INTRODUCTION
Under hydroponic systems, plants are grown in soilless media in containers, such as plastic buckets, tubs, or tanks. Containers can be arranged vertically to save space. Due to their space efficiency, hydroponic farming systems have become increasingly popular in urban communities.
Another attractive aspect of hydroponic farming is its cleanliness, because of the replacement of soil with a hydroponic growth medium, which is basically a liquid. Although hydroponic systems have advantages compared to soil systems, they still depend on a large volume of nutrient solution, or on frequent adjustment of the nutrient solution, to prevent nutrient uptake by plant roots from producing radical changes in the nutrient concentrations and pH of the medium (Taíz & Zeiger, 2010).
There are many media for hydroponic farming.
Commercial mediums, like the AB-mix medium, are widely used among hydroponic farmers. Another
medium, lab-developed to demonstrate potential inorganic solutions for plant growth, is the Hoagland medium. Among the many nutrients contained in a hydroponic medium, nitrogen is the highest in concentration. Plants require nitrogen to build many cell components, including the photosynthetic enzyme of RuBisCo in their leaves. Therefore, nitrogen deficiency, in plants, rapidly leads to chlorosis, which later inhibits plant growth. Nitrogen is usually provided in the form of ammonium (NH4+) or nitrate (NO3-). The long-term industrial production of ammonium and nitrate creates environmental burdens, because the processes use a great deal of fossil-fuel energy. In order to reduce the consumption of fabricated ammonium and nitrate, environmentally friendly nitrogen suppliers, such as N2-fixation cyanobacteria, can be used as an alternative source of nitrogen.
Nitrogen-fixing cyanobacteria are commonly found in soil (Choudhary, 2011; Deep, Bhattacharyya,
& Nayak, 2013; Singh, Kunui, Minj, & Singh, 2014).
ARTICLE INFO Keywords:
Ammonium Cyanobacteria Hydroponic Nitrate
Article History:
Received: March 20, 2018 Accepted: May 10, 2019
*) Corresponding author:
E-mail: [email protected]
ABSTRACT
For this research, an application of cyanobacteria Nostoc sp. SO-A31 as a nitrogen source for the growth of water spinach (Ipomoea aquatica L.) was carried out using a modified Deep-Water Culture (DWC) hydroponic system, outdoors. A Hoagland medium was used for the growth medium, with the absence or presence of ammonium and nitrate as the nitrogen sources. A 0.7 g fresh weight biomass of 21-day-old Nostoc sp. SO-A31 was inoculated into the system. The four treatment media for this study were HA0 (Hoagland, ammonium free+inoculant), HN0 (Hoagland, nitrate free+inoculant), HA0N0 (Hoagland, ammonium free and nitrate free+inoculant), and HI (Hoagland with ammonium and nitrate +inoculant). AB-mix and complete Hoagland media were used as controls. The result showed that water spinach cultured on HA0 had good vegetative growth, as shown by the high yield of biomass, high number of leaves, high stem growth, and long roots. Inoculation of Nostoc sp. SO-A31 elongated the root of the water spinach plants in all treatments. The presence of Nostoc sp. SO-A31 in the complete Hoagland medium, though, caused chlorosis of the water spinach leaves. This study suggests that water spinach is a nitrate-dependent leafy vegetable.
ISSN: 0126-0537Accredited First Grade by Ministry of Research, Technology and Higher Education of The Republic of Indonesia, Decree No: 30/E/KPT/2018
Application of N
2-Fixing Cyanobacteria Nostoc sp. SO-A31 to Hydroponically Grown Water Spinach (Ipomoea aquatica L.)
Andi Salamah, Nurrahmi Fadilah, Istatik Khoiriyah and Dian Hendrayanti*)
Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Indonesia, Indonesia
The vegetative cells of N2-fixation cyanobacteria are organized in a filamentous form. Vegetative cells are able to change into specialized cells, called heterocyst cells, which contain nitrogenase enzymes. Inside the heterocyst cells, catalyzed by the nitrogenase, dinitrogen is reduced into ammonium, which can later be released into the environment. Thus, the ability of Cyanobacteria to fix N2 provides ammonium in the soil (Nilsson, Bhattacharya, Rai, & Bergman, 2002;
Paudel, Pradhan, Pant, & Prasad, 2012). Moreover, the biomass of cyanobacteria also provides nitrogen when the population of cells decays.
