Promising microbial consortia for producing biofertilizers for rice fields

Two cyanobacterial cultures from rice paddies of Kyzylorda Provence, Kazakhstan were isolated and characterized: Anabaena variabilis and Nostoc calsicola. Based on these cultures, new consortia of cyanobacteria, microalgae and Azotobacter were developed: ZOB-1 (Anabaena variabilis, Chlorella vulgaris, and Azotobacter sp.) and ZOB-2 (Nostoc calsicola, Chlorella vulgaris, and Azotobacter sp.). High growth rate and photosynthetic activity of microalgae were observed in these consortia. The active consortium ZOB-1 was selected, which improved germination and growth of rice plants. ZOB-1 was recommended as a biostimulator and biofertilizer for crops.     

Mechanical stress tolerance of two microalgae

Aim of the present work is quantifying the mechanical stress generated by some major process equipment used in massive microalgae culturing plants (centrifugal and air-lift pumps, and nozzles) and highlighting its effects on the microalgal population. Two microalgal species were used as test cases: Chlorella vulgaris (unicellular) and Scenedesmus dimorphus 1237 (colonial). The evaluation of the shear effect on algal growth was carried out through measurement of absorbance, photosynthetic activity (oxygen evolution) and variable chlorophyll fluorescence. Cell aggregate development/breakage was effectuated by visual inspection and light scattering. The use of centrifugal pumps for culture recycling strongly affected the growth of C. vulgaris, while nozzles effects were confined to aggregate breakage of S. dimorphus. The analysis of experimental data is supported by the consideration of hydrodynamic stress calculated by: shear rate, shear stress, stress volumes/times, energy dissipation rates, and turbulence microscale size.     

An alternative strategy for enhancing lipid accumulation in chlorophycean microalgae for biodiesel production

Oleaginous microalgae are considered as important feedstocks for production of biodiesel. Under nutrient stress conditions, microalgae have the ability to accumulate higher amount of lipids, which can be transesterified for the production of biodiesel. In the present investigation, four different phosphate application strategies were examined in five green microalgae (Tetradesmus obliquus, Tetradesmus lagerheimii, Chlorella vulgaris, Chlorella minutissima, and Chlamydomonas sp.) to achieve higher lipid productivity. Effects of those strategies such as phosphate-sufficient (Control), phosphate-starved approach (PSA), biphasic phosphate-starved approach (BPSA), and sequential phosphate addition (SPA) were studied under batch culture mode. The BPSA emerging as the best in terms of lipid productivity consisted of two biomass harvesting phases, which would lead to an increase in the overall cost of biodiesel production. On the other hand, the SPA with a 1/200th dose of N 11 medium, i.e., 0.4 mg L^?1 of phosphate application in 3-day intervals, also resulted into higher lipid productivity which was equal to BPSA. Fatty acid composition of the biodiesel obtained from the microalgae was analyzed and the fuel characteristics were also evaluated. A profound (~14-fold) reduction in phosphorus requirements under the SPA mode with higher lipid productivity ensured qualitative biodiesel production and a lesser amount of phosphorus release, thus making the process eco-friendly.     

Comparison of <Emphasis Type="Italic">Chlorella vulgaris</Emphasis> and cyanobacterial biomass: cultivation in urban wastewater and methane production

Anaerobic digestion of microalgae is hampered by its complex cell wall. Against this background, cyanobacteria cell walls render this biomass as an ideal substrate for overcoming this drawback. The aim of the present study was to compare the growth of two cyanobacteria (Aphanizomenon ovalisporum and Anabaena planctonica) and a microalga (Chlorella vulgaris) in urban wastewater when varying the temperature (22, 27 and 32 °C). Cyanobacterial optimal growth for both strains was attained at 22 °C, while C. vulgaris did not show remarkable differences among temperatures. For all the microorganisms, ammonium removal was higher than phosphate. Biomass collected was subjected to anaerobic digestion. Methane yield of C. vulgaris was 184.8 mL CH₄ g COD in^?1 while with A. ovalisporum and A. planctonica the methane production was 1.2- and 1.4-fold higher. This study showed that cyanobacteria growth rates could be comparable to microalgae while presenting the additional benefit of an increased anaerobic digestibility.     

