Control of the Harmful Alga Microcystis aeruginosa and Absorption of Nitrogen and Phosphorus by Candida utilis
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- Kong, Y., Xu, X., Zhu, L. et al. Appl Biochem Biotechnol (2013) 169: 88. doi:10.1007/s12010-012-9946-7
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This study is aimed at controlling eutrophication through converting the nutrients such as nitrogen and phosphorus into microbial protein and simultaneously inhibiting the growth of Microcystis aeruginosa by Candida utilis. C. utilis and M. aeruginosa (initial cell density was 2.25 × 107 and 4.15 × 107 cells·mL−1) were cultured together in the absence or presence of a carbon source (glucose) during a 10-day experiment. In the absence of carbon source, the measured removal efficiencies of NH4+–N and PO43−–P were 41.39 ± 2.19 % and 82.93 ± 3.95 %, respectively, at the second day, with the removal efficiency of 67.82 ± 2.29 % for M. aeruginosa at the fourth day. In contrast, the removal efficiencies of NH4+–N and PO43−–P were increased to 87.45 ± 4.25 % and 83.73 ± 3.55 %, respectively, while the removal efficiency of M. aeruginosa decreased to 37.89 ± 8.41 % in the presence of the carbon source (C/N = 2:1). These results showed that the growth of M. aeruginosa was inhibited by C. utilis. Our finding sheds light on a novel potential approach for yeast to consume nutrients and control harmful algal during bloom events.
KeywordsEutrophicationBiological controlCandida utilisMicrocystis aeruginosaCompetition
Eutrophication has become a serious threat to many lakes and reservoirs all over the world, causing damage to biodiversity and the equilibrium of aquatic ecosystems [7, 9, 10, 13, 23, 36]. Various bloom-forming phytoplankton, such as Microcystis, Anabaena, Oscillatoria, and Aphanizomenon, release toxins into water bodies and cause illness and/or death in wildlife and humans [18, 19, 25, 30]. Among these bloom-forming phytoplankton, the most common harmful algal species that causes frequent harmful algal blooms (HABs) events is Microcystis aeruginosa, found in freshwater bodies worldwide, and the blooms caused by M. aeruginosa can persist for months or even years [8, 31].
In aquatic ecosystems, phytoplankton and bacteria are the most numerous and abundant inhabitants. Bacteria may stimulate or inhibit the growth of phytoplankton through nutrient regeneration, production of stimulative or inhibitory substances, or other means [26, 33]. Currently, many countries are seeking suitable biological control agents to moderate the M. aeruginosa blooms, and most studies have been focused on mitigating the effects of eutrophication [11, 14, 15, 20]. As a result, the nitrogen and phosphorus remain in the water and may lead to HABs afterwards. Hence, as the limiting elements of primary productivity in most freshwater ecosystems, both nitrogen and phosphorus should be considered as control targets for the restoration of eutrophic water bodies [6, 32].
Several studies have achieved effective removal of nutrients such as nitrogen and phosphorus in eutrophic water by using aquatic macrophytes or fishes [5, 12, 16, 24]. This approach (assimilating N and P from water bodies to produce biomass in aquatic plants and fishes) is somewhat effective for controlling freshwater eutrophication and can provide economic returns by generating valuable products, e.g., biogas, biofertilizer, biomaterial, and even animal food . However, there are also some shortcomings for removing nitrogen and phosphorus by aquatic macrophytes or fishes. For example, it takes a long time to remove the nutrients.
In recent years, yeast strains have been employed to treat organic wastewater and wastes in order to produce microbial biomass, and satisfactory outcomes have been achieved [2–4, 28, 29]. These studies led to our hypothesis that yeasts might be able to utilize the nitrogen and phosphorus from eutrophic water bodies and inhibit algal blooms at the same time. In this study, the hypothesis was investigated by a controlled laboratory experiment in which the blue-green alga M. aeruginosa was used as a representative of more susceptible algae.