The symbiotic, as well as free-living cyanobacteria, have been utilized in agricultural practices, especially in rice field. The water fern Azolla with its cyanobacterium symbiont Anabaena azollae released ammonium when inoculated in paddy field (Pathak et al., 2018). Free-living Anabaena, Nostoc, and Gloeotrichia were contributed to the productivity of rice in Chile (Pereira et al., 2009). In another research, several strains of Anabena were able to boost rice productivity in acidic condition when combined with nitrate-based chemical enhancers (Syiem et al., 2011). Hendrayanti, Kusmadji, Yuliana, Amanina, & Septiani (2012) showed that single inoculant Nostoc sp. GIA13a strains, isolated from rice fields in Gianyar, Bali, reduced the number of empty grains and influenced the elongation of rice roots. Fitrianti, Hendrayanti, & Kusmadji (2013) reported that, for paddy roots grown in a system of in-vitro culture, Nostoc strain GIA13a induced the formation of new lateral roots, while strain CPG24 stimulated the formation of hair roots.
N2-fixing cyanobacteria can potentially be applied as a source of nitrogen for other plants under hydroponic systems, such as vegetables. A variety of vegetable plants, such as lettuce (Lactuca sativa L.), spinach (Amaranthus spp.), and water spinach (Ipomoea aquatica L.), have been cultivated in hydroponic systems. Water spinach, known in Indonesia as kangkung, is a popular hydroponic vegetable. As a member of the Convolvulaceae (morning glory) family, the plant is characterized by long, jointed, and hollow stems. Leaves rise at the terminal nodes of the stems. Adventitious roots are formed at nodes which are in contact with water or moist soil. The young terminal shoots and leaves are used as leafy vegetables and in salads. Water spinach has high potential for commercial cultivation due to its high nutrient value (especially its iron content), agreeable taste, and fast harvest.
The two primary forms of inorganic nitrogen taken up by plants are nitrate and ammonium. In a hydroponic system, ammonium can immediately be oxidized into ammonia, which is toxic for plants.
Nitrogen, in the form of nitrate, is thus a preferable nitrogen source. Nitrogen-fixing cyanobacteria, in contrast, exhibit a preference for ammonium, which their cells readily uptake. This assimilation is sustained by a regulatory phenomenon termed nitrogen control (N control) that ensures permeases and enzymes of the assimilatory pathways for alternative nitrogen sources are not expressed when the cells are exposed to a non-limiting concentration of ammonium (Markou, Vandamme, & Muylaert, 2014). The opposing nitrogen uptake between plants and N2-fixing cyanobacteria may be an advantage for application of cyanobacteria in hydroponic systems.
The aim of this study was to examine the application of Nostoc sp. SO-A31 as a nitrogen source in hydroponic systems using Hoagland mediums for water spinach growth.
MATERIALS AND METHODS Experiment Design
The experiment was carried out in Universitas Indonesia, Depok, Indonesia from September to October 2017. The study used a modified Deep Water Culture (DWC) hydroponic system. The Deep Water Culture system allowed plants to absorb nutrients at all times, so that the plants could grow faster.
However, to minimize the loss of nutrients through precipitation, the depth of the growth medium was reduced to 1.2 L, so that only the tips of the roots were submerged into the medium. Replacement of old medium with new was carried out on day 7 of the 14-day experiment. Plant height was measured at T0, T7, and T14. Other vegetative growth parameters included root length, number of leaves, yield of biomass, and growth rate. The content of nitrate in the media was measured before and after the experiment.
Two basic media were used for plant growth: the AB-mix, as a commercial medium for hydroponic growth, and the Hoagland, as another control and for the treatment media. The biomass of Nostoc sp. for inoculum (0.7 g fresh weight) was obtained by running the 21-day-old culture through centrifugation. The treatments were as follows: AB (AB-mix medium), H (complete Hoagland medium), HA0 (Hoagland, ammonium free, with inoculant), HN0 (Hoagland, nitrate free, with inoculant),
HA0N0 (Hoagland, ammonium and nitrate free, with inoculant), and HI (complete Hoagland, with inoculant). The AB and H media served as controls.