Bioprospecting for oil producing microalgal strains: evaluation of oil and biomass production for ten microalgal strains

Microalgae have the ability to grow rapidly, synthesize and accumulate large amounts (approximately 20-50% of dry weight) of lipids. A successful and economically viable algae based oil industry depends on the selection of appropriate algal strains. In this study ten species of microalgae were prospected to determine their suitability for oil production: Chaetoceros gracilis, Chaetoceros mulleri, Chlorella vulgaris, Dunaliella sp., Isochrysis sp., Nannochloropsis oculata, Tetraselmis sp., Tetraselmis chui, Tetraselmis tetrathele and Thalassiosira weissflogii. The study was carried out in 3 L glass flasks subjected to constant aeration and controlled artificial illumination and temperature at two different salinities. After harvesting, the extraction of oil was carried out using the Bligh and Dyer method assisted by ultrasound. Results showed that C. gracilis presented the highest oil content and that C. vulgaris presented the highest oil production.     

UV-B resistance as a criterion for the selection of desert microalgae to be utilized for inoculating desert soils

The adaption capability of microalgae species to intense UV-B radiation is an important feature for their survival under the harsh growth conditions they have to face when used for inoculating unconsolidated sand soils in desert areas. In this study, the responses of photosynthetic activity, reactive oxygen species (ROS) generation, and DNA strand breaks to UV-B radiation in four microalgae isolated from artificially induced biological soil crusts were investigated. It was found that low UV-B doses easily inhibited the photosynthetic activity and induced serious DNA damage in Chlorella vulgaris. Microcoleus vaginatus showed the capability to withstand only moderate doses of UV-B, while Nostoc was capable of facing high doses of UV-B due to its lower generation of ROS and higher capability to repair photosystem II (PSII) and DNA damages. On the other hand, Scytonema javanicum showed additional strategies to survive UV-B irradiance, namely the closure of PSII when ROS generation increased rapidly, in addition to a high repair ability of PSII and DNA damage. The results obtained point out different resistance and defense mechanisms of the four microalgae in response to UV-B irradiance and suggest that the strain of Nostoc sp. tested is the most suitable for surviving under the high UV irradiation levels typical of desertified areas.     

Starvation enhances phosphorus removal from wastewater by the microalga Chlorella spp. co-immobilized with Azospirillum brasilense

In synthetic wastewater, growth and phosphorus absorption by two species of microalgae, Chlorella sorokiniana and Chlorella vulgaris, and in domestic wastewater by C. sorokiniana significantly enhanced after a starvation period of 3 days in saline solution, combined with co-immobilization with the microalgae growth-promoting bacterium (MGPB) Azospirillum brasilense Cd in alginate beads. Starvation of 5 days negatively affected the subsequent growth of C. vulgaris, but not of C. sorokiniana in fresh wastewater. Starvation of immobilized cultures of microalgae separately or microalgae with bacteria, followed by returning the immobilized cultures to the same wastewater did not enhance phosphorus absorption. However, a starvation period followed by subsequent submersion of the cultures in fresh wastewater allowed the continuation of phosphorus absorption. The best phosphorus removal treatment from a batch of synthetic or domestic wastewater was with tandem treatments of wastewater treatment with pre-starved, co-immobilized microalgae and replacement of this culture, after one cycle of phosphorus removal, with a new, similarly starved culture. This combination treatment with two cultures was capable of removing up to 72% of phosphorus from the wastewater. There was a direct correlation between the initial load of phosphorus in the domestic wastewater and the efficiency level of removal, being highest at higher phosphorus loads in co-immobilized cultures. This occurred for both immobilized and co-immobilized cultures. Further, the results showed that negative effects of starving the microalgae were mitigated by the application of the MGPB A. brasilense Cd. This is the first report of this capacity in Azospirillum sp. on a single-cell plant. This study showed that starvation periods, combined with co-immobilization with MGPB, have synergistic effects on absorption of phosphorus from wastewater and merits consideration in designing future biological treatments of wastewater.     

