The relationship between bacterial load and the microalga Chaetoceros muelleri was analyzed in a scale-up experiment. The microalga was grown during five days in a 0.4-L Erlenmeyer flask, 2-L Fernbach flask, 18-L Carboy and 400-L column, during which the cell density of C. muelleri, the abundance of heterotrophic bacteria, Vibrio spp., and total bacteria were determined. The highest specific growth rates (μ) of C. muelleri occurred during the first day of culture (0.88 to 2.29 d-1). Highest cell density was recorded on the fifth day at the 2-L (7.62 × 106 cells·mL-1) and 18-L (6.32 × 106 cells·mL-1), coinciding with the maximum counts of heterotrophic bacteria (16.55 × 105 and >30 × 105 CFU·mL-1, respectively). There was a high correlation (0.80, 0.75, 0.85; p <0.05) between microalgal cell density and total bacteria in the first three culture volumes and a low correlation (0.27; p = 0.34) at 400-L column. The highest mean concentration of total bacteria (884.13 × 105 cells·mL-1) during the five days occurred at 18-L Carboy. The concentration of total bacteria at all levels was always higher than that of heterotrophic bacteria. The average ratio of heterotrophic to total bacteria was higher in the 2-L (0.0108) and 18-L (0.0172) cultures. The high biomass of C. muelleri and the presence of Vibrio spp. at the 18-L and 400-L levels indicate that it is necessary to establish programs to prevent diseases and economic losses caused by pathogenic bacteria in penaeid shrimp farming.
An alternative approach to reduce the problem of contamination by pathogenic bacteria in the farming of molluscs, fish and crustaceans is the use of inert artificial diets, which in turn reduces production costs. In the case of shrimp farming, alternative feeds have been used to prevent the presence of pathogens, increase larval survival and reduce economic losses [
Compared with artificial diets, microalgae have high nutritional values and are easily ingested because of their size [
Several microbiological studies have focused on obtaining higher yields and survival rates in the culture of invertebrates, but bacterial load in microalgal cultures has been little studied and insufficient attention has been paid to microalgae as food and main source of contamination in culture systems. The purpose of this paper is to evaluate the abundance of Vibrio bacteria that cause diseases in cultured organisms, as well as the relationship between C. muelleri and heterotrophic and total bacteria during scale-up of the microalgal culture.
The marine diatom Chaetoceros muelleri was obtained from the collection of microalgae at the Institute of Oceanological Research of the Autonomous University of Baja California. This species was cultured at a temperature of 20˚C ± 1˚C using f/2 medium [
Heterotrophic and Vibrio bacteria were enumerated daily (in duplicate samples) by the spread plate method using Zobell Marine Agar and Thiosulfate Citrate Bile Sucrose (TCBS) agar, respectively. Both media were inoculated with 0.1 mL of serially diluted sterile aged seawater to guarantee from 30 to 300 colony forming units CFU∙mL−1. The plates were incubated at 25˚C and the colonies were counted five days after inoculation for heterotrophs and after 48 and 72 hours for Vibrio spp. using a Quebec colony counter (values expressed as CFU∙mL−1).
Total bacteria were enumerated daily (in duplicate samples) by epifluorescence microscopy. For this, 5 mL of the microalgal culture was fixed with 0.25 mL of 40% formaldehyde (final concentration of 2%) and filtered using black polycarbonate filters (0.22 μm pore size) and GF/F fiberglass filters as support. The samples were stained with DAPI (4’, 6-diamidino-2-phenylindole) at a final concentration of 2 μg∙mL−1 for 5 minutes in darkness [
The bacterial counts were performed on an Axiovert 200 (Carl Zeiss) microscope, with a 50-W lamp and UV excitation wavelength. Images were taken (25 - 40 bacterial cells per field) with an Olympus Dp70 camera adapted to the microscope. The results are expressed as number of bacteria per milliliter.
