sabrina Yeo
Abstract
Production of single cell protein (SCP) is economically profitable as only a low startup cost is required to give a large profit as a high biomass was achieved. A dilution rate between 0.016 h-1 to 0.032h-1 was idealto prevent washout of the bacteria. Undiluted substrate concentration is used to culture the bacteria as the rate of uptake is very fast. Artemia salina was used to test the product. However, the Artemia sp. did not manage to survive after a few days.
Introduction
Single Cell Protein (SCP)
The discovery of using substrates derived from renewable and nonrenewable resources for SCP first initiated soon after the production of microbial cells from hydrocarbons in the early 1960s. Several reviews had based solely on the technical and economic aspects of SCP production from a variety of substrates including carbohydrates, agricultural wastes, hydrocarbons, and petrochemicals such as methanol and ethanol (Refer to Litchfield’s paper reference [1] & [2]). This paper emphasizes the production of activated sludge SCP from acetate for animal feed use rather than human food, since foodstuff applications appear more attractive from both technical and economic standpoints in the near future. The feed is then supply to the brine shrimps, artemia sp. of, which is grown in fresh water. Hence, the research also accentuates on the production of brine shrimps, which in turn possesses economic impact on marine agricultural.
SCP in economic perspective is likely to have its major import as a world protein resource when it is produced in large scale as a bulk protein commodity (Cooney & Makiguchi from SCP journal, page 66). To fill this role, SCP must have a low manufacturing cost. The important point is to maximize the conversion of the carbon energy source to protein. Therefore, the production cost is taken into account for this experiment.
Activated sludge
The most important factor in organism selection is safety. The organism must be known to be a non-pathogen, and it must not produce toxins especially if the organism is to be used in feed or food. Nevertheless, one usually wants to select an organism with maximum growth rate and minimum maintenance requirements (SCP book, Page 12). Activated sludge fits the above criteria and small amount of the bacteria is needed for cultivation of suspended SCP.
Continuous culture system
Large-scale production of SCP as a low cost feed protein is a process ideally suited for continuous culture. Continuous culture offers the advantages of high volumetric productivity per unit of capital investment, ease of process control, and production of a consistent product under optimal steady state culture conditions (Cooney & Makiguchi from SCP journal, page 72).
The advantages of a continuous culture system over a batch culture system are less amount of time is needed to produce a certain volume and less ‘down time’ between runs, which means minimal loss of productivity and more control over final product quality (Cord-Ruwisch, 1997; Glick and Pasternak, 1994).
Artemia salina
Artemia salina, an anostracan phyllopod crustacean of approximately 12mm in length. It has especially appeared in numerous man-made salterns where salt is extracted from seawater by solar evaporation (R. M. Bond, 1937). These creatures are known to ingest SCP and have the water clean and colorless, and it has served as feed to sea animals such as whales.
Materials and Methods
A continuous chemostat was used to investigate aerobic production of nitrate from acetate medium. The chemostat was set up as shown in figure A.
The reactor vessel was placed in a water bath that was maintained at the temperature of the reactor at between 24-26C.
Table I. Composition of Acetate Medium for Bacteria Growth
Sodium Acetate / 33.3 g/2LNaHCO3 / 6.25 g/2L
NaNO3 / 2 g/2L
KH2PO4 / 2.2 g/2L
Yeast extract / 2.5 g/2L
MgSO4.7H2O / 1.25 g/2L
Trace Element Solution / 1.25 mL/L for 1x concentration
Results
The chemostat for SCP production was set up and readings of culture conditions at several dilution rates were taken daily over a period of two weeks.
