Heterocyst Cells Research Articles

Assessing the mass-culture system and proximate content of Arthrospira maxima local for biogas and biohydrogen productions

Arthrospira maxima is a type of local cyanobacteria that has shown success in being grown massively in Indonesia. This research is aimed at looking at the resilience and ability to grow in outdoor conditions using cheap media and following environmental patterns in terms of temperature, sunlight and aeration. In specific purpose is utilization of the obtained biomass for biogas or biohydrogen production. Also, media optimization research from chemical pour grade analyzes into commercial types has been carried out at laboratory scale and field scale. The results show that at the scaling up process from laboratorial to outdoor condition the amount of biomass ratio has decreased by 2–10%, whereas proximate of protein has decreased from 46% to 38%, in contrast starch tends to increase from 10% to 23% and lipid was increase from around 9%–12%. Freshwater and marine water basic media also showing difference in ratio of biomass production and main proximate. The total calory and protein seems higher if A. maxima cultivate using marine water. Cultivation time outdoors turns out to be faster growth when compared to controlled conditions indoors. Biomass analysis results show that the largest content is protein, followed by carbohydrates and lipid. Considering the composition of the proximate, the cyanobacteria biomass tends to be used for biogas conversion rather than alcohol, due to the high protein with less carbohydrate content. Moreover, Arthrospira is cyanobacteria which is capable for producing heterocyst cells that can be excite gas. It was found the cyanobacteria release hydrogen gas around 54 μL/g wet biomasses/day mature culture. However, it should be remembered that this strain is still original or wild type, and can still be developed using both environmental and molecular manipulation approaches. Survival rate from laboratory scale to field scale shows that the microalgae is strong and worthy of consideration for industrial applications. Apart from that, high pH culture conditions are very good for removing contaminants. Also changing the media from pour analyses grade chemicals to commercial chemicals grade only reduces the biomass composition by 2–5% so it is worth considering for commercial applications. Testing various reactors to make biomass as needed with high productivity will be a challenge in the future.

The Making of a Heterocyst in Cyanobacteria.

Heterocyst differentiation that occurs in some filamentous cyanobacteria, such as Anabaena sp. PCC 7120, provides a unique model for prokaryotic developmental biology. Heterocyst cells are formed in response to combined-nitrogen deprivation and possess a microoxic environment suitable for nitrogen fixation following extensive morphological and physiological reorganization. A filament of Anabaena is a true multicellular organism, as nitrogen and carbon sources are exchanged among different cells and cell types through septal junctions to ensure filament growth. Because heterocysts are terminally differentiated cells and unable to divide, their activity is an altruistic behavior dedicated to providing fixed nitrogen for neighboring vegetative cells. Heterocyst development is also a process of one-dimensional pattern formation, as heterocysts are semiregularly intercalated among vegetative cells. Morphogens form gradients along the filament and interact with each other in a fashion that fits well into the Turing model, a mathematical framework to explain biological pattern formation.

Rapid Transcriptional Reprogramming Triggered by Alteration of the Carbon/Nitrogen Balance Has an Impact on Energy Metabolism in Nostoc sp. PCC 7120.

Nostoc (Anabaena) sp. PCC 7120 is a filamentous cyanobacterial species that fixes N2 to nitrogenous compounds using specialised heterocyst cells. Changes in the intracellular ratio of carbon to nitrogen (C/N balance) is known to trigger major transcriptional reprogramming of the cell, including initiating the differentiation of vegetative cells to heterocysts. Substantial transcriptional analysis has been performed on Nostoc sp. PCC 7120 during N stepdown (low to high C/N), but not during C stepdown (high to low C/N). In the current study, we shifted the metabolic balance of Nostoc sp. PCC 7120 cultures grown at 3% CO2 by introducing them to atmospheric conditions containing 0.04% CO2 for 1 h, after which the changes in gene expression were measured using RNAseq transcriptomics. This analysis revealed strong upregulation of carbon uptake, while nitrogen uptake and metabolism and early stages of heterocyst development were downregulated in response to the shift to low CO2. Furthermore, gene expression changes revealed a decrease in photosynthetic electron transport and increased photoprotection and reactive oxygen metabolism, as well a decrease in iron uptake and metabolism. Differential gene expression was largely attributed to change in the abundances of the metabolites 2-phosphoglycolate and 2-oxoglutarate, which signal a rapid shift from fluent photoassimilation to glycolytic metabolism of carbon after transition to low CO2. This work shows that the C/N balance in Nostoc sp. PCC 7120 rapidly adjusts the metabolic strategy through transcriptional reprogramming, enabling survival in the fluctuating environment.

