Isolation, identification and ecological control ciliates in microalgal cultures
In the past decade, microalgae have emerged as a novel source of biomass that could complement the biomass supply from conventional agricultural crops and forestry for the production of food, feed, energy and chemicals. To minimize the production costs, microalgae are cultivated in large-scale open systems. However, these open pond cultivation systems are often invaded by pest species that include parasites (viruses, bacteria and fungi), protozoa (amoebae, flagellates and ciliates), microcrustaceans (rotifers, cladocerans and copepods) and undesired microalgae species. Of these, ciliates are a common group of grazers that consume microalgae. Ciliates have high growth rates compared to those of microalgae and, as a result, they can cause large losses in productivity, often leading to a collapse of an entire culture in only a few days. Moreover, ciliates produce resting cysts that can enter open ponds through air, rainfall or by the water used to make the culture medium. So far, there is very limited research into the control of ciliate contamination in large-scale microalgal cultures. The main goal of this PhD project were to isolate and identify ciliate species that contaminate microalgal cultures, and to investigate sustainable methods to control ciliate contamination.
The first aim was to develop expertise to isolate and identify algivorous ciliates to create a model system for studying and controlling ciliate grazers of microalgae (Chapter 2). Ciliates were isolated from Chlamydomonas and Chlorella enrichment cultures that were deliberately contaminated with dust (collected from a rooftop, road side and tree bark) and various non-sterile sources of water (rain water, pond water, ground water and tap water). A total of 11 ciliate strains were isolated of which 7 were herbivorous. All species preferentially fed on Chlamydomonas. None of the isolated ciliate species fed on Chlorella. These 7 herbivorous ciliate strains were identified as Tetmemena pustulata, Stylonychia notophora, Sterkiella histriomuscorum, and Paramecium biaurelia and Platyophrya sp. based on a combination of morphological observations and molecular analyses (partial 18S rDNA sequences).
The second aim of this project was to explore methods to control ciliate contamination in microalgal cultures using chemicals. In natural ecosystems, some marine microalgae produce chemicals that are believed to function as feeding deterrents against herbivores: 2,4-decadienal, dimethyl sulfoniopropionate (DMSP), proline and glycine betaine. We evaluated whether these chemicals could be used to control contamination of the ciliate Sterkiella in cultures of the microalgae Chlamydomonas (Chapter 3). In addition, we also tested a chemical analogue of DMSP, (methyl 3-(methylthio)propionate or MMP. All chemicals tested were able to kill the ciliate after 30 minutes exposure, but also had a negative impact on the microalgae at a higher dose. However, ciliates could be completely controlled with minimal losses in biomass productivity at the optimal doses of 0.13 mM decadienal, 4.75 mM DMSP, 10 mM MMP, 250-300 mM proline and 250-300 mM glycine betaine. Moreover, all chemicals tested were also effective against other ciliates (Stylonychia notophora and Paramecium biaurelia). Of these 5 chemicals tested, DMSP and MMP are the most promising because they kill ciliates at a relatively low dose without impacting microalgal biomass productivity.
One of the chemicals tested in chapter 3 was glycine betaine, a compound that contains a quaternary ammine group. Quaternary amine groups are known to be toxic towards bacteria, protozoa and crustaceans. Therefore, in chapter 4, we also tested whether an industrially important quaternary amine compound could be used to control biological contamination in microalgal cultures, cetyltrimethyl ammonium bromide or CTAB. CTAB was tested not only against a herbivorous ciliate (Sterkiella), but also against other types of herbivores: the rotifer Brachionuscalyciflorus and flagellate Paraphysomonas sp. CTAB rapidly eradicated (within 1 – 2 d) the three types of grazers at very low doses (≤ 3 µM) without causing a reduction in microalgal productivity. However, care should be taken when using CTAB as doses higher than 5 µM also caused a negative effect on the growth of both Chlamydomonas and Chlorella. CTAB has the potential to be used as a fast-acting, low-cost pesticide against a wide range of grazers in microalgal mass cultures. However, CTAB is a synthetic chemical that has environmental impact. Hence, before a practical application in large-scale cultivation systems, environmental testing and biodegradability of CTAB has to be carefully assessed.
It is well known that extracts from some plant species can be used as anti-protozoal agents in human disease. Because ciliates are protozoa, in chapter 5, we decided to evaluate if extracts of 3 plant species can be used to control ciliate contamination in microalgal cultures: garlic oil (Allium sativum), clove oil (Syzygium aromaticum) and walnut leaf extract (Juglansregia). We analysed the chemical compositions of the plant extracts using GC-MS and tested the activity of the main compounds in the plant extracts: diallyl disulfide from garlic oil, eugenol and β-caryophyllene from clove oil and juglone from walnut leaves. All 3 extracts and 4 compounds were capable of controlling the ciliate Oxytricha sp. in a Chlamydomonas culture. For most compounds, either the dose required was high, or specificity was low (i.e. the LD50 for Chlamydomonas was close to the LD50 for Oxytricha). Only garlic oil and its active compound diallyl disulfide were effective at a low dose and had a relatively high specificity. Garlic oil required a lower dose (about 5 ppm) than the pure compound diallyl disulfide, which indicates that the activity of garlic oil is not due to diallyl disulfide but to other chemicals or a combination of chemicals present in garlic oil. The low toxicity of garlic oil to microalgae combined with the low cost makes this an attractive product for controlling ciliate contamination in microalgal cultures.
Another approach to control ciliate contamination was based on the principle of classical biological control (Chapter 6). In natural ecosystems, cyclopoid copepods are important grazers of ciliates. We tested if a cyclopoid copepod could be used to eliminate ciliates from microalgal cultures. We isolated and identified a carnivorous cyclopoid copepod, Acanthocyclops robustus, and used it as a biological control agent to control contamination by the ciliate Sterkiella in cultures of Chlamydomonas. Our experiments showed that the copepod Acanthocyclops robustus possesses high predation rate, consuming up to 400 ciliates copepod-1 day-1. A complete eradication of ciliates within 1 day resulted from addition of 0.07 copepods mL-1 to a culture contaminated with 10 ciliates mL-1. The copepods and the juveline stages (copepodites and nauplii) did not feed on the microalgae Chlamydomonas. These laboratory-scale experiments indicated that copepods have potential to be used as a biological agent to control ciliate contamination of large-scale microalgal cultures.