The stock solution of Hoagland medium recipe is as follows: 101.1 g/L KNO3, 236.16 g/L Ca(NO3)2.4H2O, 246.49 g/L MgSO4.7H2O and 115.08 g/L NH4H2PO4; micronutrients: 74.5 g/L KCl, 64 g/L Fe(III)-EDTA, 12.5 g/L H3BO4, 1 g/L MnSO4, 1 g/L ZnSO4, 0.25 g/L CuSO4 and 0.25 g/L H2MoO4. To make 1 L of medium add each compound as follows respectively: 6, 4, 1, 2 mL and 2 mL for micronutrient solution. Acidity of the solution is adjusted to 6.5 pH (Taíz & Zeiger, 2010).
Selection of Cyanobacterial Strain
Selection of the strain of cyanobacteria for the inoculant was based on the observed growth of potential strains. Ten strains of cyanobacteria were cultured on Blue Green 11 liquid medium, without a nitrogen source (BG110), and placed on a rack at room temperature of 28-30 ˚C. The rack culture was provided with 300 lux intensity of continuous light.
Cultures were incubated for 3 weeks. Biomass was measured twice a week, for fresh and dry weights.
The strain with the maximum weight was selected for the inoculant.
Before application to the hydroponic system, the inoculant of cyanobacteria was habituated on a Hoagland medium compatible with each treatment.
This process shortened the adaptation phase of the culture when inoculated to the hydroponic system.
Co-cultivation of Water Spinach in the Hydroponic System
The plant grains used for the experiment were selected by soaking the potential grains in water. Floating grains were eliminated, and the submerged grains were preserved. The selected grains were then placed on rockwool to let them germinate. Three grains were put onto each section of rockwool. The seedling growth took place for 5 days at a room temperature of 30 ˚C. On the sixth day, the rockwools with growing seedlings were co- cultivated in the hydroponic system.
Rockwools were put inside the net pots and placed on the trays for each treatment. 12 rockwools (36 individual plants) were provided for each treatment (HA0, HN0, and HA0N0); 15 rockwools (45 individual plants) for each control (AB and H); and 4 rockwools for HI (12 individual plants). Growth media of 1.2 L for each treatment was poured into the corresponding tray, and the system was placed outdoors. Replacement of media was carried out
after 7 days of co-cultivation. On day 14, the plants were harvested.
Data Analyses
The plants’ vegetative parameters were analyzed by measuring the growth rate of each plant. Nostoc culture condition was observed using a stereo microscope, Olympus SZX16, and a digital microscope, Hirox KH-8700. For nitrate measurement, the samples were measured with Horiba Compact Water Quality Meter LAQUAtwin-NO3-11.
RESULTS AND DISCUSSION
The abilities of water spinach to grow in AB-mix and Hoagland media was first tested. Ten individual plants were cultivated on each media and placed in the system for two weeks. At the end of this part of the experiment, the water spinach was harvested, and the biomass was measured.
The data showed that the water spinach was able to grow in both media (Table 1). Plants in the complete Hoagland medium had plant height and biomass higher than those in the AB-mix medium;
meanwhile, the root length of plants in the AB-mix was longer than for those in the Hoagland.
Table 1. Growth of water spinach in Hoagland and AB-mix Media
Media Plant height
(cm)
Root length
(cm)
Fresh weight
(g)
weight Dry (g)
Hoagland 26 9.06 35.3 3.85
AB Mix 21.58 10.72 56.28 6.04 Isolates from the culture collection were purified and tested for their capabilities as inoculants.
Comparison of 10 strains showed that strain SO-A31 had the highest biomass after incubation for 21 days (Fig. 1). The yield of biomass from the 10 strains ranged from 0.009 to 0.809 g/mL (fresh weight). The dry weight of the biomasses fluctuated over a greater range. All strains showed a lag phase during the first 3 days of incubation. After that, the growth was enhanced for ca. 7 days before the majority of the strains entered a stationary phase, except for strains SO-A31, SO-B2, and SO-311.
The growth of these three strains was still exponential at day 21. Among these three strains, SO-A31 offered the highest yield (0.809 g/mL fresh weight; 0.0321 g/mL dry weight). Based on the stability of the culture and its capacity for rapid growth, strain SO-A31 was selected as the inoculant for this study.
Preliminary study showed that one considered medium, BG11, was not suitable for plant growth.