Molecular circuit of heterocyst differentiation in cyanobacteria.

Differentiation commitment is one of the most complex mechanisms to study in biological science. One of the model systems used for understanding differentiation complexity is heterocyst development in cyanobacteria. Cyanobacteria have the capability of biological nitrogen fixation due to highly differentiated heterocyst cells. Once the nitrogen deficiency signal is perceived by the cyanobacteria, few of its vegetative cells commit toward the development of heterocyst. Heterocyst provides a microoxic environment that is essential for the nitrogenase complex to fix the atmospheric dinitrogen. The entire process of development of heterocyst can be divided into different steps, such as (a) sensing signal and differentiation induction, (b) positional (pattern) determination of heterocyst in the filament, (c) formation of extracellular thick heterocyst-specific layers, and (d) assembly of nitrogen-fixing machinery. Many of the key regulators that are essential for heterocyst formationin these different steps have been identified. Recently, the role of small RNA and interruption DNA elements that influence the heterocyst formation and function has also been identified. In this review article, we have outlined the current understanding of the entire molecular circuit of heterocyst development in a simplistic way. This article focuses on explaining key concepts related to heterocyst development and discusses recent discoveries in this line.

Functional Dissection of Genes Encoding DNA Polymerases Based on Conditional Mutants in the Heterocyst-Forming Cyanobacterium Anabaena PCC 7120.

The filamentous cyanobacterium Anabaena sp. PCC 7120 develops N2-fixing heterocyst cells under condition of combined-nitrogen deprivation and constitutes an excellent model for studying cell differentiation. The mechanism of heterocyst development has been extensively investigated and a network of regulating factors has been identified. A few studies have showed that the process of heterocyst differentiation relates with cell cycle events, but further investigation is still required to understand this relationship. In a previous study, we created a conditional mutant of PolI encoding gene, polA, by using a CRISPR/Cpf1 gene-editing technique. Here, we were able to create another conditional mutant of a PolIII encoding gene dnaENI using a similar strategy and subsequently confirmed the essential roles of both polA and dnaENI in DNA replication. Further investigation on the phenotype of the mutants showed that lack of PolI caused defects in chromosome segregation and cell division, while lack of DnaENI (PolIII) prevented bulk DNA synthesis, causing significant loss of DNA content. Our findings also suggested the possible existence of a SOS-response like mechanism operating in Anabaena PCC 7120. Moreover, we found that heterocyst development was differently affected in the two conditional mutants, with double heterocysts/proheterocysts found in PolI conditional mutant. We further showed that formation of such double heterocysts/proheterocysts are likely caused by the difficulty in nucleoids segregation, resulting delayed, or non-complete closure of the septum between the two daughter cells. This study uncovers a link between DNA replication process and heterocyst differentiation, paving the way for further studies on the relationship between cell cycle and cell development.

Reconstruction of a regulated two-cell metabolic model to study biohydrogen production in a diazotrophic cyanobacterium Anabaena variabilis ATCC 29413.

Anabaena variabilis is a diazotrophic filamentous cyanobacterium that differentiates to heterocysts and produces hydrogen as a byproduct. Study on metabolic interactions of the two differentiated cells provides a better understanding of its metabolism especially for improving hydrogen production. To this end, a genome-scale metabolic model for Anabaena variabilis ATCC 29413, iAM957, was reconstructed and evaluated in this research. Then, the model and transcriptomic data of the vegetative and heterocyst cells were applied to construct a regulated two-cell metabolic model. The regulated model improved prediction for biomass in high radiation levels. The regulated model predicts that heterocysts provide an oxygen-free environment and then, this model was used to find strategies for improving hydrogen production in heterocysts. The predictions indicate that the removal of uptake hydrogenase improves hydrogen production which is consistent with previous empirical research. Furthermore, the regulated model proposed activation of some reactions to provide redox cofactors which are required for improving hydrogen production up to 60% by bidirectional hydrogenase.

