Liquid organic fertilizer production
pH, EC, total N, total P and total K, organic matter organic carbon, C/N ratio and E. coli in the original substrates (e.g., molasses, distillery slop and sugarcane leaves) were measured and the results of chemical properties of the original substrates are shown in Table 1. The average EC values of molasses and distillery slop were 39.10 and 32.86 dS/m, respectively. These values are extremely high, indicating a large salt content.
The pH (Fig. 1a) of liquid organic fertilizer formulas 1 (F1), 3 (F3) and 5 (F5) proved most suitable, whereas those of F2, F4 and F6 were slightly acidic. These results were similar to those of Saelee (2004), who found that most liquid organic fertilizers suitable for agricultural production had pH in the range 3–5. These acidic pH values were due to the amount of sugars in the substrate which are converted to lactic acid or acetic acid by the activities of microorganisms. All formulas had very high values of EC (Fig. 1b) due to the high levels in the natural substrate such as molasses and distillery slop. Good quality liquid organic fertilizers should normally have a pH less than 5 and an EC value less than 20 dS/m. Such values are the standards for liquid organic fertilizer in Thailand (National Bureau of Agricultural Commodity and Food Standards, Ministry of Agriculture and Cooperatives 2005). The values reported here were consistent with those recorded by Saouy (2002), who showed that some liquid organic fertilizers had high electrical conductivity (0.03–93.1 dS/m). The results of the chemical analysis of liquid organic fertilizer after periods of fermentation are presented in Table 2. After 15 days of fermentation, liquid organic fertilizer formula 4 (F4) had the highest content of total N (0.21%), total P (0.015%), total K (1.08%), OM (5.89%), OC (3.43%) and C/N ratio (16.41). After 30 days of fermentation, the highest total N (0.33%) and total K (1.18%) were found in F6. In contrast, F3 had very high of C/N ratio (17.92) compared to all other formula. Total N content increased with time of fermentation due to the activity of nitrogen-fixing microorganisms. The microorganism decomposed organic nitrogen materials in the fermentation process (Moonrat 2010). The results showed that the dissolution of phosphate was high due to the large population of phosphate dissolving microorganisms which had an important role in mineralizing P (Sureshkumar et al. 2013). The total K content, however, mostly remained stable. Liquid organic fertilizer had low nutrient content due to decomposition by microorganisms converting the substrates to nutrient content. The total amounts of N and P were lower than those present in the liquid organic fertilizer standard of Thailand, which are greater than 0.5%. However, total K showed higher value than the standard (0.5%) as the substrates used in this study contained large amounts of phosphorus. The organic carbon content decreased when the fermentation process was complete. During microbial fermentation, microorganisms used organic carbon as a source of energy and nutrients (Yusran 2008). Organic carbon was converted to carbon dioxide, resulting in reduced carbon content due to loss of atmosphere (Alexander 1977). Measuring C/N ratio in liquid organic fertilizer during fermentation found that the C/N ratio at 30 days of fermentation showed the C/N ratio value was lower than 20. According to the report Leblanc et al. (2007), if the C/N ratio of a fertilizer was lower than 20, the substrate was easily degraded, initially immobilized by microbes. In addition, the C/N ratio is an important requirement for microorganisms, which use carbon as an energy source and nitrogen as a basic element for the formation of protein and other constituents of the cell protoplasm and microbial and organic carbon. This activates microbial cellulolytic and microbial proliferation which immobilize N (Satisha and Devarajan 2007).
A nutrient solution is used in normal hydroponic cultivation for plant nurseries and commercial greenhouses. This commercial nutrient solution consists of two concentrated solutions, namely “solution A” and “solution B”, which is typical of any hydroponic recipe (Jones 2016). The chemical composition of the liquid chemical fertilizer (A and B) used in this study has the pH: 6.20–6.41, EC: 3.01–3.25 dS/m, Total N (0.139–0.198%), P (0.005–0.310%), K (0.002–2.530%) (Table 3). This shows that the nitrogen, phosphorus and potassium content in chemical fertilizer was higher than in the liquid organic fertilizer product. Furthermore, nitrate levels were also higher in chemical fertilizers (32,240 mg/l). These nitrates accumulate in the plant and consequently can be harmful to both human health and plant growth (Ikemoto et al. 2002; Ishiwata et al. 2002; Anjana and Iqbal 2007).