Absence of negative allelopathic effects of cylindrospermopsin and microcystin-LR on selected marine and freshwater phytoplankton species

Cyanobacterial toxins have been regarded by some researchers as allelopathic substances that could modulate the growth of competitors. Nevertheless, often the concentrations of toxins used are too high to be considered ecologically relevant. In this work we tested the hypothesis that microcystin-LR (MC-LR) and cylindrospermopsin (CYN) at ecologically relevant concentrations have no allelopathic effects on some species of phytoplankton. Extracts containing the toxins as well as pure MC-LR and CYN toxins were used to assess their effects on the growth rates of Nannochloropsis sp., Chlamydomonas reinhardtii, and Chlorella vulgaris. Cyanobacterial crude extracts induced more pronounced effects on growth rates than pure toxins. Microcystis aeruginosa and Aphanizomenon ovalisporum crude extracts containing MC-LR and CYN at 0.025–2.5 mg l^?1 stimulated growth rates of microalgae, whereas A. ovalisporum crude extracts containing 2.5 mg l^?1 of CYN strongly inhibited growth rates of microalgae after 4 and 7 days of exposure. MC-LR and CYN at environmentally occurring concentrations were unable to affect negatively the growth of microalgae, and therefore these molecules may play roles other than allelopathy in natural ecosystems.     

Effects of <Emphasis Type="Italic">Ascophyllum nodosum</Emphasis> extract on growth and antioxidant defense systems of two freshwater microalgae

Microalgae in genus Chlorella and Scenedesmus are common in aquatic ecosystems and are widely used for various studies on algal growth and applications. Macroalgae may play an important role for control of microalgal growth, attributable to their rich content of bioactive compounds. In this study, the brown seaweed Ascophyllum nodosum was extracted with 70% acetone and the extract was used to treat the green microalgae, Chlorella vulgaris and Scenedesmus sp. Cell density and chlorophyll a concentration were used as growth indexes to evaluate the effects of A. nodosum extract (ANE) on the microalgae. The ANE with concentrations > 1% exhibited significant capability of inhibition of the growth of microalgae by over 80%. On the contrary, 1% ANE caused varying degrees of acceleration of cell proliferation and chlorophyll a synthesis in C. vulgaris and Scenedesmus sp., respectively. Analysis of antioxidant activities of the enzymes superoxide dismutase (SOD) and catalase (CAT) revealed the impact of ANE on the antioxidant defense system of the microalgae. The SOD and CAT activities were significantly depressed by high concentrations (> 2%) ANE, while a slight increase of the enzyme activities was observed with 1% ANE at the early period, which could be correlated to the growth response. Therefore, the mechanism of microalgae control could be related to the interaction between the ANE and the antioxidant defense systems. Phlorotannins are proposed as the principal algistatic components in the ANE which could be utilized in controlling microalgae growth.     

Effects of UV-C radiation on <Emphasis Type="Italic">Chlorella vulgaris</Emphasis>, a biofilm-forming alga

Photosynthetic biofilms proliferating on heritage monuments represent a major threat for curators leading to biodegradation and esthetic issues. Previous studies demonstrated that UV-C, used as a tool for biofilm eradication, is a promising avenue to combat microbial proliferation. In this study, this environmentally friendly method was tested on biofilm-forming Chlorella vulgaris suspension. Algal physiological response to UV-C was then assessed. Results showed that >?10 kJ m^?2 UV-C exposure was enough to directly kill cells whereas low UV-C exposure reduced quantum yield of photosystem II and inhibited both respiration and photosynthesis. Clear relationships between UV-C exposure times and physiological responses were found. In addition, the use of VIS-light after UV-C treatment enhances chlorophyll bleaching. Our findings contribute to a better understanding of the physiological responses of Chlorella vulgaris to UV-C radiation allowing thus an optimization of the UV-C treatment reported in our previous studies.     

Biomass productivity and productivity of fatty acids and amino acids of microalgae strains as key characteristics of suitability for biodiesel production

Microalgae are discussed as an alternative source for the production of biofuels. The lipid content compared to cultivation time of used species is the main reason for any choice of a special strain. This paper reviews more analytical data of 38 screened microalgae strains. After the cultivation period, total content of lipids was analysed. The extracted fatty acids were quantified as fatty acid methyl esters by GC analysis. The amino acids were analysed by HPLC. Chlorella sp., Chlorella saccharophila, Chlorella minutissima and Chlorella vulgaris were identified as species with the highest productivity of fatty acids relevant to transesterification reactions. The components were mainly linoleic acid, palmitic acid and oleic acid. To increase productivity of highly saturated fatty acids, cultivation parameters light intensity and temperature were varied. In this manner, the ideal conditions for biodiesel production were defined in this publication.     

