Microalgae bio-stimulating effect on traditional tomato agriculture

MICROALGAE BIO-STIMULATING EFFECT ON TRADITIONAL TOMATO AGRICULTURE MÁSTER UNIVERSITARIO EN OCEANOGRAFÍA FACULTAD DE CIENCIAS DEL MAR UNIVERSIDAD DE LAS PALMAS DE GRAN CANARIA Facultad de Ciencias del Mar CURSO 2012 - 2013 AUTORA : TUTOR : STEPHANIE GARCÍA-SENÍN Dr. J.L. GÓMEZ PINCHETTI Microalgae bio-stimulating effect on traditional tomato agriculture Author/Autora: Stéphanie García Senín Abstract Soil care, organic agriculture and preservation of the environment are among the current interests of sustainable agriculture. Numerous plants’ stimulators are tested in the world as an alternative to organic agriculture. In the Canary Technology Institute (ITC) of Gran Canaria and under greenhouse conditions this study was carried out in order to analyze the effect of microalgae culture as irrigation water on Solanum lycopersicum plant. Through evaluations from days 7, 14, 21 and 28 the crop growth was analyzed. A bio-stimulating effect of Chlorella pyrenoidosa and Chlorella sp was established. Keywords: Chlorella pyrenoidosa and Chlorella sp, Microalgae, Open ponds, Solanum lycopersicum var. Charony, Agriculture, Bio-stimulants, Seed, Substrate, Irrigation water. Resumen El cuidado del suelo, la agricultura orgánica y la conservación del medio ambiente son algunos de los intereses actuales de la agricultura sostenible. Estimuladores para plantas son analizados en el mundo como alternativa en la agricultura orgánica. En el Instituto Tecnológico de Canarias (ITC) de Gran Canaria y bajo condiciones de invernadero se llevó a cabo este estudio con el objeto de analizar el efecto del uso de un agua de riego procedente del cultivo de microalgas sobre el crecimiento en plantas de Solanum lycopersicum. Mediante evaluaciones realizadas a partir de los días 7, 14, 21 y 28 se analizó la dinámica de crecimiento del cultivo. Un efecto bio-estimulante procedente del uso de Chlorella pyrenoidosa y Cholrella sp fue determinado. Palabras claves: Chlorella pyrenoidosa and Chlorella sp , Microalgas, Raceways, Solanum lycopersicum var. Charony, Agricultura, Bio-estimulantes, Sustrato, Cytokininas, Agua de riego. 1 2 INDEX 1. Introduction 3 2. Objectives 5 3. Materials and methods 6 3.1. Study location 3.2. Biological material 3.3. Irrigation water solutions 3.4. Culture scale up process 3.4.1. Culture media 3.4.2. Semi-continuous culture at pilot plant scale 3.4.3. Harvesting process 3.5. Obtaining irrigation water 3.6. Seed irrigation process 3.7. Seed treatment in petri dishes 4. Results 11 4.1. Cell density curve 4.2. Calibration curve 4.3. Emergency % analysis 4.4. Germination rate analysis 4.5. Green leaves rate 4.6. Dry weight analysis 4.7. Stem growth analysis 5. Discussion 21 6. Conclusions 24 7. Acknowledgments………………………………………………………..……..25 8. References 25 3 1. INTRODUCTION Seaweeds have been applied as soil conditioning agents for decades (Chapman and Chapman 1980; Guiry and Blunden 1981; Blunden 1986; Temple and Bomke 1988; Metting et al. 1988, 1990; Hong et al. 2007). About 15 million metric tonnes of seaweed products are produced annually (FAO 2006). A considerable portion of them is used as bio-stimulants to increase plant growth and yield. Numerous studies have revealed a wide range of beneficial effects of seaweed extract applications such as improved crop performance and yield, elevate resistance to biotic and abiotic stress, enhance postharvest shelf-life of perishable products, etc. (Hankins and Hockey 1990; Blunden 1991; Zhang and Schmidt 1997; Norrie and Keathley 2006). Besides eliciting a growth-promoting effect on plants, seaweeds also affect the properties of soil which in turn, influence plant growth (Jeannin and others 1991; Eyras and others 1998; Gandhiyappan and Perumal 2001; Moore 2004 ;). Although many of the chemicals components of seaweed