Change of microbial population in liquid organic fertilizer during fermentation
Molasses or sugar was used as raw materials for the fermentation of liquid organic fertilizer utilizing the plasmolysis process. The plant cell was autolysis in this condition and released organic matter such as amino acids, and carbohydrates inside the cell. These substances were degraded by microorganisms with natural contaminates in the substrate, producing amino acids, hormones, and enzymes (Wiparwin 2006).
The process of producing organic fertilizer was directly related to microbial decomposition of the substrate. The population of plant growth-promoting microorganisms in liquid organic fertilizer was determined for all formulations. The results showed that plant growth-promoting bacteria (such as nitrogen fixer bacteria) were present in the system and led to an increase in biomass yield. Phosphate solubilizing bacteria and potassium solubilizing bacteria were also present in the fertilizer. Plant growth-promoting bacteria assist plant growth, and the inclusion of such bacteria in liquid organic fertilizer could increase organic N and nutrient content. Ratneetoo (2012) reported that microbes were beneficial to plant growth, helping to circulate nutrients to plants. F6 has the highest in total bacteria in the sample as compared to other liquid organic fertilizers (P < 0.05) (Fig. 2). The beneficial microbes in the fertilizer could improve product quality, through the application of a sustainable fermentation process. All fertilizer standards were tested for human pathogen contamination and importantly E. coli was not found in any of the liquid organic fertilizers.
Indole acetic acid (IAA)
Pizzeghello et al. (2001) and Glick (2003) explained that plant growth hormones are produced within the plant through synthesis of the microorganism in the fertilizer. At the end of the fermentation process, microorganisms play an important role producing phytohormones such as auxin and cytokinin, organic acids and plant growth promoters which appear in liquid organic fertilizers. Therefore, the plant growth-promoting activity of microorganisms can be related to the production of auxins (Ortíz-Castro et al. 2009). The microbial population produces auxins as part of their metabolism including indole-3-acetic acid (IAA) (Patten and Glick 1996). According to Lwin et al. 2012, IAA hormone was detected in 80% of bacteria isolated from the rhizosphere. Consequently, IAA production was a determinant trait both for plant growth-promoting rhizobacteria (PGPR) (Patten and Glick 1996, 2002). The assay of indole acetic acid (IAA) in liquid organic fertilizers found that all formula contained IAA (Table 4). The IAA in liquid organic fertilizers was significantly different (P < 0.05). After 30 days of fermentation, the IAA content values increased in all liquid organic fertilizers. According to Kamla’s (2007) research on bio-extract fermentation, such bio-extracts might serve as a food source for microorganisms in the process of plant hormone synthesis. These plant hormones were then able to stimulate plant growth. Standard liquid organic fertilizer (National Bureau of Agricultural Commodity and Food Standards, Ministry of Agriculture and Cooperatives 2005) requires a content of more than 0.1 mg/l of IAA and our fertilizer had an IAA content greater than 0.1 mg/l which is the standard level of Thai fertilizers.