Investigating different yeast strains on the effect of removing the nitrogen and phosphorus;
The removal and growth inhibition of algae by C. utilis in synthetic water;
Study of the influence of C/N ratio of water samples on the removal of N/P and the inhibition of M. aeruginosa.
Materials and Methods
Strains and Culture Conditions
The Saccharomyces cerevisiae 609, Saccharomyces cerevisiae A7, and Candida utilis F87 that can utilize nitrogen source effectively were obtained from Laboratory of Resource and Environmental Microbiology (Wuhan Polytechnic University). Potato dextrose agar (PDA) medium (potato extract 1,000 mL and dextrose 20 g) was used for pre-cultivation. The yeasts were cultured under a constant temperature of 28 °C and shaken at 100 revolutions per minute (rpm) for 2 days before use.
M. aeruginosa FACHB-905 was purchased from the Freshwater Algae Culture Collection of Institute of Hydrobiology, Chinese Academy of Sciences (Wuhan, China) and cultured under standard conditions: sterilized BG11 medium , 2,000 lx white light, light/dark = 14 h:10 h, 25 °C, for about 7 days to reach the log phase before used as inoculants.
The basic parameters of lake water quality
Dissolved total phosphorus (DTP) (mg·L−1)
Dissolved total nitrogen (DTN) (mg·L−1)
Ammonium nitrogen (NH4+–N) (mg·L−1)
Chemical oxygen demand (CODCr) (mg·L−1)
Concentration of chlorophyll a (mg·L−1)
Algal density (cells·L−1)
2.5 × 105
Total bacteria (cfu·L−1)
1.0 × 106
2.5 × 104
The Removal Efficiency of Nitrogen and Phosphorus
To test the removal efficiency of nitrogen and phosphorus with different yeasts, water samples were prepared individually by placing 100 mL of freshly collected water in 250-mL sterile Erlenmeyer flasks stoppered with cotton wool bungs. Two milliliters of S. cerevisiae 609, S. cerevisiae A7, or C. utilis F87 (with the initial cell densities of 6.0 × 107, 4.0 × 107, and 5.45 × 107 cells·mL−1, respectively) was added to each flask aseptically. Two milliliters of sterile water was added to the flask as control. The samples were cultured at 28 °C and shaken at 100 rpm for 2 days. Triplicate samples were prepared for all experiments and controls.
To test the effect of the initial density of C. utilis on the removal efficiency of nitrogen and phosphorus, the initial density of C. utilis F87 were diluted into four different concentrations: 5.0 × 107, 5.0 × 106, 5.0 × 105, and 5.0 × 104 cells mL−1. Two milliliters of each dilute solution was added to a flask with 100 mL water sample aseptically. The samples were cultured at 28 °C and shaken at 100 rpm for 2 days. Triplicate samples were prepared for all experiments and controls.
The effects of different C/N ratios (1:2, 1:1, 2:1, and 3:1) on the removal efficiency of nitrogen and phosphorus by C. utilis F87 were tested by using glucose as a carbon source (measured as carbon or nitrogen atoms); Two milliliters C. utilis F87 and 5 mL sterile M. aeruginosa were added to each flask aseptically with the initial cell densities of 2.25 × 107 and 4.15 × 107 cells·mL−1, respectively. All experiments were carried out in parallel batch culture (100 mL) using 250-mL Erlenmeyer flasks, and the incubation conditions were similar to that as described in the “Strains and Culture Conditions” section. All the experiments and controls were carried out in triplicate.
Effects of C. utilis on the Growth of M. aeruginosa
Two milliliters C. utilis F87 and 5 mL sterile M. aeruginosa were added to each flask aseptically with the initial cell densities of 2.25 × 107 and 4.15 × 107 cells·mL−1, respectively. To study the effects of C. utilis on the growth of M. aeruginosa, the samples were divided into two groups (condition I—cultured at 28 °C and shaken at 100 rpm without white light; condition II—cultured under the conditions of 28 °C, 2,000 lx white light, light/dark = 14 h:10 h). Two milliliters of aseptic water instead of C. utilis F87 was added to each control sample.