extracts and their modes of action remain unknown (Fornes and others 2002; Vernieri and others 2005). The beneficial effects of Cyanophyta on improving the growth and yield of the crop plants cannot be explained by elevated nitrogen levels alone (Boussiba, 1988; Whitton, 2000). It has been suggested that plant hormones synthesized by Cyanophyta may also play a role in promoting growth (Pedurand and Reynaud 1987). It is probably that secondary metabolites present in the Cyanophyta such as cytokinins, will also be released into the soil during cell decomposition and so, will become available for uptake by plant roots. The cytokinins effect on various physiological processes is well documented with many practical applications in tissue culture and agriculture (Mok 1994; Kamı´nek et al. 1997; Schmu¨lling, 2002; Sakakibara 2006). Cytokinins have also been identified in Chlorophyta. Cytokinin-like activity was measured in strains Chlorella sp. and others isolated from soil samples collected in Brazil (Stirk et al. 2002; O¨ rdo¨g et al. 2004). The diversity of cytokinin forms detected, in vitro deuterium-labeling experiments and others studies related with cytikinin-like activity into their growth media, suggest that microalgae are able to synthesis their own cytokinins (Stirk et al. 2006; Burkiewicz 1987). 4 Plants grown in soils treated with seaweed or extracts applied either to the soil or foliage, exhibit a wide range of responses that have been well documented. Positive responses include improved germination, root development, leaf quality, general plant vigor and resistance to pathogens (Myklestad 1964; Booth 1965, 1966, 1969, 1981; Stephenson 1974; Blunden 1977; Senn and Kingman 1978; Abetz 1980; Verkleij 1992; Kahn et al. 2009). Tomato “Solanum Lycopersicum” is considered one of the most important vegetable in many countries (Nuez, F. 1995). In 1885 was established in Canary Island the first crops of tomatoes. Juliano Bonny Gómez S.L., in those early years, was one of the pioneers and promoters of tomatoes crop, essential, even our days, for Canary Island economy (EDEI, 1996). The recent challenges in food production due to the increasing occurrence of biotic and abiotic stress as likely due to climate change will further reduce yields and will have an impact on crops in the 21st century. Therefore, research into developing sustainable methods to alleviate these stresses should be a priority (IPCC 2007). Bio-stimulants used in tomato crops have accomplished effects on root development by improving lateral and longitudinal root formation, roots nutrient uptake, increasing total volume and vigor of the root system, plant chlorophyll content enhanced so at the same time general plant growth (Finnie and van Staden 1985; Crouch and van Staden 1992; Atzmon and Van Staden 1994; Blunden and others 1997; Thompson 2004; Sla`vik 2005; Mancuso and others 2006). However, bio-stimulants are unable to provide all the nutrients needed by a plant in required quantities (Schmidt and others 2003). Studies with radiolabelled tomato seedlings show clear evidence that roots are able to take up cytokinins from their external environment, metabolize them and then release some back into the growth medium (Van Staden 1976b ;Arthur et al. 2001a). High quantity of microalgae biomass is produced to cover industry demand and several systems of microalgae culture are used and studied for culturing in an extensive scale. Photo-biorreactors are closed systems where microalgae are growing under artificial, natural or combined illumination. They have been successfully used for producing large quantities of microalgae biomass (Molina Grima et al., 1999; Tredici, 1999; Pulz, 2001; Carvalho et al., 2006). Open pond (raceways) are made of a closed loop recirculation channel, built in concrete blocks or compacted earth. Mixing and culture circulation