Determining the quality and maturity liquid fertilizer is important. The presence of phytotoxicity substances in the product would render it unsafe for use. Immature fertilizer is often contaminated with intermediate compounds such as ammonia and organic acids that cause toxicity in the plant. These compounds inhibit germination of root length. Fertilizer maturity can be evaluated in many ways, such as by detection on C/N ratio, NH4+/NO3− ratio humicfication, Cation Exchange capacity (CEC), and germination index (GI) (Yang, and Chang, 1998). However, GI is the only means to easily measure, through a simple process to yield fast and reliable results. This method aims to measure toxic (phytotoxic substance) in plants resulting from incomplete maturity of the fertilizer by extracting the organic matter in fertilizer with a melting texture, organic acid salt group, phenolic group and other toxins (Tiquia et al., 1996). If the fertilizer is toxic, these elements will have a direct effect on seed germination and plant root length (Wong et al. 2001). Under Thailand’s standards for liquid organic fertilizer (National Bureau of Agricultural Commodity and Food Standards, Ministry of Agriculture and Cooperatives 2005), maturity is indicated by GI value which is greater than 80%. According to CCQC (2001), a GI value higher than 90% indicates the phytotoxic-free property of the tested fertilizers which could then be safely applied for plant growth. The effect of liquid organic fertilizer on the seed germination of Green Cos Lettuce is presented in Fig. 3. The liquid organic fertilizer samples, such as non-diluted and those diluted with distilled water in the ratio 1:10, showed lower germination index values (data not shown), as high concentrations of fertilizer had negative effect on seed germination. The inhibitory effect of liquid fertilizer on germination and growth of young seedlings was probably due to the high EC and heavy metal content (Lwin et al. 2012), and other toxic compounds such as ammonia (Ells et al. 1991), ethylene oxide (Wong et al. 1983) and phenolic compounds. Liquid organic fertilizer obtained at 30 days of fermentation and diluted at 1:100 gave a germination index greater than 100% which indicated that diluted liquid fertilizer of all formulas were without phytotoxicity to seed germination. The value of germination index was found highest in F3 (147.9%) and was significantly different (P < 0.05) compared to that of the control (Fig. 3). Our result yielded data similar to that of Malaviya and Sharma (2011), who worked on the impact of distillery effluent on seed germination of Brassica napus L. They found decreasing values of germination index when diluted samples of fertilizers were used. Ramana et al. (2002), studying the effects of distillery effluent on the germination of some vegetable seeds, showed similar results, and in their findings the germination index was lower when the concentration of effluent was higher.
Growth of Green Cos Lettuce in the hydroponics cultivation
In this experiment, liquid organic fertilizer contained only small amounts of total N and P, which was lower than the Thai agricultural standard. However, it was a rich source of total K due to the substrates used in production. Moreover, the liquid organic fertilizer contained micro and supplement nutrients and also beneficial microorganisms such as plant growth-promoting rhizobacteria (PGPR) not found in chemical fertilizers. Most importantly, hormone IAA product was found in the liquid organic fertilizers. The IAA content was 59.53 mg/l. This hormone was produced by microorganisms, with some found in the plant substrate. IAA stimulates the growth of root hairs and increases the root surface area. This advantage was unique to the liquid organic fertilizer, as these nutrients are not found in chemical fertilizer. Increased root growth means that the plant can better absorb water and nutrients. The combination of chemical fertilizer and organic fertilizer indicated a possible decrease in the population of beneficial microorganisms in hydroponic systems (2006). These microorganisms (such PGPRs) have been used in agriculture as biofertilizers, bio-control agents, and bioremediations. PGPR have been introduced into both soil and hydroponic systems with positive effects on plant quality and quantity (Kidoglu et al. 2009; Lee and Lee 2015). PGPR act through: N2 fixation, control of plant stress, extracting nutrients from soil, competition with pathogens, production of various kinds of plant hormones and biological controls, and promotion of plant growth (Bull et al. 1991; Freitas et al. 1993; Gaskins et al. 1985; Kloepper 1993; Lugtenberg and Kamilova 2009). The result from this work indicated that hydroponic systems that used liquid organic fertilizer were a suitable medium for observing interactions between rhizobacteria and roots. After the germination experiment, an experiment was carried out to confirm the effects of the liquid organic fertilizer on the growth of the vegetable. The optimum dilution ratio (1:100 ratio liquid organic fertilizer: water) for organic fertilizers, which was indicated by the above results, was used in this experiment. Hydroponic cultivation (NFT) was selected for growing the lettuce. The pH (Fig. 4a) and EC (Fig. 4b) values of diluted (1:200) liquid organic fertilizers (T2–T7) and diluted (1:200) liquid chemical fertilizer AB changed slightly from day 0 to day 28 of planting. The highest electrical conductivity was found in T1 (chemical fertilizer AB) (Fig. 4b). Chemical fertilizers A and B were fed to the plants as a control treatment. Effects of liquid organic fertilizers (T2–T7) and liquid chemical fertilizer AB (T1) on the growth of Green Cos Lettuce varied according to the treatments (Tables 5, 6, 7). After 14 days of cultivation (Table 5), T1 (chemical fertilizer AB) showed the greatest plant height (11.60 cm), leaf length (10.33 cm) and leaf width (4.66 cm). However, T6 (F5) gave the highest Green Cos Lettuce growth among the liquid organic fertilizer treatments. The highest growth parameters of lettuce grown in T6 (F5) were root length (24.03 cm), leaf width (4.36 cm), shoot fresh weight (2.42 g) and shoot dry weight (0.15 g) which were significantly different (P < 0.01) from those of other treatments. However, after 21 days of cultivation, T4 (F3) gave the highest growth parameters, including root length (37.66 cm), plant height (17.33 cm), leaf width (18.83 cm) and shoot fresh weight (21.65 g) (Table 6). After 28 days of cultivation, T4 (F3) still gave the highest growth among the liquid organic fertilizers. Plants cultured in T4 (F3) had a significantly higher number of leaves and greater root length than those grown with T1, whereas all other growth parameters were similar to or lower than those in T1 (Table 7). The liquid organic fertilizer was without pathogen, indicated by a negative result on detecting E. coli. The study of Shinohara et al. (2011) presented a novel and practical hydroponic culture method using microorganisms to degrade organic substrate and utilizing fish-based soluble matter in the hydroponic solution which has been developed as an organic fertilizer. In addition, this research found that the lettuce seedlings grew well when the microbial culture solution was used. Nelson (2013) compared organic and inorganic fertilizers for hydroponic lettuce production. In this study, it was found that liquid organic fertilizer formula 3 and 5 showed no significant difference in all growth parameters measured when compared to the results using chemical fertilizer as a treatment (Fig. 5). Nitrate content was determined in the liquid organic fertilizer formula 3 which was sampled at 15 and 30 days of fermentation. It was found that the nitrate contents were 19.07 and 19.95 mg/l, lower amounts than in the chemical fertilizer AB (type A 32,240 and type B 27,115 mg/l, respectively). The inorganic fertilizer was found to contain more nitrates. However, it was discovered that the amount of nitrogen must be in a balanced form. In this research, toxins and heavy metals in liquid organic fertilizers of all formula were below the limits mandated by Thai Agricultural standards. The research of Mavrogianopoulos et al. (2002) used wastewater for hydroponic cultivation and examined the efficiency of giant reed (Arundo donax L.) as a source of biomass production and bio-based filtering device for sewage effluents. Giant reed populations showed a positive response to wastewater applications, in terms of growth and biomass production and gave no visual sign of detrimental or toxic effect. It can be concluded that organic hydroponics based on this method is a practical solution using organic sources as a fertilizer (Shinohara et al. 2011).
Chlorophyll is essential to photosynthesis and plant leaves with high chlorophyll content will gain energy and produce food more efficiently (National Bureau of Agricultural Commodity and Food Standards, Ministry of Agriculture and Cooperatives 2005). The main nutrients required are nitrogen, phosphorus, and potassium. Nitrogen is an important nutrient for chlorophyll production (Tancho 2013). Leaf chlorophyll content is related to the amount of nitrogen, which is a component of the chlorophyll structure. Cabrera (1998) found that the nitrogen content of a leaf was related to its color. Chlorophyll is the main pigment in plants and a greener leaf indicates more pigment and, therefore, more nitrogen. According to Lesing and Aungoolprasert (2016), organic fertilizer was the source of nitrogen, and similarly it presented higher chlorophyll content in kale leaves. Previous research showed that the pigment in green cos lettuce contained the chlorophyll A, B and total chlorophyll. In general, leaf chlorophyll content is affected by environmental conditions such as light volume and salinity changes. According to Romero-Aranda et al. (2001), as the value of the EC of the solution increased, the amount of chlorophyll within the leaves of the tomato also increased. The effects of liquid chemical fertilizer and liquid organic fertilizer on the chlorophyll contents of Green Cos Lettuce are presented in Table 8. The variation in chlorophyll content was found to be significantly different in all treatments. The highest chlorophyll a, chlorophyll b and total chlorophyll were found in T6 which were similar to T1. The lowest chlorophyll contents were found in T2.