The growth of Chaetoceros muelleri during the scale-up process is shown in
Mean growth of heterotrophic bacteria in C. muelleri cultures is shown in
Bacterial growth at 2-L Fernbach was similar to that at 0.4-L Erlenmeyer, the concentration of heterotrophic bacteria increasing from 0.15 × 105 CFU∙mL−1 on the day of inoculation (Day 0) to 16.55 × 105 CFU∙mL−1 on Day 5, with a bacteria to microalga ratio of 0.046 and 0.217 for the respective days. The inoculum size (0.3 × 106
Time | V | HB | HB:M | DSB | HB:DS | Vibrio |
---|---|---|---|---|---|---|
(Day) | (L) | (CFU∙mL−1 × 105) | (Cell∙mL−1 × 105) | CFU | ||
0 | 0.4 | 0.02 | 0.101 | 7.01 | 0.0032 | 0 |
2 | 0.15 | 0.046 | 45.24 | 0.0033 | 0 | |
18 | 3.43 | 0.322 | 104.49 | 0.0328 | 0 | |
400 | 1.19 | 0.362 | 139.03 | 0.0085 | 0 | |
1 | 0.4 | 0.20 | 0.192 | 19.29 | 0.0102 | 0 |
2 | 0.17 | 0.013 | 74.49 | 0.0023 | 0 | |
18 | >3.00 | 0.117 | 423.53 | 0.0071 | 0 | |
400 | 2.69 | 0.235 | 271.91 | 0.0099 | 0 | |
2 | 0.4 | 0.23 | 0.023 | 24.62 | 0.0095 | 0 |
2 | 3.07 | 0.125 | 96.74 | 0.0317 | 0 | |
18 | >3.00 | 0.087 | 576.03 | 0.0052 | 10 | |
400 | >3.00 | 0.149 | 574.08 | 0.0052 | 0 | |
3 | 0.4 | 0.20 | 0.012 | 44.37 | 0.0044 | 0 |
2 | 1.45 | 0.028 | 231.63 | 0.0062 | 0 | |
18 | 19.53 | 0.376 | 1028.69 | 0.0190 | 0 | |
400 | >3.00 | 0.131 | 956.10 | 0.0031 | 30 | |
4 | 0.4 | 1.24 | 0.061 | 191.88 | 0.0006 | 0 |
2 | 1.75 | 0.026 | 328.99 | 0.0053 | 0 | |
18 | 26.38 | 0.424 | 1053.07 | 0.0250 | 0 | |
400 | 9.60 | 0.412 | 480.25 | 0.0200 | 25 | |
5 | 0.4 | 1.74 | 0.082 | 125.40 | 0.0138 | 0 |
2 | 16.55 | 0.217 | 1036.37 | 0.1600 | 0 | |
18 | >30.00 | 0.474 | 2118.95 | 0.0142 | 0 | |
400 | 1.55 | 0.060 | 307.04 | 0.0050 | 0 |
V = Volume Per Liter; HB = Heterotrophic Bacteria; CFU = Colony Formers Units mL−1 × 105; HB:M = Ratio Hetrotrophic Bacteria Microalga; DSB = Dapi Stain Bacteria; HB:DS = Ratio Heterotrophic Bacteria Dapi Stain.
cells∙mL−1) was one order of magnitude higher than at 0.4-L Erlenmeyer, and consequently there was a greater concentration of both bacteria and microalgae.
Mean initial concentration of heterotrophic bacteria at 18-L Carboy was 3.43 × 105 CFU∙mL−1, increasing to >30 × 105 CFU∙mL−1 on the fifth day, with a bacteria to microalga ratio of 0.322 and 0.474 for Days 0 and 5, respectively. The concentration of heterotrophic bacteria was one order of magnitude higher than that recorded at 2-L Fernbach and two orders of magnitude higher than at 0.4-L Erlenmeyer.
The concentration of heterotrophic bacteria at 400-L column was 1.19 × 105 CFU∙mL−1 on Day zero. It increased to 9.60 × 105 CFU∙mL−1 on Day 4 and decreased to 1.55 × 105 CFU∙mL−1 on Day 5, the latter concentration being lower than that observed at the 18-L level. The bacteria to microalga ratio were 0.362 and 0.412 for Days 0 and 4, respectively.
Mean initial concentration of bacteria at 0.4-L Erlenmeyer was 7.01 × 105 cells∙mL−1, increasing to a maximum of 191.88 × 105 cells∙mL−1 on day 4 of the culture (
The concentrations of total bacteria in all the cultures were always higher than those of heterotrophic bacteria. The mean heterotrophic to total bacteria ratios during the five days of culture were higher in the Fernbach (0.0108) and Carboy (0.0172) assays than in the Erlenmeyer (0.0079) and column (0.0086) assays cultures.
The highest mean concentration of Vibrio spp. during the culture of C. muelleri occurred at 18-L Carboy on day 2 (10 CFU∙mL−1), and on days 3 (30 CFU∙mL−1) and 4 (25 CFU∙mL−1) at 400-L column (
Cell densities of C. muelleri were similar to the values reported by other authors [
The culture of C. muelleri was not axenic since the bacterial population increased over time and during the scale-up process. Also other authors have indicated that it is difficult to maintain microalgal cultures free of bacteria because of their epiphytic relationship [
The maximum values of heterotrophic bacteria in all the cultures (
volumes, the concentration of heterotrophic bacteria increased as microalgal biomass increased, whereas at 400- L column both cell density and the number of heterotrophic bacteria decreased. The abundance of heterotrophic bacteria suggests a symbiotic relationship with microalgae, possibly because of the exudates released by the latter. According to [
The decrease in the growth rate of C. muelleri at 400-L column can be associated with the decrease in bacterial biomass. The high correlation values (0.80, 0.75, 0.85; p ˂ 0.05) obtained by direct counts for C. muelleri and total bacteria in the first three culture volumes indicate some type of dependency between these microorganisms. The low correlation coefficient (0.27, p = 0.34) found at 400-L column can be attributed to environmental variables like temperature, due to use incandescent light which modify bacterial growth.
Vibrio bacteria were not detected at 0.4 L or 2 L due to seawater treatment (filtration, ultraviolet radiation) and because the culture medium was sterilized in autoclave. Similar results were obtained by [
The high biomasses of C. muelleri and the presence of Vibrio bacteria detected in this study indicate that it is necessary to establish prevention and control systems in aquaculture laboratories since there have been worldwide reports of diseases attributed to Vibrio spp. in fish [
This research comprised part of the projects Production of Microalgae and Bacteriological Shellfish Studies in Baja California at Instituto de Investigaciones Oceanológicas of the Universidad Autónoma de Baja California, with support from the UABC.