Cycle 1 / Cycle 2Days / Day 1 / Day 2 / Day 3 / Day 4 / Day 5 / Day 6 / Day 7 / Day 8 / Day 9 / Day 10
Date / 11-Sep / 12-Sep / 13-Sep / 16-Sep / 19-Sep / 20-Sep / 23-Sep / 24-Sep / 25-Sep / 26-Sep
Time / 12pm / 11am / 12.30pm / 11.30am / 9.30am / 10.55am / 10.10am / 9.10am / 11.40am / 10am
Time (hr) / 19.5 / 23 / 25.5 / 71 / 19 / 24.25 / 71.25 / 23 / 25.5 / 22.33
Temp (*C) / 25 / 25 / 25 / 19 / 20 / 23 / 25 / 23 / 22 / 23
pH / 8.86 / 9.55 / 9 / 9.09 / 9 / 9.48 / 9.5 / 9.31 / 9.74 / 9.7
Airflow (L/h) / 125 / 125 / 125 / 150 / 135 / 200 / 200 / 200 / 200 / 200
Reactor vol (mL) / 1250 / 1250 / 1250 / 1250 / 1250 / 1250 / 1250 / 1250 / 1250 / 1250
Recirculation rate (L/h) / 0.05 / 0.05 / 0.05 / 0.05 / 0.05 / 0.05 / 0.05 / 0.05 / 0.05 / 0.05
Vol of feed used (mL) / 800 / 800 / 1000 / 3600 / 1000 / 700 / 2000 / 1000 / 800 / 850
Vol of harvest (mL) / 800 / 800 / 1000 / 3600 / 1000 / 700 / 2000 / 1000 / 800 / 850
Medium flowrate (L/h) / 0.04 / 0.04 / 0.04 / 0.04 / 0.05 / 0.03 / 0.02 / 0.04 / 0.03 / 0.03
Wtg of filter paper (g) / 1.86 / 1.85 / 1.85 / 1.86 / 1.8 / 2.06 / 1.91 / 1.91 / 1.93 / 1.88
Dry wtg (g) / 1.92 / 1.9 / 1.91 / 1.88 / 1.87 / 2.09 / 1.94 / 1.96 / 2 / 1.95
Biomass (g/10ml) / 0.06 / 0.05 / 0.06 / 0.02 / 0.03 / 0.03 / 0.03 / 0.05 / 0.07 / 0.07
Biomass (g/L) / 6 / 5 / 6 / 2 / 3 / 3 / 3 / 5 / 7 / 7
Optical density (600nm) / 0.37 / 0.845 / 0.661 / 0.245 / 0.292 / 0.323 / 1.379 / 1.474 / 1.53 / 1.496
Gas chromatography analysis
Acetic conc. in reactor (M) / 33.0211 / 12.74326 / 1.529669 / 1.436546 / 23.47776 / 11.68319 / 2.152396 / 1.716846 / 0.721094 / 0.668525
Observations
Outflow observation / chalky / chalky / yellow / yellow / chalky / yellow / chalky / chalky / chalky / yellow chalk
Reactor observation / chalky / chalky / yellow / yellow / slight yellow / yellow / chalky / chalky / chalky / yellow chalk
Dilution rate D h-1 / 0.032 / 0.032 / 0.032 / 0.032 / 0.04 / 0.024 / 0.016 / 0.032 / 0.024 / 0.024
Biomass yield coe Y (g/gS) / 0.182 / 0.392 / 3.922 / 1.392 / 0.128 / 0.257 / 1.394 / 2.912 / 9.707 / 10.471
OD (600nm) of tank H2O / 0.063 / 0.063 / 0.046 / 0.021
OD after addition of harvest / 0.106 / 0.063 / 0.055
OD after consumption / 0.108 / 0.052
Survivors count / 129 / 115 / 95 / 60 / 31 / 0 / 0 / 0 / 0 / 0
Product production rate RP (g/L h) is determine at a later stage where GC is involved.