For experimental plants in laboratories, Hoagland is a common growth medium (Taíz & Zeiger, 2010). However, the ability of cyanobacteria to live in Hoagland must be tested. Although most of the macronutrients in Hoagland are similar to BG11, the difference in concentrations may affect the morphology and physiology of cyanobacterial strains. Hence, examining the adaptation of culture Nostoc sp. SO-A31 in the Hoagland medium, before applying the culture into the system was important. Fig. 2 shows the morphological changes of the culture in the Hoagland media with the various nitrogen sources. When cultured in BG110, the vegetative cells of strain SO-A31 were green, indicating that the thylakoid membrane filled the entire cell. When cultured in Hoagland, the thylakoid membrane was sequestered, making the vegetative cells look pale. However, the growth of filaments was still occurred and the culture became denser.
Physiological change was shown by the formation of heterocyst cells. Heterocyst cell count was based on 5 photo frames, with 3 replications of culture sampling. The average number of heterocyst cells in the cultures in the complete Hoagland, Hoagland without ammonium (HA0), Hoagland without nitrate (HN0), and Hoagland without ammonium or nitrate (HA0N0) media were 16, 45,
44, and 50, respectively. The data showed that when strain SO-A31 grew on a medium containing ammonium and nitrate (as in the complete Hoagland medium), the heterocyst number was reduced by more than 50%. If the medium was deprived of either or both nitrogen sources (as in HA0, HN0, HA0N0), numerous heterocyst cells formed.
It seems that ammonium and nitrate, independently, have the same influence on the formation of heterocyst cells. Strains of SO-A31 grown on the medium containing nitrate (HA0) formed a roughly equal number of heterocyst cells to the strain grown on the medium containing ammonium (HN0). According to Kumar, Mella- Herrera, & Golden (2010), the formation of heterocyst cells is influenced by nitrogen (usually in the form of ammonium or nitrate). Depletion of ammonium or nitrate in the environment will trigger the production of 2-oxoglutarate, which later binds the NtcA protein, a receptor protein from the CAMp family. This macromolecule then binds to the promoter region on the het-operon that regulate the synthesis of heterocyst cells. Heterocyst cells form gradually, as the nitrogen in a medium is consumed during the growth of a culture. Horn (2008) reported that cultures of N. commune began to form numerous heterocyst cells after 41 days on BG11 media, provided with 17.65 mM NaNO3 as the only source of nitrogen. In this study, strain SO-A31 had already formed heterocyst cells at 21 days.
Fig. 1. Growth of 10 isolates on BG11N0 medium for 21 days experiment
Table 2 shows the vegetative growth of water spinach after 14 days of incubation. The plants on the AB-mix and complete Hoagland media had very similar lengths of 23.5 and 23.9 cm, respectively.
This result was slightly different from the previous result. On the preliminary research, the plants planted on the Hoagland medium grew taller (26 cm) than those on the AB-mix medium (21.58 cm).
However, the data on root length was consistent with the previous experiment; in both cases, the plants on the Hoagland medium had shorter roots than those on the AB-mix. Similar to the plants in the control groups (complete Hoagland and AB-mix), the plants planted on HA0 were 24.06 cm in height. The other treatments showed lower plant heights, following the pattern HN0 > HI > HA0N0. The growth rate between days 7 and 14 showed which plants had good growth. Plants planted on the control media and in groups HA0, and HN0 showed increases in growth rate, while groups HA0N0 and HI decreased. It means that the metabolism of the plants in HA0N0 and HI
was distracted, causing growth to slow. The nitrogen missing from the HA0N0 treatment was supposed to be replaced by the Nostoc inoculant. However, the inoculant could not provide the nitrogen needed by the plants, which then slowed the plants’ growth.
Plants in the inoculant-treated groups showed longer root lengths. The root length of plants on HN0 reached 12.96 cm, followed by HA0 (12.24 cm), HI (10.38 cm) and HA0N0 (9.88 cm). Although the root length on HA0N0 was shorter than on other treatment groups, it was still longer for this group than for plants on complete Hoagland and was almost the same as the root length of plants on AB- mix. In previous research, a deficiency of NO3- was reported to increase the length of root hair in spinach and tomato plants, while NH4+ was reported to have no effect on root development (Vatter, Neuhäuser, Stetter, & Ludewig, 2015). This study showed the same result, the root length of water spinach in the nitrate-free medium (HN0) was longer than in the ammonium-free medium (HA0).
Fig. 2. Growth of Nostoc strain SO-A31 in Hoagland medium with a variety of nitrogen sources. A. BG11N0; B. Hoagland, ammonium free; C. Hoagland, nitrate free; D. Hoagland, ammonium free and nitrate free.
(scale: 100 μm). Arrow indicates heterocyst cell.