A novel Ca2+-binding protein influences photosynthetic electron transport in Anabaena sp. PCC 7120

Ca2+ is a potent signalling molecule that regulates many cellular processes. In cyanobacteria, Ca2+ has been linked to cell growth, stress response and photosynthesis, and to the development of specialist heterocyst cells in certain nitrogen-fixing species. Despite this, the pathways of Ca2+ signal transduction in cyanobacteria are poorly understood, and very few protein components are known. The current study describes a previously unreported Ca2+-binding protein which was called the Ca2+ Sensor EF-hand (CSE), which is conserved in filamentous, nitrogen-fixing cyanobacteria. CSE is shown to bind Ca2+, which induces a conformational change in the protein structure. Poor growth of a strain of Anabaena sp. PCC 7120 overexpressing CSE was attributed to diminished photosynthetic performance. Transcriptomics, biophysics and proteomics analyses revealed modifications in the light-harvesting phycobilisome and photosynthetic reaction centre protein complexes.

Anabaena sp. strain PCC 7120: Laboratory Maintenance, Cultivation, and Heterocyst Induction.

Anabaena sp. strain PCC 7120 is a multicellular, filamentous, freshwater cyanobacterium that is capable of differentiating specialized heterocyst cells for nitrogen fixation. This unit includes protocols for the growth and maintenance of Anabaena appropriate for a research or teaching laboratory. Controlled induction and assessment of heterocyst development is also covered. © 2018 by John Wiley & Sons, Inc.

Phenotypic Assessment Suggests Multiple Start Codons for HetN, an Inhibitor of Heterocyst Differentiation, in Anabaena sp. Strain PCC 7120.

Multicellular organisms must carefully regulate the timing, number, and location of specialized cellular development. In the filamentous cyanobacterium Anabaena sp. strain PCC 7120, nitrogen-fixing heterocysts are interspersed between vegetative cells in a periodic pattern to achieve an optimal exchange of bioavailable nitrogen and reduced carbon. The spacing between heterocysts is regulated by the activity of two developmental inhibitors, PatS and HetN. PatS functions to create a de novo pattern from a homogenous field of undifferentiated cells, while HetN maintains the pattern throughout subsequent growth. Both PatS and HetN harbor the peptide motif ERGSGR, which is sufficient to inhibit development. While the small size of PatS makes the interpretation of inhibitory domains relatively simple, HetN is a 287-amino-acid protein with multiple functional regions. Previous work suggested the possibility of a truncated form of HetN containing the ERGSGR motif as the source of the HetN-derived inhibitory signal. In this work, we present evidence that the glutamate of the ERGSGR motif is required for proper HetN inhibition of heterocysts. Mutational analysis and subcellular localization indicate that the gene encoding HetN uses two methionine start codons (M1 and M119) to encode two protein forms: M1 is required for protein localization, while M119 is primarily responsible for inhibitory function. Finally, we demonstrate that patS and hetN are not functionally equivalent when expressed from the other gene's regulatory sequences. Taken together, these results help clarify the functional forms of HetN and will help refine future work defining a HetN-derived inhibitory signal in this model of one-dimensional periodic patterning.IMPORTANCE The proper placement of different cell types during a developmental program requires the creation and maintenance of a biological pattern to define the cells that will differentiate. Here we show that the HetN inhibitor, responsible for pattern maintenance of specialized nitrogen-fixing heterocyst cells in the filamentous cyanobacterium Anabaena, may be produced from two different start methionine codons. This work demonstrates that the two start sites are individually involved in a different HetN function, either membrane localization or inhibition of cellular differentiation.

Adaptations of cyanobacterium Nostoc commune to environmental stress: Comparison of morphological and physiological markers between European and Antarctic populations after rehydration

Availability of water may influence activities of all living organisms, including cyanobacterial communities. Filamentous cyanobacterium Nostoc commune is well adapted to wide spectrum of ecosystems. For this reason, N. commune had to develop diverse protection strategies due to exposition to regular rewetting and drying processes. Few studies have been conducted on activities, by which cyanobacteria are trying to avoid water deficit. Therefore, the present study using physiological and morphological parameters is focused on comparison between European and Antarctic ecotypes of N. commune during rewetting. Gradual increase of FV/FM ratios, as the markers of active PS II, demonstrated the recovery processes of N. commune colonies from Europe as well as from Antarctica after time dependent rehydration. During the initial hours of rewetting, there was lower content of soluble proteins in colonies from Antarctica in comparison to those from Europe. Total content of nitrogen was higher in European ecotypes of N. commune. Significantly higher frequency of occurrence of heterocysts in Antarctic ecotypes was observed. The heterocyst cells were significantly longer in Antarctic ecotypes rather than European ecotypes of N. commune.