is provided by the 5 paddlewheels. Microalgae cultures of raceways are exposed under atmospheric or greenhouse conditions where the temperature fluctuates during the diurnal cycle and the seasons (Richmond A., 1991). Microalgae are relatively easily grown in mass culture and they provide a source of biological material with potential application in agricultural industries. (Moore, 2004) In an attempt to develop a cost-effective methodology for Chlorella sp mass cultivation, the company Juliano Bonny Gómez S.L. is studying the Chlorella culture in open systems (raceways) to obtain high value synergies between traditional tomato crop and microalgae culture. To reach this goal Juliano Bonny Gómez S.L. and the Canary Technological Institute (ITC) signed a collaboration agreement to develop a new project: "Feasibility study on microalgae culture as a complementary activity to traditional agriculture”, based on a previous ITC project called MAXPRUA. Chlorella pyroneidosa and Chlorella sp cultures were placed in the southeast of Gran Canaria (Canary Island), in the pilot plant facilities of the biotechnology department of the Canary Technological Institute (ITC). These organisms provide the author with their support, knowledge and facilities for the development of her research. The aim of this paper is to show the first results of a series of tests carried out with the intention to endorse the hypothesis of a possible bio-stimulating effect in tomato crop using Chlorella pyr and Chlorella sp culture growth media as irrigation water. 2. OBJECTIVES 1. Demonstrate the possibility of bio-stimulant effect on Solanum lycopersicum plant using microalgae culture growth media as irrigation water. 2. To identify possible different bio-stimulant affects between strains of Chlorella pyrenoidosa) and Chlorella sp. (Hydroponic water isolated). 3. To analyze behavior differences between two substrates (peat and vermiculite) under the bio-stimulant influence. 6 3. MATERIALS AND METHODS 3.1. Study location The study was conducted in March-May of 2010 at the Canary Technological Institute (ITC) within the premises of the Biotechnology’s Department, located in Pozo Izquierdo in Gran Canaria. 3.2. Biological material - Tomato seeds Solanum lycopersicum var. charony was obtained from Juliano Bonny Gómez S.L. - The strain of Chlorella pyrenoidosa (BNA-ITC-Chlo-pyr-01) and Chlorella sp. (Hydroponic water isolated) (ITC-Chlo-hidrop-09) were facilitated by the research group of ITC biotechnology department. - As substrates, were chosen, Substrate T: peat (mixture of black and light pet; Juliano Bonny Gómez S.L.) as organic substrate and substrate V: vermiculite as mineral substrate (Vermiculita Extendida; Perindustria S.L.). 3.3. Irrigation water solutions A = Water from Chlorella Pyr culture + macro and micro nutrients to reach 100% of its initial concentration according to modified prescription of Tamiya, 1968 recipe. B = Water from Chlorella hy culture + macro and micro nutrients to reach 100% of its initial concentration according to modified prescription of Tamiya, 1968 recipe. C = Water control (sterilized freshwater) + macro and micro nutrients to reach the same concentration according to chlorella supernatant ones. 7 3.4. Culture scale up process 3.4.1. Culture media Culture was grown in filtered (0.2 m) and sterilized freshwater (121ºC during 22 min), enriched with M/M modified medium (Table 1). Table 1: M/M medium for laboratory culture. SOLUTION 1 (g/L): (S.Beijerink) (25ml/L) SOLUTION 2(g/L): (25ml/L) MICRO-NUTRIENTS(g/l): (1ml/L) MgSO4*7H2O = 4.00g KH2PO4 = 14.52g CoCl2* 6H2O = 1.61g CaCl2 = 1.54g K2HPO4 = 37.42g EDTA = 50.00mg NH4Cl = 16.00g. MnCl2 * 4H2O = 5.06 g CuSO4 * 5H2O = 1.57g H3BO3 = 11.40g ZnSO4*7H2O = 22.00g (NH4)Mo7O24 = 1.10g FeSO4 * 7H2O = 4.99g Recipients of different sizes were used for the scale up process. After