Cyanobacterial biofilm on zeolite substrate: potential biofertilizer (ZeoCyan)
The ZeoCyan project is funded by the Romanian Ministry of Research, Innovation and Digitalization: UEFISCDI - PNCDI III/Programme 2/PED2021
Project duration: 24 months (July 2022 - June 2024)
Project budget: 124.152 Euro
Principal investigator: Dr. Bogdan Drugă
Project description
This project aims at producing cyanobacterial biofilm using zeolite as substrate and wastewater as growth medium, and to test the usability of zeolite/cyanobacteria biofilm compound as soil fertilizer. The activities will be done as a joint effort of a team of scientists specialized on microalgae and plant research. Zeolites are inorganic aluminosilicate materials with extraordinary ion-exchange and adsorption potential. For such properties, they can be used to remove substances such as ammonium, orthophosphate or heavy metals from wastewaters. Due to their slow-release of water and nutrients, they can also be used as soil fertilizers. Filamentous cyanobacteria are photosynthetic microalgae that can grow in wastewater effluent (as photogranules), as they uptake the nutrients (mainly N and P) usually present in excess in these environments. They are also known to improve soil quality to plants, due to their high content in N and P. In the first part of the project we will attempt to develop cyanobacterial biofilm on zeolite granules, using multiple candidate strains from a culture collection. Next, the zeolite/biofilm compound will be dried and used ad soil fertilizers for tomato plants. The project requires only resources that are naturally available or even in excess (zeolite, microalgae, wastewater), and the resulting product (zeolite-based photogranules) will be used as soil fertilizer. Therefore, the methodology proposed here falls under the idea of circular economy.
Main objective
To produce cyanobacterial biofilm using zeolite granules as substrate and wastewater as growth medium, and to test the usability of zeolite/cyanobacteria compound as soil fertilizer.
RESULTS
Summary
The ZeoCyan project aimed to develop a biofilm of cyanobacteria on a zeolite substrate with potential use as a biofertilizer. This research was conducted over two years and included the identification and selection of suitable cyanobacteria strains, the establishment of optimal growth conditions, and the production of significant quantities of biofilm for agricultural testing.
Initially, 36 strains of cyanobacteria were selected from the Algae and Cyanobacteria Collection of the Biological Research Institute in Cluj-Napoca. These strains were characterized morphologically and molecularly, and their toxic potential was assessed. The strain Anabaena oscillarioides (AICB 1382) was identified as the most efficient for biofilm formation on zeolite in both artificial growth media and municipal wastewater effluent. The project successfully established a stable cultivation system for Anabaena oscillarioides AICB 1382, which was tested multiple times in 11.4-liter aquariums using zeolite substrate. The optimal growth period was determined to be approximately 14 ± 5 days with partial exposure to natural and fluorescent light. This system produced large quantities of biofilm, which were harvested and stored for further testing on tomato plants.
Throughout the project, all initial objectives were met, and additional activities were performed, such as extended toxicity analysis and further testing of zeolite-based photogranules in wastewater effluents. These supplementary activities reinforced the project's outcomes, demonstrating the broader applicability of cyanobacteria and zeolite-based biofertilizers. Furthermore, dissemination efforts included the publication of four scientific articles, participation in international conferences, and outreach activities to high school students through the "Științescu" program. The ZeoCyan project has thus provided significant advancements in the use of cyanobacteria for environmental and agricultural applications, showcasing the potential for sustainable and efficient biofertilizers.
MATERIAL AND METHODS
Selection and Taxonomic Affiliation of Cyanobacterial Strains: A total of 36 cyanobacterial strains were selected for the study. Among them, 28 strains belong to the order
Oscillatoriales, and eight strains are affiliated with the order Nostocales, known for their nitrogen-fixing capabilities. The strains were verified for purity using microscopy and cultivated in BG11 medium under natural light and ambient temperature. Cultures were reinoculated to obtain an exponential inoculum with minimal bacterial and cellular debris, which was then used for genomic DNA extraction, imaging, and zeolite experiments.
Genomic DNA Extraction and Sequencing: DNA was extracted using a commercial kit and verified spectrophotometrically. The 16S rRNA gene was amplified using universal primers for cyanobacteria. The purified fragment was sequenced, and the resulting sequences were assembled and compared with the GenBank database for taxonomic determination.
Cultivation Systems and Biofilm Formation: Three cultivation models were implemented based on container volume. The first model used 100 mL Erlenmeyer flasks with wet biomass, liquid, and zeolite. The second model utilized 300 mL Erlenmeyer flasks with adjusted quantities of biomass, liquid, and zeolite. The surface area of the zeolite was crucial for biofilm formation.