The effect of carbon source for C. utilis F87 on the growth of M. aeruginosa was studied by adding glucose (2 g carbon/1 g nitrogen, C/N ratio = 2:1, measured as carbon or nitrogen atoms); controls consisting M. aeruginosa received the same volume of the PDA medium instead of 2 mL C. utilis F87. All experiments were carried out in parallel batch culture (100 mL) using 250-mL Erlenmeyer flasks, and the incubation conditions were similar to that as described in the “Strains and Culture Conditions” section. All the experiments and controls were carried out in triplicate.
The Removal Efficiencies of Nitrogen and Phosphorus with Different Yeasts
The removal efficiencies of ammonia nitrogen and phosphorus with different yeast strains
NH4+–N removal efficiency (%)
PO43−–P removal efficiency (%)
11.28 ± 0.84
6.89 ± 0.27
7.65 ± 0.33
3.84 ± 0.15
S. cerevisiae 609
84.66 ± 3.55
53.23 ± 1.85
32.56 ± 0.94
36.24 ± 1.76
S. cerevisiae A7
88.59 ± 3.04
59.72 ± 1.17
38.78 ± 1.22
44.85 ± 1.55
C. utilis F87
91.26 ± 2.55
62.48 ± 2.01
42.93 ± 1.68
59.48 ± 2.11
Effect of Initial Density of C. utilis F87 on Nitrogen and Phosphorus Removing
Effects of C/N Ratio on the Removing Refficiency of Nitrogen and Phosphorus
For C/N ratio of 1:2, the removal efficiency of NH4+–N was 42.13 ± 1.85 % on the second day and then declined linearly afterwards, while the removal efficiency of PO43−–P reached 54.67 ± 2.11 %. That means it is easier to remove PO43−–P than NH4+–N with the C/N ratio of 1:2. For C/N ratio of 1:1, the removal efficiencies of NH4+–N and PO43−–P were 71.16 ± 2.98 % and 69.60 ± 3.95 %, respectively, which were much higher than those with the C/N ratio of 1:2, but the removal efficiency of NH4+–N was still unsteady after the second day. The reason might be that the carbon source was not enough for the growth of C. utilis F87 while the experimented cell density of C. utilis F87 could remove PO43−–P effectively with the removal efficiency of more than 60 %. For C/N = 2:1, the removal efficiency of NH4+–N was nearly up to 100 % on the second day, and the removal efficiency of PO43−–P was 81.6 ± 3.44 % on the fourth day. For C/N = 3:1, both NH4+–N and PO43−–P removal efficiencies were lower than the ratio of 2:1. Based on the results above, C. utilis F87 should be operated at an optimum C/N ratio of 2:1 for removing NH4+–N and PO43−–P.
Effects of C. utilis F87 on the Growth of M. aeruginosa
These phenomena suggested that C. utilis F87 were more able to absorb ammonia nitrogen and phosphorus than M. aeruginosa in water at the first 2 days, and the growth of M. aeruginosa was inhibited due to the lack of nutrients. Six days later, the C. utilis F87 cells could not be detected by hemocytometer (date not shown) while M. aeruginosa grew well. One possible reason for this was that nutrients such as ammonia nitrogen and phosphorus were released into the water after the lysis of C. utilis F87 cells, and then absorbed by M. aeruginosa.