Increasing Hydrogen Production Efficiency of N2-Fixing Cyanobacterium Anabaena siamensis TISTR 8012 by Cell Immobilization

H2 produced by cyanobacteria is one of the interesting alternative energy carriers for the future. The filamentous N2-fixing cyanobacterium Anabaena siamensis TISTR 8012 is a potential microorganism for H2 production. It can produce H2 via both photosynthesis and nitrogen fixation processes. This study aimed to increase the efficiency of H2 production by immobilization of A. siamensis cells. The result showed that H2 production rate by A. siamensis grown in BG110 medium was higher than that in BG11 and Allen-Arnon media due to an increase of the heterocyst cells. The sulfur deprivation during adaptation period for 24 hours increased its H2 production rate. The optimal conditions for H2 production by immobilized cells were immobilization with alginate and using 150 gel beads in 20 mL glass vial. In immobilized cells, an addition of 0.5% fructose resulted in a 2-fold increase of H2 production rate. Finally, the reducing agents β–mercaptoethanol and methylviologen enhanced H2 production rate with 3.092 and 2.426 µmolH2 mg chl a-1 h-1, respectively, in A. siamensis immobilized cells whereas NADH, dithiothreitol and sodium dithionite were not capable of increasing H2 production rate.

Thành phần loài vi khuẩn Lam (Cyanobacteria) trong đất trồng lúa ở một số xã thuộc huyện Nghĩa Đàn, tỉnh Nghệ An

THE SPECIES COMPOSITION OF CYANOBACTERIA IN RICE FIELDS OF NGHIA DAN DISTRICT, NGHE AN PROVINCE
 Nguyen Canh Hieu et al.
 Nghia Dan is a mountainous district of Nghe An province, stretching from 19013’ to 19033’ north latidute and between 105018’ to 105035’ east longitude. In August and December 2014 and in May 2015 we have investigated the species composition of Cyanobacteria distributing in rice fields of Nghia Dan district. We have found 40 species and subspecies in soil samples collected in 4 communes: Nghia Hung, Nghia My, Nghia Lam and Nghia Thuan. The most diverse genera were Oscillatoria (14 species and subspecies), Anabaena (6), Phormidium (5). There are 10 species and subspecies cyanobacteria contain heterocyst cells. Sorenxen coefficients between the sampling phases were from 0.82 to 0.88. Our study also showed that the numbers of species distributing at sampling sites were from 19 to 29 species.

Elevated Atmospheric CO2 and Warming Stimulates Growth and Nitrogen Fixation in a Common Forest Floor Cyanobacterium under Axenic Conditions

The predominant input of available nitrogen (N) in boreal forest ecosystems originates from moss-associated cyanobacteria, which fix unavailable atmospheric N2, contribute to the soil N pool, and thereby support forest productivity. Alongside climate warming, increases in atmospheric CO2 concentrations are expected in Canada’s boreal region over the next century, yet little is known about the combined effects of these factors on N fixation by forest floor cyanobacteria. Here we assess changes in N fixation in a common forest floor, moss-associated cyanobacterium, Nostoc punctiforme Hariot, under elevated CO2 conditions over 30 days and warming combined with elevated CO2 over 90 days. We measured rates of growth and changes in the number of specialized N2 fixing heterocyst cells, as well as the overall N fixing activity of the cultures. Elevated CO2 stimulated growth and N fixation overall, but this result was influenced by the growth stage of the cyanobacteria, which in turn was influenced by our temperature treatments. Taken together, climate change factors of warming and elevated CO2 are expected to stimulate N2 fixation by moss-associated cyanobacteria in boreal forest systems.

Mutation of the murC and murB Genes Impairs Heterocyst Differentiation in Anabaena sp. Strain PCC 7120.