Table 2. Vegetative growth of water spinach after 14 days Treatment Plant height
(cm)1 Root length
(cm)1 Number of
leaf1 Fresh weight
(g)2 Dry weight
(g)2 Growth rate (H7-H14)
Complete Hoagland 23.9 6.5 9.2 72.66 5.64 1.28
AB Mix 23.5 10.44 9.6 72.48 6.91 1.22
HN0 21.54 12.96 7.8 30.56 2.7 1.14
HA0 24.06 12.24 8.2 49.52 3.4 1.29
HA0N0 11.58 9.88 5.6 15 1.71 0.52
HI 20.76 10.38 8.3 14.64 1.17 0.90
Remarks: HN0 = Hoagland, nitrate free + inoculant SO-A-3-1; HA0= Hoagland, ammonium free + inoculant SO-A-3-1;
HA0N0 = Hoagland, nitrate & ammonium free + inoculant SO-A-3-1; HI = Complete Hoagland + inoculant SO-A-3-1; 1 5 individual random samples; 2 15 individual samples
The measurements of fresh and dry weight biomass showed that plant biomass on the control media were higher than the plants in the treatment groups (Table 1). The fresh weight of plant biomass on the AB-mix and complete Hoagland media reached 72.48 and 72.66 g, respectively, and the biomass dry weight reached 6.91 g for plants on the AB-mix and 5.64 g for plants on the complete Hoagland. The fresh weight of plant biomass on HA0 was higher than on other treatments; the weights were 49.52 g (HA0), 30.56 g (HN0), 15 g (HA0N0), and 14.64 g (HI). The total number of leaves per plant, based on the biomass samples, was highest on HA0 (41), followed by HN0 (39), HA0N0 (28), and HI (27).
Among the treatments, HA0 showed results most similar to the control groups. Interestingly, the water content of the plants on HA0 was higher than the content of the control plants. After biomass drying, the reduction from the fresh weight in HA0 was 93%, while the control groups showed reductions of 91
% (AB-mix) and 92% (complete Hoagland). This suggests that water absorption in HA0-plants was better than in control plants. The water content in plants like water spinach makes the plants juicier and gives the harvested plants a fresher look, which is an advantage for the sale on the market.
Performance measures for plants grown on the AB-mix, complete Hoagland, and HA0 media were stronger than those of the HN0, HA0N0, and HI groups (Fig. 3). The stems were strong, and the leaves were green. Overall, the plants looked fresh and healthy. The performance of plants on HN0 was better than on HA0N0 and HI. Although the stems were slim, the leaves were green The plant conditions on HA0N0 and HI were poor. The plants were either slim (HI) or short (HA0N0), and the leaves were yellowish, indicating chlorosis. Slow growth and chlorosis have also been reported in cucumber (C. sativus) and spinach (S. esculentum) plants when grown in nitrate-limited media (Górska, Lazor, Zwieniecka, Benway, & Zwieniecki, 2010). All plants have their specific preferences and dependencies in response to different nutrients. For example, aspen plants were reported to have greater efficiency in nitrate acquisition than conifers (Kalcsits, Min, &
Guy, 2015). The fern Nephrolepsis exaltata and the black cottonwood Populus trichocarpa are low- nitrate need plants, so low-nitrate environments have no effect on their growth (Górska, Lazor, Zwieniecka, Benway, & Zwieniecki, 2010). Some leafy-vegetables, like lettuce and rocket (Eruca
sativa), tend to accumulate nitrate in their leaves in order to maintain high turgor pressure (Petropoulos, Chatzieustratiou, Constantopoulou, & Kapotis, 2016). This study suggests that water spinach is a nitrate-dependent plant. The presence of nitrate on the treatment medium of HA0, compared to HN0, promoted good vegetative growth: high stems, more leaves, high biomass, and high water content.
The measurement of media pH was carried out every week (Fig. 4). pH changing can be classified into three phenomena: A). pH was continuously decreased until the end of experiment;
B). pH was first decreased, then increased at the end of experiment; C). pH was first increased, then decreased at the end of experiment. pH changing is common in hydroponic cultivation. Basically, composition of ion H3O+ and OH- on the medium role the changing of pH. However, absorption of cations and anions in medium like K+ and NO3- will also shift the direction of pH equilibrium. Plant nutrient uptake is very related with pH regulation as plants always compensate ion absorption by releasing a pH altering ion of the same charge. For example, when water spinach’s roots absorbed NO3-, it released OH- in order to balance the charge. The same happen if plants absorb NH4+ as they release H3O+ to compensate ion absorption.