Effects of Carbon Source for C. utilis F87 on the Growth of M. aeruginosa
Water remediation of rivers, lakes, and reservoirs has become an increasing problem due to the increase in eutrophication. As is well known, HABs are mainly caused by the accumulation of nutrients such as nitrogen and phosphorus. It is a basic viewpoint to transform or get rid of the accumulation of nutrients in lakes (or reservoirs) and control the nutrients from the external pollution resource by effective interception . To date, it is generally recognized that the most applicable and representative techniques to remove nitrogen and phosphorus from eutrophic water are biological control methods, such as constructed wetlands, floating island, and submerged hydrophytes [20, 23, 27]. Previous study reported that nitrogen, nitrite, and ammonium were removed with the removal efficiency of 70.2 %, 92.2 %, and 50.9 %, respectively, by the microbial–plant integrated system using Eichhornia crassipes and Elodea nuttallii . The ecological quality of Danish lake improved by fish removal has also been published in another recent study, and the concentrations of chlorophyll a, total phosphorus, total nitrogen, and suspended solids decreased to 50–70 % of the level prior .
Despite biological control methods for removing nitrogen and phosphorus from eutrophic water by using aquatic macrophytes and fishes are viable, little is known about the ecological safety and secondary contamination of excrement produced by fishes. It was pointed out that nutrients such as phosphorus could be removed from high-concentration organic wastewater by the yeast [28, 29]. Compared with previous reports, we designed a new way to remove nitrogen and phosphorus by C. utilis F87, which could convert the nutrients into microbial biomass as a food source for fish, while inhibiting the growth of M. aeruginosa at the same time. The results of the current study clearly demonstrate that nitrogen and phosphorus could be removed from synthetic eutrophic water effectively by C. utilis F87 during the first 2 days, while both the removal efficiencies of nitrogen and phosphorus decreased afterwards. The result was related to the C. utilis F87 cell density; thus, the cell density of C. utilis F87 might be responsible for the removal efficiencies.
For two wastewater treatment yeasts, Hansenula fabianii J640 and Hansenula anomala J224-1, it was found that 50 % of dissolved total phosphorus was removed from Shochu wastewater , while the strain C. utilis F87 used in the present study obtained nearly 60 % at the first 2 days for unsterilized water (Table 2). Our study also showed that both C. utilis F87 and M. aeruginosa appeared to play a major role in the competition for nutrients. As M. aeruginosa is a kind of photoautotrophic microorganisms, while C. utilis F87 is a kind of chemoheterotrophy microorganisms, the carbon source (glucose) was added to the solution in order to maintain the activity of C. utilis F87. Our results suggest that the carbon source has a positive impact on removing nitrogen and phosphorus while a negative impact on the inhibition growth of M. aeruginosa over the first 4 days. The positive impact confirms the conclusions of many previous studies that the addition of carbon source was conducive to remove nitrogen and phosphorus [3, 4, 28, 29]. It is interesting that the removal efficiency of M. aeruginosa for the culture condition with glucose was not as good as that for the culture condition without glucose. The reason may be that the glucose could be used effectively for photosynthesis of M. aeruginosa. From these results, we conclude that the inhibited effect of M. aeruginosa seem to be controlled not only by C. utilis F87 cell density but also by the abiotic factors such as carbon source. Furthermore, the removal efficiency of phosphorus was not affected by the presence of M. aeruginosa, as the growth of M. aeruginosa was inhibited by C. utilis F87, either in condition I or in condition II (Fig. 3).
The major finding of this study is that controlling algal blooms by converting the nutrients into microbial biomass and inhibiting the growth of M. aeruginosa at the same time is a potential method for eutrophication control. This biological control method, which removes nitrogen and phosphorus combined with the inhibited growth of M. aeruginosa by C. utilis F87, seems well suited for short-term eutrophication control. However, the scope of this study is limited in terms of control M. aeruginosa and some questions arise. For example, there are many other bacteria and zooplanktons in natural conditions; the existence of these microorganisms may influence the effects of C. utilis F87. Additionally, some questions still exist; more researches such as how to keep the activity of C. utilis F87 under natural conditions and how to make it easy for fish to eat are still needed in the future.
This study was financially supported by a grant from the Science and Technology R & D Program of Wuhan, China (No. 200960223065 and No. 201120637175-4). We would like to thank Dr. Nathan Moore (Department of Geography, Michigan State University) for the help of checking English.