Harvesting and Drying Photogranules: Biomass was harvested by centrifugation. The supernatant was used to wash the zeolite, removing adherent biomass until all residues were eliminated. The wet biomass was weighed and stored for future experiments. The entire system was scaled up to aquariums with a capacity of 11.4 liters, using wet biomass, zeolite, and effluent, distributed uniformly. Control aquariums included setups with biomass and effluent without zeolite and zeolite with effluent without biomass. The optimal cultivation period was approximately 14 days.
Biomass Sampling for DNA and Nutrient Analysis: Samples were taken from three distinct zones within the cultivation system, both from test and control aquariums. In Zone 1, characterized by a consistent biofilm on the effluent surface, samples were collected, centrifuged, and stored. Zone 2 provided filtered samples, and Zone 3 consisted of biofilm on and among the zeolite particles. Biomass was collected and washed from the zeolite, then stored for analysis.
Quantitative Determination of Nutrients: Nitrate and phosphate concentrations in the effluent were measured at the beginning and end of the cultivation experiments. Samples were filtered, and concentrations were determined using a multiparameter device with specific reagent kits for total nitrogen and phosphate.
Metagenomic DNA Extraction and Sequencing: For assessing taxon abundance in the aquarium cultivation systems, metagenomic DNA was extracted, quantified, and sequenced. Seqtrains from the genus Oscillatoria were evaluated for the presence of toxin-coding genes, with specific focus on genes for saxitoxin and microcystin. Although some genes were detected, the synthesis of toxins was deemed unlikely, making these strains suitable for further applications.
RESULTS AND DISCUSSION
PART 1
Screening of the AICB Collection for Candidate Cyanobacterial Strains:
During the first three months, the project focused on selecting and characterizing cyanobacterial strains from the AICB Collection. Strains predominantly from the genus Phormidium were selected based on initial morphological observations. However, due to evolving taxonomic criteria, these strains underwent a thorough review using both morphological and genetic criteria, specifically the 16S rRNA gene.
Morphological and Taxonomic Characterization: Out of the strains investigated, 34 were highlighted, including those capable of nitrogen fixation (Fig. 1). Genomic DNA was extracted from these strains for sequencing, confirming their taxonomic affiliations through BLAST comparisons with the GenBank database. This process ensured accurate identification and selection of suitable strains for further experiments.
PART 1
Screening of the AICB Collection for Candidate Cyanobacterial Strains:
During the first three months, the project focused on selecting and characterizing cyanobacterial strains from the AICB Collection. Strains predominantly from the genus Phormidium were selected based on initial morphological observations. However, due to evolving taxonomic criteria, these strains underwent a thorough review using both morphological and genetic criteria, specifically the 16S rRNA gene.
Morphological and Taxonomic Characterization: Out of the strains investigated, 34 were highlighted, including those capable of nitrogen fixation (Fig. 1). Genomic DNA was extracted from these strains for sequencing, confirming their taxonomic affiliations through BLAST comparisons with the GenBank database. This process ensured accurate identification and selection of suitable strains for further experiments.
Fig. 1 Macroscopic appearance of the 34 investigated cyanobacterial strains, exemplified by 14 strains and 4 of the 8 nitrogen-fixing strains from the AICB collection (the AICB code accompanies each phenotype)
Evaluation of Biofilm Formation on Zeolite: The ability of 28 selected strains to form biofilms on zeolite was tested. Among these, nine strains were identified for their high biofilm-forming capacity and were further evaluated (Fig. 2-3) The effectiveness of biofilm formation was assessed in both individual and mixed cultures, focusing on strains with the potential for photogranule production.
Fig. 2 Degree of photogranule formation of cyanobacteria-zeolite in the 9 selected AICB strains. The days when photographs were taken and the AICB codes of the strains are indicated by numbers.
Fig. 3 Degree of cyanobacteria-zeolite biofilm elevation in 4 AICB strains. The numbers indicate the interval (in days) when the photographs were taken and the AICB codes of the strains.
Cultivation in Mixed Cultures: Four additional strains were selected to be cultivated in combination with the previously chosen five strains. These mixed cultures were tested in BG11 medium and on zeolite, with optimal performance observed in strains AICB 1265 and AICB 404. This stage aimed to enhance the robustness and productivity of biofilms in varied conditions.
Data Analysis: This activity spanned months 3 to 9 and included data from both the initial and subsequent stages. It involved assessing the toxic potential of selected strains, morphological characterization, and molecular analysis. Nine strains from the genus Oscillatoria were evaluated for the presence of toxin-coding genes, with specific focus on genes for saxitoxin and microcystin. Although some genes were detected, the synthesis of toxins was deemed unlikely, making these strains suitable for further applications.
Data Analysis: This activity spanned months 3 to 9 and included data from both the initial and subsequent stages. It involved assessing the toxic potential of selected strains, morphological characterization, and molecular analysis. Nine strains from the genus Oscillatoria were evaluated for the presence of toxin-coding genes, with specific focus on genes for saxitoxin and microcystin. Although some genes were detected, the synthesis of toxins was deemed unlikely, making these strains suitable for further applications.