being, sterilized, they were filled with the volume of MM medium necessary in each step, 24 h previously to the inoculation day. The scaling up was carried out from stock cultures of the growth chamber conducted in containers of 500ml borosilicate Erlenmeyer, containers were stirred with filtered air, enrichment with 0.3-0.5% CO2 in air (V / V). The stock was maintained in exponential growth by sub culturing weekly series. (Carmona, D. L. 2007; Said, M.M. 2010). Laboratory cultures were grown under 20˚C temperature and 55% humidity conditions. Photon flux (PAR) of 250 mmol photon m -2 s -1 was continuously provided by fluorescent tubes (Philips Master TLD 36W/840). 14 Figure 3. Emergency % Analysis in Petri dishes versus time (days). EVPY(Water irrigation solution A with vermiculite (V) substrate Emergency %) yellow represented, EVC (Water irrigation solution C with vermiculite (V) substrate Emergency %) red represented, EVHI (Water irrigation solution B with vermiculite (V) substrate Emergency %) green represented, ETPY(Water irrigation solution A with peat (T) substrate Emergency %) violet represented, ETC (Water irrigation solution C with peat (T) substrate Emergency %) blue represented and ETHI (Water irrigation solution B with peat (T) substrate Emergency %) orange represented. Table nº 3: Average of emergency % rate in Petri dishes. DAY EVPY EVC EVHI ETPY ETC ETHI 1 0,00% 0,00% 0,00% 0,00% 0,00% 0,00% 2 3,00% 3,00% 7,00% 3,00% 0,00% 3,00% 3 43,00% 23,00% 43,00% 33,00% 17,00% 27,00% 4 60,00% 33,00% 50,00% 47,00% 43,00% 43,00% 5 67,00% 57,00% 67,00% 60,00% 57,00% 53,00% 6 87,00% 80,00% 83,00% 73,00% 73,00% 60,00% 7 93,00% 100,00% 97,00% 93,00% 80,00% 67,00% 8 97,00% 100,00% 97,00% 100,00% 97,00% 80,00% 9 100,00% 100,00% 97,00% 100,00% 97,00% 80,00% 15 4.4. Germination rate analysis For vermiculite substrate (V) no significant variances were found among the irrigation solutions (Figure 4). Nevertheless, a decrease of 4.76% and 9.52 % in germination rate value with irrigation solutions A and B were found in peat substrate (T). Figure 4. Germination rate versus time (days). TGVPY(Water irrigation solution A with vermiculite (V) substrate Germination rate) dark blue resented, TGVC (Water irrigation solution C with vermiculite (V) substrate Germination rate) red represented, TGVHI (Water irrigation solution B with vermiculite (V) substrate Germination rate) green represented, TGTPY(Water irrigation solution A with peat (T) substrate Germination rate) violet represented, TGTC (Water irrigation solution C with peat (T) substrate Germination rate ) blue represented and TGTHI (Water irrigation solution B with peat (T) substrate Germination rate) orange represented. GERMINATION RATE Germination % Days 16 Table nº 4: Average of the percent of emergency rate. DAY TGVPY TGVC TGVHI TGTPY TGTC TGTHI 1 0,00% 0,00% 0,00% 0,00% 0,00% 0,00% 2 0,00% 0,00% 0,00% 0,00% 0,00% 0,00% 3 0,00% 0,00% 0,00% 0,00% 0,00% 0,00% 4 0,00% 0,00% 0,00% 0,01% 0,00% 0,03% 5 11,43% 13,33% 9,52% 12,38% 8,57% 9,52% 6 16,19% 21,90% 16,19% 15,24% 12,38% 17,14% 7 38,09% 48,57% 40,00% 53,33% 38,09% 44,76% 8 47,62% 49,52% 41,90% 56,19% 45,71% 55,24% 9 50,47% 49,52% 41,90% 56,19% 49,52% 56,19% 10 64,76% 62,86% 58,10% 65,72% 60,00% 60,95% 11 64,76% 66,67% 64,76% 72,38% 69,52% 65,71% 12 64,76% 66,67% 64,76% 76,19% 84,76% 69,52% 13 66,66% 67,62% 66,67% 77,14% 85,71% 71,43% 14 66,66% 67,62% 66,67% 77,14% 85,71% 71,43% 15 66,66% 68,57% 66,67% 78,10% 85,71% 71,43% 16 66,66% 68,57% 66,67% 78,10% 85,71% 71,43% 17 66,66% 68,57% 66,67% 80,00% 85,71% 72,38% 18 66,66% 68,57% 67,62% 80,00% 85,71% 73,33% 19 66,66% 68,57% 67,62% 80,95% 85,71% 74,28% 20 67,62% 68,57% 69,52% 80,95% 85,71% 76,19% 21 67,62% 68,57% 70,48% 80,95% 85,71% 76,19% 22 67,62% 68,57% 70,48% 80,95% 85,71% 76,19% 17 4.5. Green leaves rate For peat substrate (T) a decrease of 8.58% in green leaves rate