Management of Warm- and Cool-Season Grasses for Biomass on Marginal Lands: II. Composition and Nutrient Balance

Organic fertilizers can improve soil health while providing nutrients for perennial grass growth for bioenergy feedstock, particularly under marginal soil conditions. The impact of organic fertilizer application on perennial grass composition needs clarification. Our objective was to evaluate feedstock composition, and N, P, and K dynamics of switchgrass (Panicum virgatum L.), tall fescue [Lolium arundinaceum (Schreb.)], and reed canarygrass (Phalaris arundinacea L.) provided with either inorganic or organic fertilizer sources. Grasses were established on a sandy soil and a clay soil at the Cornell University Willsboro Research Farm in Willsboro, NY. The experiment was a split-split plot randomization of a randomized block design with six replicates. Sites were whole plots, grass species were subplots, and fertility treatments were sub-subplots. Six treatments were (1) 168 kg ha^?1 of N fertilizer for cool-season grasses; 84 kg ha^?1 for switchgrass, (2) 56 kg ha^?1 of 0-46-0 P fertilizer plus N (#1), (3) 112 kg ha^?1 of 0-0-60 K fertilizer plus N (#1), (4) 89.6 Mg dairy manure ha^?1, (5) 44.8 Mg dairy manure compost ha^?1, and (6) a control without fertilizer. Organic fertilizers produced a net positive P and K balance, while other treatments had negative balances. Organic fertilizer treatments resulted in lower lignin and gross energy values, and higher total ash and Cl, compared to inorganic fertilizer treatments. Switchgrass biomass had higher fiber and gross energy, lower total ash, and much lower Cl content under organic fertilizer applications than cool-season grasses, making switchgrass a more desirable feedstock regardless of conversion process.     

Oil production by six microalgae: impact of flocculants and drying on oil recovery from the biomass

Oil production in batch photoautotrophic cultures of the following microalgae is reported: the freshwater microalgae Chlorella vulgaris, Choricystis minor, and Neochloris sp.; the marine microalgae Nannochloropsis salina and Cylindrotheca fusiformis; and C. vulgaris grown in a full-strength seawater medium. In all cases, the solvent extraction of lipids from freeze-dried biomass is compared with extraction from the fresh biomass paste. For all algae, the oils could be extracted equally effectively from freeze-dried samples and the paste samples (67–88 % moisture by weight). Moisture content determinations of the biomass using the freeze-drying method and the high-temperature oven drying were found to be equivalent for all algae. The biomass recovered by flocculation with metal salts (aluminum sulfate, ferric chloride) followed by centrifugation had a certain amount of the flocculant irreversibly bound to it. Washing failed to remove the adsorbed flocculants. For all algae, the adsorbed flocculants did not interfere with oil recovery by solvent extraction. The solvent system of chloroform–methanol–water proved highly effective for quantitative extraction of the lipids from all algae.     

Nostoc cyanobacterial inoculation in South African agricultural soils enhances soil structure, fertility, and maize growth

Many soils in South Africa have low nutrient supply, poor structural stability and are prone to soil erosion due to susceptibility to surface sealing and crusting. Two crusting soils from the Eastern Cape Province, South Africa were used to evaluate the effects of inoculation with a strain of Nostoc on soil structure, fertility and maize growth. The Nostoc suspension was uniformly applied over potted soils at a rate of 6g (dry weight) per square meter soon after maize germination. Nostoc inoculation increased soil N by 17% and 40% in Hertzog and Guquka soils, respectively. Soil C was also increased significantly and this increase was strongly associated with that of soil N (R ² = 0.838). The highest contents of soil C, soil N and mineral N, however, were found in non-cropped Nostoc inoculated soils. Nostoc inoculation increased maize dry matter yields by 49% and 40% in Hertzog and Guquka soils, respectively. Corresponding increases in maize tissue N were 23% and 14%, respectively. Scanning electron microscopy (SEM) revealed that soil particles and fragments of non-cropped inoculated soils had coatings of extracellular polymeric substances (EPS) with other particles enmeshed in networks of filaments, whilst by contrast little or no EPS and/or filaments were observed on cropped and/or non-inoculated soils. This was consistent with chemical analysis which showed that Nostoc caused significant increases in the EPS and soil C contents of non-cropped soils. The proportion of very stable aggregates was increased by inoculation with Nostoc possibly due to the greater quantities of soil C and EPS observed in inoculated soils. Inoculated soils cropped with maize had a lower proportion of stable aggregates presumably due to their low soil C and EPS contents compared to non-cropped soils. The results suggested that Nostoc could improve the fertility and structural stability of the studied degraded soils.     