PART 2
Biofilm Formation in Individual Cyanobacterial Cultures in Wastewater: Strains AICB 404 and AICB 1265 were evaluated in municipal wastewater effluent across several experimental setups, including 100 and 300 mL Erlenmeyer flasks and 11.4-liter aquariums. While some strains showed reduced growth, AICB 404 and AICB 1265 demonstrated consistent biofilm formation, making them prime candidates for subsequent phases (Fig. 4).
Biofilm Formation in Individual Cyanobacterial Cultures in Wastewater: Strains AICB 404 and AICB 1265 were evaluated in municipal wastewater effluent across several experimental setups, including 100 and 300 mL Erlenmeyer flasks and 11.4-liter aquariums. While some strains showed reduced growth, AICB 404 and AICB 1265 demonstrated consistent biofilm formation, making them prime candidates for subsequent phases (Fig. 4).
Fig. 4 Measuring the growth of AICB 404 and AICB 1265 strains on zeolite (A, B - individual cultures, C - mixed culture) in effluent collected from the municipal wastewater treatment plant, in an experimental setup conducted in aquariums (cultivation system 3). Details were highlighted from the beginning of the experiment (day 1) (top panels) and from the end of the cultivation period (day 35) (bottom panels). The control (D) was not inoculated with biomass.
Biofilm Formation in Mixed Cultures: Initial experiments with mixed cultures were discontinued in favor of focusing solely on strain AICB 1382, which showed superior performance. This decision streamlined the process and ensured the best results for biofilm formation.
Harvesting and Drying Photogranules: Biomass samples were harvested, dried, and stored for further testing. This stage confirmed the feasibility of producing sufficient quantities of photogranules for practical applications.
Nutrient Measurement: Nutrient analysis revealed a significant reduction in nitrate and phosphate concentrations in aquariums with AICB 1382, highlighting the strain's potential for wastewater remediation.
Molecular Analysis of Photogranules: A total of 52 biomass samples were processed, yielding satisfactory concentrations of metagenomic DNA. This analysis identified a diverse range of microorganisms within the biofilm, including various cyanobacteria and heterotrophic bacteria such as Pseudomonas and Bacillus, which play essential roles in nutrient recycling and biofilm stability.
Harvesting and Drying Photogranules: Biomass samples were harvested, dried, and stored for further testing. This stage confirmed the feasibility of producing sufficient quantities of photogranules for practical applications.
Nutrient Measurement: Nutrient analysis revealed a significant reduction in nitrate and phosphate concentrations in aquariums with AICB 1382, highlighting the strain's potential for wastewater remediation.
Molecular Analysis of Photogranules: A total of 52 biomass samples were processed, yielding satisfactory concentrations of metagenomic DNA. This analysis identified a diverse range of microorganisms within the biofilm, including various cyanobacteria and heterotrophic bacteria such as Pseudomonas and Bacillus, which play essential roles in nutrient recycling and biofilm stability.
PART 3
Obtaining Photogranules as Fertilizer:
This activity spanned approximately 10 months and involved selecting and characterizing biological material, establishing growth parameters (light, temperature, zeolite quantity, inoculum amount, effluent volume), and testing the cultivation system's productivity. Following these steps, cultivation was conducted in at least five aquariums simultaneously to produce significant quantities of photogranules. These were harvested and stored for future testing on tomato plants. The activities led to the establishment of a stable growth system for the Anabaena oscillarioides AICB 1382 strain, which was tested multiple times in zeolite aquariums.
Cultivation of Plants in the Presence of Photogranules as Fertilizer:
The activity involved testing the effect of AICB 1382 cyanobacteria on tomato seed germination. Two types of biomass were used: from the effluent surface (Zone 1) and the zeolite surface (Zone 3). Three concentrations were tested, alongside control treatments with distilled water and effluent Fig. 5).Visual evaluation showed more seeds germinated with cyanobacterial biomass than with control treatments. Seeds treated with biomass had a higher germination percentage, with Zone 3 biomass slightly more effective than Zone 1. This suggests the zeolite surface biofilm has more nutrients, aiding germination.
Obtaining Photogranules as Fertilizer:
This activity spanned approximately 10 months and involved selecting and characterizing biological material, establishing growth parameters (light, temperature, zeolite quantity, inoculum amount, effluent volume), and testing the cultivation system's productivity. Following these steps, cultivation was conducted in at least five aquariums simultaneously to produce significant quantities of photogranules. These were harvested and stored for future testing on tomato plants. The activities led to the establishment of a stable growth system for the Anabaena oscillarioides AICB 1382 strain, which was tested multiple times in zeolite aquariums.