value with irrigation solution B was found as well as no significant variances were recognized among the irrigation solutions A and C. Referring to green leaves rate should be emphasized that vermiculite substrate had lower value than the obtained in peat substrate regardless of the irrigation water used. Figure 5. Green leaves rate versus time (days). THVVPY(Water irrigation solution A with vermiculite (V) substrate Green leaves rate) red resented, THVVC (Water irrigation solution C with vermiculite (V) substrate Green leaves rate) green represented, THVVHI (Water irrigation solution B with vermiculite (V) substrate Green leaves rate) violet represented, THVTPY(Water irrigation solution A with peat (T) substrate Green leaves rate) blue represented, THVTC (Water irrigation solution C with peat (T) substrate Green leaves rate) orange represented and THVTHI (Water irrigation solution B with peat (T) substrate Green leaves rate) grey represented. 18 Table nº5: Average of the percent of green leaves rate. DAY THVVPY THVVC THVVHI THVTPY THVTC THVTHI 1 0,00% 0,00% 0,00% 0,00% 0,00% 0,00% 2 0,00% 0,00% 0,00% 0,00% 0,00% 0,00% 3 0,00% 0,00% 0,00% 0,00% 0,00% 0,00% 4 0,00% 0,00% 0,00% 0,00% 0,00% 0,00% 5 0,00% 0,00% 0,00% 1,90% 1,90% 0,00% 6 0,00% 0,00% 0,00% 3,81% 3,81% 0,00% 7 17,14% 29,52% 11,43% 29,52% 29,52% 20,00% 8 34,29% 40,95% 27,62% 34,28% 34,28% 39,05% 9 35,24% 40,95% 27,62% 43,81% 43,81% 42,86% 10 50,47% 50,48% 28,57% 45,71% 45,71% 42,86% 11 59,05% 54,29% 62,86% 54,29% 54,29% 50,47% 12 61,90% 65,71% 64,76% 80,95% 80,95% 67,62% 13 63,81% 66,67% 65,71% 82,86% 82,86% 68,57% 14 64,76% 66,67% 65,71% 82,86% 82,86% 69,52% 15 64,76% 66,67% 66,67% 82,86% 82,86% 69,52% 16 64,76% 67,62% 66,67% 82,86% 82,86% 69,52% 17 64,76% 67,62% 66,67% 82,86% 82,86% 69,52% 18 64,76% 67,62% 66,67% 82,86% 82,86% 70,47% 19 64,76% 67,62% 67,62% 82,86% 82,86% 71,43% 20 65,71% 67,62% 69,52% 82,86% 82,86% 74,28% 21 65,71% 67,62% 70,48% 82,86% 82,86% 74,28% 22 65,71% 67,62% 70,48% 82,86% 82,86% 74,28% 4.6. Dry weight analysis: A decrease on dry weight was present in peat substrate with irrigation water solution B. But in the other hand it appears that an increase in the growth was found using irrigation water solution A and B in vermiculite substrate. Referring to the dry weight rate should be emphasized that the vermiculite substrate has a lower rate than the obtained with peat substrate with the exception of irrigation water solution B. 19 Figure 6. Dry weight versus time (days). PSVPY(Water irrigation solution A with vermiculite (V) substrate Dry weight) dark blue resented, PSVC (Water irrigation solution C with vermiculite (V) substrate Dry weight) red represented, PSVHI (Water irrigation solution B with vermiculite (V) substrate Dry weight) green represented, PSTPY(Water irrigation solution A with peat (T) substrate Dry weight) violet represented, PSTC (Water irrigation solution C with peat (T) substrate Dry weight) blue represented and PSTHI (Water irrigation solution B with peat (T) substrate Dry weight) orange represented. Table nº6: Average of the percent of dry weight. DAY D7 D7 D14 D14 D21 D21 D28 D28 PSR PSP PSR PSP PSR PSP PSR PSP PSVPY 0,038 0,024 0,133 0,039 0,23 0,19 0,463 0,19 PSVC 0,033 0,022 0,114 0,037 0,119 0,141 0,4 0,141 PSVHI 0,038 0,018 0,154 0,039 0,201 0,473 0,579 0,473 PSTPY 0,055 0,016 0,181 0,025 0,169 0,48 0,846 0,48 PSTC 0,034 0,01 0,211 0,019 0,224 0,614 0,818 0,614 PSTHI 0,025 0,01 0,083 0,021 0,11 0,3 0,228 0,3 20 4.7. Stem growth analysis (length and width) No significant variances were recognized among the irrigation solutions AB or C with peat substrate but lower growth trend was found in solution B with vermiculite substrate. Results obtained in peat substrate were higher than in vermiculite, highest values occurring with irrigation water solution A Figure 7.1. Growth stem (width) versus time (days). AVPY(Water