To stabilize cellular integrity in the face of environmental perturbations, most bacteria, including cyanobacteria, synthesize and maintain a strong, flexible, three-dimensional peptidoglycan lattice. Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium capable of differentiating morphologically distinct nitrogen-fixing heterocyst cells in a periodic pattern. While heterocyst development has been shown to require proper peptidoglycan remodeling, the role of peptidoglycan synthesis has remained unclear. Here we report the identification of two peptidoglycan synthesis genes, murC (alr5065) and murB (alr5066), as required for heterocyst development. The murC and murB genes are predicted to encode a UDP-N-acetylmuramate:L-alanine ligase and a UDP-N-acetylenolpyruvoylglucosamine reductase, respectively, and we confirm enzymatic function through complementation of Escherichia coli strains deficient for these enzymes. Cells depleted of either murC or murB expression failed to differentiate heterocysts under normally inducing conditions and displayed decreased filament integrity. To identify the stage(s) of development affected by murC or murB depletion, the spatial distribution of expression of the patterning marker gene, patS, was examined. Whereas murB depletion did not affect the pattern of patS expression, murC depletion led to aberrant expression of patS in all cells of the filament. Finally, expression of gfp controlled by the region of DNA immediately upstream of murC was enriched in differentiating cells and was repressed by the transcription factor NtcA. Collectively, the data in this work provide evidence for a direct link between peptidoglycan synthesis and the maintenance of a biological pattern in a multicellular organism. Multicellular organisms that differentiate specialized cells must regulate morphological changes such that both cellular integrity and the dissemination of developmental signals are preserved. Here we show that the multicellular bacterium Anabaena, which differentiates a periodic pattern of specialized heterocyst cells, requires peptidoglycan synthesis by the murine ligase genes murC (alr5065) and murB (alr5066) for maintenance of patterned gene expression, filament integrity, and overall development. This work highlights the significant influence that intracellular structure and intercellular connections can have on the execution of a developmental program.

ABC Transporter Required for Intercellular Transfer of Developmental Signals in a Heterocystous Cyanobacterium.

In the filamentous cyanobacterium Anabaena, patS and hetN encode peptide-derived signals with many of the properties of morphogens. These signals regulate the formation of a periodic pattern of heterocysts by lateral inhibition of differentiation. Here we show that intercellular transfer of the patS- and hetN-dependent developmental signals from heterocysts to vegetative cells requires HetC, a predicted ATP-binding cassette transporter (ABC transporter). Relative to the wild type, in a hetC mutant differentiation resulted in a reduced number of heterocysts that were incapable of nitrogen fixation, but deletion of patS or hetN restored heterocyst number and function in a hetC background. These epistasis results suggest that HetC is necessary for conferring self-immunity to the inhibitors on differentiating cells. Nine hours after induction of differentiation, HetC was required for neither induction of transcription of patS nor intercellular transfer of the patS-encoded signal to neighboring cells. Conversely, in strains lacking HetC, the patS- and hetN-encoded signals were not transferred from heterocyst cells to adjacent vegetative cells. The results support a model in which the patS-dependent signal is initially transferred between vegetative cells in a HetC-independent fashion, but some time before morphological differentiation of heterocysts is complete, transfer of both signals transitions to a HetC-dependent process. How chemical cues that regulate pattern formation in multicellular organisms move from one cell to another is a central question in developmental biology. In this study, we show that an ABC transporter, HetC, is necessary for transport of two developmental signals between different types of cells in a filamentous cyanobacterium. ABC transporters are found in organisms as diverse as bacteria and humans and, as the name implies, are often involved in the transport of molecules across a cellular membrane. The activity of HetC was shown to be required for signaling between heterocysts, which supply fixed nitrogen to the organism, and other cells, as well as for conferring immunity to self-signaling on developing heterocysts.

Involvement of thioredoxin on the scaffold activity of NifU in heterocyst cells of the diazotrophic cyanobacterium Anabaena sp. strain PCC 7120.

The diazotrophic cyanobacterium Anabaena sp. strain PCC 7120 (A.7120) differentiates into specialized heterocyst cells that fix nitrogen under nitrogen starvation conditions. Although reducing equivalents are essential for nitrogen fixation, little is known about redox systems in heterocyst cells. In this study, we investigated thioredoxin (Trx) networks in Anabaena using TrxM, and identified 16 and 38 candidate target proteins in heterocysts and vegetative cells, respectively, by Trx affinity chromatography (Motohashi et al. (Comprehensive survey of proteins targeted by chloroplast thioredoxin. Proc Natl Acad Sci USA, 2001; 98: , 11224-11229)). Among these, the Fe-S cluster scaffold protein NifU that facilitates functional expression of nitrogenase in heterocysts was found to be a potential TrxM target. Subsequently, we observed that the scaffold activity of N-terminal catalytic domain of NifU is enhanced in the presence of Trx-system, suggesting that TrxM is involved in the Fe-S cluster biogenesis.

Cyanobacterial Oxygenic Photosynthesis is Protected by Flavodiiron Proteins.