Cultivation of Plants in the Presence of Photogranules as Fertilizer:
The activity involved testing the effect of AICB 1382 cyanobacteria on tomato seed germination. Two types of biomass were used: from the effluent surface (Zone 1) and the zeolite surface (Zone 3). Three concentrations were tested, alongside control treatments with distilled water and effluent Fig. 5).Visual evaluation showed more seeds germinated with cyanobacterial biomass than with control treatments. Seeds treated with biomass had a higher germination percentage, with Zone 3 biomass slightly more effective than Zone 1. This suggests the zeolite surface biofilm has more nutrients, aiding germination.
Fig. 5 Representation of the testing protocol for the stimulating capacity of AICB 1382 strain biomass on the germination percentage of tomato seeds. The reference conditions (top row) are represented by treatments with distilled water, effluent before cultivation (Effluent Start), and effluent after cultivation (Effluent End). Biomass treatments were conducted at three concentrations for Zone 1 (middle row) and Zone 3 (bottom row).
There were no significant differences in germination rates between different biomass concentrations. However, biomass treatments increased seed weight, especially from Zone 3. For epicotyl development, biomass-treated seeds had longer epicotyls compared to controls. The most significant improvement was in seeds treated with biomass, regardless of the source zone.
Overall, AICB 1382 biomass positively affected germination, seed weight, and epicotyl growth, indicating its potential as a biofertilizer.
Overall, AICB 1382 biomass positively affected germination, seed weight, and epicotyl growth, indicating its potential as a biofertilizer.
Main Conclusions and Findings of the ZeoCyan Project
The ZeoCyan project successfully achieved its goals by demonstrating the efficiency of a cyanobacterial biofilm on a zeolite substrate as a biofertilizer. The resulting product, cyanobacteria-zeolite photogranules, significantly improved seed germination rates qualifies for as potent helper for plant growth as well. This innovative approach aligns with the principles of circular economy, where cyanobacteria abilities to consume nutrients from the effluent and zeolite's nutrient retention capacity are harnessed to enhance soil fertility and plant health.
A key outcome of the project was the successful identification and cultivation of the cyanobacterium Anabaena oscillarioides AICB 1382, which proved highly efficient in forming biofilms on zeolite. The cultivation process, optimized in several experimental setups, consistently produced substantial quantities of photogranules. These photogranules were then tested on tomato seeds, showing a significant increase in germination rates compared to control conditions.
Furthermore, the project included extensive testing in municipal wastewater effluent, proving the dual benefit of these photogranules in both fertilizing plants and reducing nutrient concentrations in wastewater. This dual functionality highlights the potential of cyanobacteria-zeolite photogranules in sustainable agriculture and environmental protection.
Dissemination of the project results included the publication of scientific papersand participation in various international conferences. These efforts ensured that the findings reached a broad audience, fostering further research and collaboration in the field of biofertilizers. Additionally, the project engaged in educational outreach through programs like "Științescu," promoting scientific knowledge among high school students.
In conclusion, the ZeoCyan project demonstrated that cyanobacteria-zeolite photogranules are a promising biofertilizer with significant applications in sustainable agriculture. The project successfully met its objectives, providing a foundation for future research and practical applications in the field.
The ZeoCyan project successfully achieved its goals by demonstrating the efficiency of a cyanobacterial biofilm on a zeolite substrate as a biofertilizer. The resulting product, cyanobacteria-zeolite photogranules, significantly improved seed germination rates qualifies for as potent helper for plant growth as well. This innovative approach aligns with the principles of circular economy, where cyanobacteria abilities to consume nutrients from the effluent and zeolite's nutrient retention capacity are harnessed to enhance soil fertility and plant health.
A key outcome of the project was the successful identification and cultivation of the cyanobacterium Anabaena oscillarioides AICB 1382, which proved highly efficient in forming biofilms on zeolite. The cultivation process, optimized in several experimental setups, consistently produced substantial quantities of photogranules. These photogranules were then tested on tomato seeds, showing a significant increase in germination rates compared to control conditions.
Furthermore, the project included extensive testing in municipal wastewater effluent, proving the dual benefit of these photogranules in both fertilizing plants and reducing nutrient concentrations in wastewater. This dual functionality highlights the potential of cyanobacteria-zeolite photogranules in sustainable agriculture and environmental protection.
Dissemination of the project results included the publication of scientific papersand participation in various international conferences. These efforts ensured that the findings reached a broad audience, fostering further research and collaboration in the field of biofertilizers. Additionally, the project engaged in educational outreach through programs like "Științescu," promoting scientific knowledge among high school students.
In conclusion, the ZeoCyan project demonstrated that cyanobacteria-zeolite photogranules are a promising biofertilizer with significant applications in sustainable agriculture. The project successfully met its objectives, providing a foundation for future research and practical applications in the field.