Product production rate (g/L/ h) / 0.31 / 0.22 / 0.24 / 0.028 / 0.16 / 0.12 / 0.042 / 0.217 / 0.27 / 0.3
Volumetric Productivity (g/L cell/h) / 0.192 / 0.16 / 0.192 / 0.064 / 0.12 / 0.072 / 0.048 / 0.16 / 0.168 / 0.168
Production Cost
Assuming the cost of sludge is $0.2/kg/L, we managed to produce 19g/L for the first cycle (Day1 to 4) and 28g/L for the second cycle (Day 5 to 10)
Production Cost for Cycle 1 ($) / 0.010526
Production Cost for Cycle 2 ($) / 0.007143
We target to cell the Single Cell Protein at $5/kg/L
STANDARD CURVE
[acetate] / Area counts
0 / 0
1.4 / 117627
2.8 / 229435
4.2 / 303248
5.6 / 419401
GC AREA COUNTS FOR SAMPLES
GC sample / SWW
Area counts / [acetate] / [acetate] / [acetate]
mM / mM / mM
OLD D1 / 497496 / 6.604221 / OLD D1 / 6.604221 / NEW D1 / 4.695553 / OLD D1 / 33.02111 / NEW D1 / 23.47776
OLD D2 / 191990 / 2.548653 / OLD D2 / 2.548653 / NEW D2 / 2.336639 / OLD D2 / 12.74326 / NEW D2 / 11.68319
OLD D3 / 23046 / 0.305934 / OLD D3 / 0.305934 / NEW D3 / 0.430479 / OLD D3 / 1.529669 / NEW D3 / 2.152396
OLD D4 / 21643 / 0.287309 / OLD D4 / 0.287309 / NEW D4 / 0.343369 / OLD D4 / 1.436546 / NEW D4 / 1.716846
NEW D1 / 353716 / 4.695553 / NEW D5 / 0.144219 / NEW D5 / 0.721094
NEW D2 / 176019 / 2.336639 / NEW D6 / 0.133705 / NEW D6 / 0.668525
NEW D3 / 32428 / 0.430479
NEW D4 / 25866 / 0.343369
NEW D5 / 10864 / 0.144219
NEW D6 / 10072 / 0.133705
Discussion
From the results obtained (refer to graph 1), there was an increase in the volumetric productivity with increase in dilution rate from 0.016 h-1 to 0.032h-1. At 0.04h-1, there was a decrease in the volumetric productivity. Theoretically, there should be an increase in the volumetric productivity with respect to an increase in the dilution rate. However, the decrease in volumetric productivity at 0.04h-1 could be due to washout of biomass.
There was a huge drop in the biomass concentration from day 3 to 4 (Cycle 1). This could be due to the lack of nutrients, as an initial 5X substrate concentration was used. Therefore, a more concentrated substrate concentration (2X) was used when reculturing (Cycle 2). In cycle 1, we started with a high biomass concentration but we had a low substrate concentration, therefore, the cells were depleted of nutrients, hence, causing rapid decrease in biomass concentration. In addition, there was a fluctuation in the biomass concentration from day 1 to 3. This was most likely due to experimental errors. In cycle 2, a low biomass concentration and higher substrate concentration was used to start the chemostat, hence, cell growth with an initial lag phase followed by exponential and stationary phase was observed.
Gas chromatography (GC) was performed to determine the acetate concentration in the reactor. This could in turn be used to determine the uptake rate of acetate by the bacteria. The acetate concentration in the reactor was calculated to be approximately 33mmol/L. The results obtained from GC for cycle 1, 32.48mmol/L, corresponded with that of the calculated value. However, the GC results from cycle 2 was 23.4776mmol/L. This could be explained by the fact that a lower amount of acetate was added during preparation.
The graph of acetate uptake rate by the bacteria for cycle 1 and 2 were approximately identical. This means that the degradation rate of acetate by the bacteria is the same for the 2 cycles, thus, metabolism is the same.
Artemia salina which are known to ingest SCP and in turn makes the water clean and colorless were used to test our product. The experiment started out with 129 Artemia sp. however, the Artemia sp. slowly died off. On the tenth day, there were no Artemia sp. alive. This could be due to unsuitable water conditions and temperature. There was a great decrease between day 3 to 4 (weekend) and day 4 to 5 (started a new chemostat culture). This great decrease could be explained by the lack of feed.
This experiment is economically profitable because a low cost is required for the chemostat. Large profits can be made from selling SCPs and brine shrimps.
Recommendations for future work
-Diluted substrate concentration should not be used when starting the chemostat
-High biomass concentration should be avoided when starting the chemostat
-Concentrated sludge suspension should not be added into the reactor vessel
-Low air flow rate should not be used
-Dilution rate of above 0.032h-1 should be avoided.
Figure A