irrigation solution A with vermiculite (V) substrate Growth stem (width)) dark blue resented, AVC (Water irrigation solution C with vermiculite (V) substrate Growth stem (width)) red represented, AVHI (Water irrigation solution B with vermiculite (V) substrate Growth stem (width)) green represented, ATPY(Water irrigation solution A with peat (T) substrate Growth stem (width)) violet represented, ATC (Water irrigation solution C with peat (T) substrate Growth stem (width)) blue represented and ATHI (Water irrigation solution B with peat (T) substrate Growth stem (width)) orange represented. Figure 7.2. Growth stem (length) versus time (days). LVPY(Water irrigation solution A with vermiculite (V) substrate Growth stem (length)) dark blue resented, LVC (Water irrigation solution C with vermiculite (V) substrate Growth stem (length)) red represented, LVHI (Water irrigation solution B with vermiculite (V) substrate Growth stem (length)) green represented, LTPY(Water irrigation solution A with peat (T) substrate Growth stem (length)) violet represented, LTC (Water irrigation solution C with peat (T) substrate Growth stem (length)) blue represented and LTHI (Water irrigation solution B with peat (T) substrate Growth stem (length)) orange represented. STEM WIDTH Stem widht (cm) STEM LENGTH Stem Lenght (cm) 21 Table nº7: Average of growth stem (length and width) DAY D7 D7 D14 D14 D21 D21 D28 D28 LV AV LV AV LV AV LV AV VVPY 1,883 0,078 3,161 0,102 4,193 0,127 5,497 0,268 VVC 1,867 0,073 3,006 0,095 4,411 0,128 5,269 0,224 VVHI 1,633 0,077 2,829 0,098 3,839 0,123 4,846 0,193 VTPY 2,133 0,09 3,083 0,103 4,437 0,108 5,4 0,271 VTC 1,817 0,082 3,394 0,102 4,789 0,137 5,93 0,252 VTHI 1,95 0,055 3,111 0,105 4,773 0,119 5,863 0,274 In order to confirm the previous results variance analysis (ANOVA) were performed. (Massart, 1997) Considering the results obtained, null hypothesis having a calculated F value bigger than the F tabulated (α = 0,02,) was accept, demonstrating that data were significant. 5. DISCUSSION Microalgae mass production optimization has been investigated in closed and open outdoor systems (Grobbelaar 2007; Sandnes et al. 2005; Zou and Richmond 2000). For commercial objectives, microalgal cultivation is carried out in open, outdoor systems (Huang et al. 2010). The present work shows the possibility of scale Chlorella pyr and Chlorella sp (hy) culture from the laboratory to the greenhouse. Two raceways of 3 m2 have been successfully used for scale Chlorella pyr and Chlorella sp (hy) cultivation, under greenhouse conditions. Open raceways, however, present several difficulties. High cell densities and efficient control of principal culture parameters had to be achieved, to ensure a sustainable cultivation process (Tredici and Chini Zittelli, 1998). Biomass requires large ground areas and becomes easily contaminated by microalgae and zooplankton (Richmond, 1986) demanding therefore long experience to maintain it successfully. During 90 days both cultures worked in semi-continuous system. Cultures were inoculated on March 2010 with 250 L monoalgal inoculum for each raceway. They were grown at the same conditions and under the same 22 management. Under temperature and irradiance condition in Gran Canaria Island, from March to May 2010, scale Chlorella sp (hy) cultures flocculate and cells fall down the bottom of the raceway at the same time than polysaccharidelike segregation was observed. Cultures were harvesting at the beginning of the stationary phase (1-1.2 optical density) where cultures were nutrients consuming with low growth rate. Semi-continuous system was working during three months, until the culture collapsed, due to the appearance of algal predators and contaminating algal species (Sukenik et al., 1993). For the first phase of germination, seedlings were placed during three days in ITC garage to avoid