Flavodiiron proteins (FDPs, also called flavoproteins, Flvs) are modular enzymes widely present in Bacteria and Archaea. The evolution of cyanobacteria and oxygenic photosynthesis occurred in concert with the modulation of typical bacterial FDPs. Present cyanobacterial FDPs are composed of three domains, the β-lactamase-like, flavodoxin-like and flavin-reductase like domains. Cyanobacterial FDPs function as hetero- and homodimers and are involved in the regulation of photosynthetic electron transport. Whilst Flv2 and Flv4 proteins are limited to specific cyanobacterial species (β-cyanobacteria) and function in photoprotection of Photosystem II, Flv1 and Flv3 proteins, functioning in the “Mehler-like” reaction and safeguarding Photosystem I under fluctuating light conditions, occur in nearly all cyanobacteria and additionally in green algae, mosses and lycophytes. Filamentous cyanobacteria have additional FDPs in heterocyst cells, ensuring a microaerobic environment for the function of the nitrogenase enzyme under the light. Here, the evolution, occurrence and functional mechanisms of various FDPs in oxygenic photosynthetic organisms are discussed.

Fatty alcohols can complement functions of heterocyst specific glycolipids in Anabaena sp. PCC 7120

Heterocyst glycolipid synthase (HglT) catalyzes the final step of heterocyst glycolipid (Hgl) biosynthesis, in which a glucose is transferred to the aglycone (fatty alcohol). Here we describe the isolation of hglT null mutants. These mutants lacked Hgls under nitrogen-starved conditions and instead accumulated fatty alcohols. Differentiated heterocyst cells in the mutants were morphologically indistinguishable from those of the wild-type cells. Interestingly, the mutants grew under nitrogen starvation but fixed nitrogen with lower nitrogenase activity than did the wild-type. The mutants had a pale green phenotype with a decreased chlorophyll content, especially under nitrogen-starved conditions. These results suggest that the glucose moiety of the Hgls may be necessary for optimal protection against oxygen influx but is not essential and that aglycones can function as barriers against oxygen influx in the heterocyst cells.

Expanding the Direct HetR Regulon in Anabaena sp. Strain PCC 7120

In response to a lack of environmental combined nitrogen, the filamentous cyanobacterium Anabaena sp. strain PCC 7120 differentiates nitrogen-fixing heterocyst cells in a periodic pattern. HetR is a transcription factor that coordinates the regulation of this developmental program. An inverted repeat-containing sequence in the hepA promoter required for proheterocyst-specific transcription was identified based on sequence similarity to a previously characterized binding site for HetR in the promoter of hetP. The binding affinity of HetR for the hepA site is roughly an order of magnitude lower than that for the hetP binding site. A BLAST search of the Anabaena genome identified 166 hepA-like sites that occur as single or tandem sites (two binding sites separated by 13 bp). The vast majority of these sites are present in predicted intergenic regions. HetR bound five representative single binding sites in vitro, and binding was abrogated by transversions in the binding sites that conserved the inverted repeat nature of the sites. Binding to four representative tandem sites was not observed. Transcriptional fusions of the green fluorescent protein gene gfp with putative promoter regions associated with the representative binding sites indicated that HetR could function as either an activator or repressor and that activation was cell-type specific. Taken together, we have expanded the direct HetR regulon and propose a model in which three categories of HetR binding sites, based on binding affinity and nucleotide sequence, contribute to three of the four phases of differentiation.

Optimation of Nitrogen and Phosphorus in Azolla Growth as Biofertilizer

Nitrogen is one of the most important minerals for living organisms. Azolla is known as a small water fern which can fix nitrogen through the symbiotic association with the blue green algae Anabaena. Experimental studies were carried out in the glass house, investigating the growth of Azolla using different levels of nitrogen and phosphorus in the media. The experiment used 12 treatments with 3 levels of nitrogen (0, 5, and 10 mg atom/L) and 4 levels of phosphorus (0, 10, 20, and 30 mg atom/l) in a factorial design with 3 replicates. The results show that the highest biomass for fresh weight (13.28 g), dry weight (1,126 g), and the fastest doubling time (7.71 days) were found in combination of 10 mg atom/L N and 30 mg atom/L P. Meanwhile, the highest protein content was found in combination of 5 mg atom/L N and 20 mg atom/L P. After the third day heterocyst cells in Anabaena could only be found in those two combinations, but the highest heterocyst cells was found in the lower N and P combination similar to the highest protein content. Further experiment is suggested to introduce Azolla as bio-fertilizer for acid water system, such as peat land water.