On the presence of ammonium, all treatments tended to experience the same pattern of pH changing. As water spinach quickly absorbed NH4+ on the medium, they released H+ (in the form of H3O+) which, in turn, lowered pH level. After first week, the pH level of treatments of AB-mix, H, and HN0 (Fig.
4, curve A) continued to decrease, while pH level of HI (Fig. 4, curve B) was increased at the end of experiment. Different phenomenon was showed in treatment of HA0 and HA0N0 (Fig. 4, curve C). pH was 6 at the beginning then increased to 7. At the end of experiment, the pH decreased drastically to 5. Most likely, water spinach in HA0 treatment absorbed NH3- and released OH- rising the pH up to 7. On the case of HA0N0 where ammonium and nitrate were zero, the plants could not absorb nitrogen. Plants absorbed phosphor in the form of HPO42-, in order to gain more energy for nitrogen substitution. As plants continue to grow, more addition of Nostoc biomass at the second week made pH decreased. The decay of Nostoc biomass from first week might lowered the pH level. Decomposition of Nostoc biomass served as ammonium source for plants.
Fig. 3. Growth of water spinach after 15 days of experiment. A. AB-mix; B. Complete Hoagland; C. HI (Complete Hoagland inoculated with strain SO-A31); D. HA0 (Hoagland, ammonium free, inoculated with strain SO-A31); E. HN0 (Hoagland, nitrate free, inoculated with strain SO-A31); F. HA0N0 (Hoagland, ammonium free and nitrate free, inoculated with strain SO-A31)
A B
D C
E F
Inoculation of strain SO-A31 was supposed to provide nitrogen in the medium to support the growth of water spinach. Proliferation of filaments of SO-A31 and formation of heterocyst cells was observed on HA0 and HN0. In the absence of nitrogen (HA0N0), filaments of SO-A31 proliferated and formed heterocyst cells. This indicates that SO- A31 can survive with or without nitrogen available in the environment (medium). For water spinach, the absence of nitrogen will stunt the growth of plants, as shown in Fig. 3f. Interestingly, when water spinach was grown with the inoculum on the complete Hoagland medium, the performance of plants was good, but there was a significant weight loss of biomass (Fig. 3c). There is evidence that the preferential order of nitrogenous compounds for cyanobacteria is ammonia, followed by nitrate, then urea (Chaffin & Bridgeman, 2014). Ammonium plays an important role on the GS-GOGAT cycle, as a metabolic pathway of nitrogen assimilation in N2-fixing cyanobacteria (Zhang & Zhao, 2008).
Ammonium is incorporated into glutamine to synthesize glutamate, one of the amino acids essential for cell metabolism. In the presence of nitrate or urea, utilization of these minerals takes a longer metabolic pathway. First, nitrate or urea has to be actively transported through the membrane cell. Second, it is reduced by nitrate reductase (for nitrate) or urease (for urea) into ammonium. Whether strain SO-A31 competes with water spinach for ammonium needs further study. Certainly, this
study showed that, although heterocyst cells form numerously when the strain was habituated on a medium containing a single nitrogen source of ammonium or nitrate, the product of the N2-fixation was consumed by the cyanobacteria alone. Another interesting phenomenon was that the presence of strain SO-A31 elongates the roots of water spinach plants, no matter what kinds of nitrogen sources were used in the environment.
CONCLUSION
The growth of water spinach in a medium containing nitrate as the single source of nitrogen resulted in good vegetative growth, as shown by the plants’ high biomass, high number of leaves, high stem growth, and long roots. The inoculation of Nostoc strain SO-A31 elongates the roots of water spinach grown in all treatments, but the effect on the shoots is still not clear.
ACKNOWLEDGEMENT
We would like to thank the Directorate General of Higher Education of Indonesia and the Directorate of Research and Community Engagement of Universitas Indonesia for supporting this research through The International Publication Research Grant (PITTA) 2017 to AS under contract No. 617/UN2.R3.1/HKP.05.00/2017. We also thank the Department of Biology Faculty of Mathematics and Natural Sciences Universitas Indonesia for their support.
Fig. 4. pH pattern on the treatments during experiment. A. Treatment of Hoagland, AB-mix, and HN0; B. HI;
C. Treatment of HA0 and HA0N0
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