The role of chemical and organic fertilizers on yield, yield variability and carbon sequestration— results of a 19-year experiment

Fertilization practice in the North China Plain has been changing since the late 1970s. To evaluate how organic and chemical fertilizers contribute to yield, yield variability and soil carbon sequestration, we analyzed wheat (Triticum aestivum L.) yield data in a long-term fertilization experiment that began in 1989, conducted pot experiments using soils from the long-term fertilization experiment plots, and simulated the soil organic carbon (SOC) dynamics of individual treatments in the long-term experiments. Wheat yield results showed that when organic fertilizer was used as an alternative nutrient source for chemical fertilizers, it was neither directly beneficial to crop yield, nor decreased yield variability when compared to a balanced chemical fertilizer. However, there was a linear relationship between yield trend and SOC change rate (r = 0.951, P?<?0.01). The use of organic fertilizer increased SOC and soil fertility and consequently resulted in a larger yield trend when compared to a balanced chemical fertilizer. Roth-C model simulation and pot experimental results indicated that soils with higher SOC had a higher root/shoot ratio. Therefore, the long-term use of organic fertilizer not only directly increases SOC, but indirectly contributes to carbon sequestration by favoring root development. We found that yield variability was determined by the relative contributions of soil fertility and fertilizer to yield (the contribution of fertilizer to yield is the yield difference between fertilized and unfertilized treatments). The contribution of balanced chemical fertilizer to yield was higher than that of organic fertilizer, resulting in less yield variability in balanced chemical fertilizer treatment. However, if organic fertilizer was used as a complementary nutrient source with chemical fertilizers, it would increase the contribution of fertilizers to yield, thus decreasing yield variability.     

Effects of arbuscular mycorrhiza on community composition and seedling recruitment in temperate forest understory

Arbuscular mycorrhizal (AM) fungal communities can influence the species composition of plant communities. This influence may result from effects of AM on seedling recruitment, although the existing evidence is limited to experimental systems. We addressed the impact of AM fungi on the plant community composition and seedling recruitment of two species – Oxalis acetosella and Prunella vulgaris – in a temperate forest understory. We established a field experiment over two years in which soil fertility (using fertilizer to enhance and sucrose to decrease fertility) and the activity of AM fungi (using fungicide) was manipulated in a factorial design. Species richness, diversity and community composition of understory plants were not influenced by soil fertility or AM fungal activity treatments. However, plant community composition was marginally significantly affected by the interaction of these treatments as the effect of AM fungal activity became evident under enhanced soil fertility. Suppression of AM fungal activity combined with decreased soil fertility increased the number of shoots of herbaceous plants. Unchanged activity of AM fungi enhanced the growth of O. acetosella seedlings under decreased soil fertility, but did not influence the growth of P. vulgaris seedlings. We conclude that the role of AM fungi in structuring plant communities depends on soil fertility. AM fungi can have a strong influence on seedling recruitment, especially for those plants that are characteristic of the habitat.     

Microalgae and Cyanobacteria: How Exploiting These Microbial Resources Can Address the Underlying Challenges Related to Food Sources and Sustainable Agriculture: A Review

For thousands of years, crop production has almost entirely depended on conventional agriculture. However, the reality is changing. The ever-growing population, global climate change, soil degradation and biotic/abiotic stresses are a growing threat to food production and security. Thus, sustainable alternatives to increase crop production for a population projected to reach 9.8 billion by 2050 are a major priority. In addition to vertical and soilless farming, innovative products based on bioresources, including plant growth stimulants, have been a target for sustainable food production. Such solutions have led to the exploitation of microorganisms, including microalgae and cyanobacteria as potential bioresources for food and plant biostimulant products. Microalgae (eukaryotic) and cyanobacteria (prokaryotic) are photosynthetic microorganisms with the capacity to synthesize a vast array of bioactive metabolites from atmospheric CO₂ and inorganic nutrients. The present review outlines the nutritional value of microalgae and cyanobacteria as alternative food resources. The potential aspects of microalgae and cyanobacteria as stabilizers of the net change in soil organic carbon (C) levels for reduced farmland degradation are also highlighted. The applications of microalgae and cyanobacteria as remedies for improved soil structure and fertility, and as enhancers of crop productivity and abiotic stress tolerance in agricultural settings are outlined. This review also discusses the co-cultivation of crops with microalgae or cyanobacteria in hydroponic systems to favor optimum root CO₂/O₂ levels for optimized crop production.