sunlight. Alveoli (holes) where seeds would be placed later were identified for future monitoring and control. Each of the alveoli seeds were buried in 0.5 - 1 cm depth. After three days of phase 1, the alveoli plates were taken outside outside (greenhouse) to start the emergency phase. Seed daily control was done. Growth phase was carried out simultaneously with the emergency phase. To perform the various parameters analysis random of three seedlings of each treatment were chosen. For each substrates and treatments, seedlings were placed in transparent bags identified with their code, after being removed from the alveoli in a delicate way to prevent breakage. Measurements were carried out immediately after extraction in 7, 14, 21 and 28 day of each trial (Bewley and Blanck, 1982). Three experiments replicated were carried out using representative samples in which irrigation water solution A-B and C was used. Tomato seedling during the different growth phases showed a healthy appearance deprived of visible parasites or diseases. The plant growth analysis has been developed over the decades as a discipline related to the ecophysiology and agronomy. (Diez, C. et al, 2001). Peat is one of global seedling production substrate more used, its physical, chemical and biological characteristics provide excellent germination and seedling growth, but its high cost and unsustainable exploitation, has begun to restrict it use. This has motivated the search for other alternatives substrates like vermiculite substrate among others (Abad et al. 1998; Budgee, GJ et al. 1989; Harz, K. et al 1986; Peña 2005). Numerous products from organic and mineral as an alternative to organic agriculture are tested in the world. These are foliar applied as stimulators of plant growth and some extent the ground, having effects on soil 23 biota and physico-chemical properties. (Pohl J., 2002). Substances such as hormones, vitamins and amino acids had been studied in bacteria during the decomposition of organic matter (Coyne M.; 2000). Microorganisms and lichenlike were found in our soils after 10 day of growth, without affecting the growth of plants, compounds might be part of the bio-stimulant effect. The presence of microalgae culture water irrigation favors soil fertility, improves organic matter and makes phosphorus available for plants and edaphic medium. The effects achieved by Chlorella pyr and Chlorella hy culture as source of irrigation water, corroborate the points made by scientists who have devoted serious efforts to related to the synthesis, biological activity and practical applications of a new class of plant growth regulators (Laugart and Romero (2003), Casanova et al., 2003). Germination rate is an important factor related with agriculture industrial production because of the significance uniformity of plans for bidding (Bridges et al. 1997). No substantial differences were attained in germination test with V substrate although the results achieved, showed a 10% decrement in relation to substrate T. Uniformity was observed in seedlings as a favorable indicator about the future growth of tomato plants. At that point, it’s important to underline that germination rate results in peat substrate were more efficient than vermiculite ones. These marks prompts us to say that peat has best physical, chemical and nutrition conditions which promotes germination rate and confirm peat substrate capacity of moisture retention during microbiological processes, temperature control, pH, etc. Similar data were obtained by Jacob et al. 1993. With an average of 80.90 % and 68.89%, substrate T and V respectively, seedlings presented two fully deployed cotyledon leaves, which designate their level of development, indicative of speed and uniformity seed germination (FernandezBravo, C. et al. 2000). 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