     

Magnetophoretic harvesting of freshwater microalgae using polypyrrole/Fe<Subscript>3</Subscript>O<Subscript>4</Subscript> nanocomposite and its reusability

A magnetophoretic harvesting agent, a polypyrrole/Fe₃O₄ magnetic nanocomposite, is proposed as a cost and energy efficient alternative to recover biomass of the microalgae Botryococcus braunii, Chlorella protothecoides, and Chlorella vulgaris from their culture media. The maximal recovery efficiency reached almost 99 % for B. braunii, 92.4 % for C. protothecoides, and 90.8 % for C. vulgaris. The maximum adsorption capacity (Q ₀) of the magnetic nanocomposite for B. braunii (63.49 mg dry biomass mg^?1 PPy/Fe₃O₄) was higher than that for C. protothecoides (43.91 mg dry biomass mg^?1 PPy/Fe₃O₄) and C. vulgaris (39.98 mg dry biomass mg^?1 PPy/Fe₃O₄). The highest harvesting efficiency for all the studied microalgae were at pH 10.0, and measurement of zeta-potential confirmed that the flocculation was induced by charge neutralization. This study showed that polypyrrole/Fe₃O₄ can be a promising flocculant due to its high efficacy, low dose requirements, short settling time, its integrity with cells, and with great potential for saving energy because of its recyclability.     

Potential yields and properties of oil from the hydrothermal liquefaction of microalgae with different biochemical content

A range of model biochemical components, microalgae and cyanobacteria with different biochemical contents have been liquefied under hydrothermal conditions at 350 °C, ∼200 bar in water, 1 M Na₂CO₃ and 1 M formic acid. The model compounds include albumin and a soya protein, starch and glucose, the triglyceride from sunflower oil and two amino acids. Microalgae include Chlorella vulgaris,Nannochloropsis occulata and Porphyridium cruentum and the cyanobacteria Spirulina. The yields and product distribution obtained for each model compound have been used to predict the behaviour of microalgae with different biochemical composition and have been validated using microalgae and cyanobacteria. Broad agreement is reached between predictive yields and actual yields for the microalgae based on their biochemical composition. The yields of bio-crude are 5-25 wt.% higher than the lipid content of the algae depending upon biochemical composition. The yields of bio-crude follow the trend lipids > proteins > carbohydrates.     

Interactive effects of molybdenum and phosphorus fertilizers on photosynthetic characteristics of seedlings and grain yield of Brassica napus

A pot experiment with acid yellow–brown soil was conducted to investigate the interactive effects of molybdenum (Mo) and phosphorus (P) fertilizers on the photosynthetic characteristics of seedlings and grain yield of Brassica napus which is sensitive to soil P and Mo deficiency. Both Mo and P fertilizers were applied at three levels (0 mg Mo kg^?1, 0.15 mg Mo kg^?1, 0.30 mg Mo kg^?1 soil; 0 mg P kg^?1, 80 mg P kg^?1, 160 mg P kg^?1 soil). The results showed that P fertilizer application increased grain yield, soluble sugar concentrations of seedling leaves, DM and P accumulation of seedling shoots of Brassica napus in the absence or presence of Mo fertilizer. In contrast, Mo fertilizer increased these parameters only in the presence of P fertilizer. Mo accumulation in shoots, chlorophyll concentrations and net photosynthesis rate (P _n) of seedling leaves were increased by both Mo and P fertilizers, particularly with the combination of the two fertilizers. The results also showed that the Mo and P fertilizers increased photosynthetic rate through two different mechanisms, with Mo increasing photosynthetic activity of mesophyll cells, and P increasing stomatal conductance. The results demonstrate that there was a synergetic effect on photosynthesis and grain yield between Mo and P fertilizers and it is conducive for Brassica napus growth to co-apply the two fertilizers.