Enrichment of composition of collected water hyacinth biomass
Water hyacinth samples were collected from the lake of Sholinganallur 12.9010 o N,80.2279oE Chennai, Tamil Nadu, India (Fig. 1a,b,c). Pre-treatment of the collected biomass was done by the soluble alkali. Leaves were expelled from the stalk and the gathered leaf material was washed with tap water followed by distilled water. Washed material was slashed into littler pieces, soaked in known volume of sodium hydroxide solution ( 1.5 % ) for 48 hours. Following 48 hours of alkali treatment, the drenched pieces were dried in a hot air oven for over night to expel the dampness content. The dried material was finely powdered utilizing residential blend (Fig. 1d, e). Homogenized material was sieved through 22 work and the sieved powder was stored in separate container, utilized for further studies. Scanning electron microscopy (SEM) studies was carried out to determine structural changes of WHB before and after alkali treatment. SEM micrographs were taken to determine the structural changes.
Ethanol production from the processed biomass
In the case of bioethanol production, solid state fermentation (SSF) was adopted for the preparation of hydrolysate- which consists of digested simple sugar from the biomass treated with cellulolytic fungal co culture. Hydrolysate thus obtained was allowed to fermentation under batch condition using yeast Saccharomyces cerevisiae. Total yield and concentration of final fermented bioethanol was measured.
Solid state fermentation was adopted for the hydrolysate preparation from the alkali treated biomass. Processed biomass (60 g) was transferred to 250 ml of conical flasks, followed by sterilization using autoclave. After sterilization, the moisture content of the substrate was adjusted to 0, 1, 2.5, 5.0 7.5 &10.0 % using sterile distilled water under aseptic condition. Cellulolytic fungal strains used in the present study, Trichoderma sp and Aspergillus niger were isolated from agriculture field soil samples. Isolated fungal strains were identified based on morphological and cultural characteristics on solid media (Fig. 3).Screening of cellulolytic activity of the fungal strains was done by the method of Vyas & Vyas (2005).Pure culture of the respective fungal strain was maintained on potato dextrose agar (PDA) slant. 1.0% (w/v) of co culture of cellulolytic fungal (Trichoderma sp and Aspergillus niger) inoculum were inoculated into the conical flasks respectively. Inocula of the respective fungal strain was prepared in mineral broth medium supplemented with substrate solution (0.1 % carboxyl methyl cellulose). Seeded flasks were incubated for five days at 35oC under static condition. After the incubation period, the respective flask was flooded with sterile distilled water, kept, under shaking condition at ambient temperature for one hour. Content was filtered through the whatman No 1 filter paper, the collected filtrate was centrifuged at 10,000 rpm. Supernatant thus obtained was used as the source of hydrolysate for bioethanol production.The concentration of total reducing sugars was estimated using the DNS method (Miller 1959).
Conversion of biomass derived hydrolysate into ethanol by yeast mediated fermentation
Batch fermentation study was adopted for ethanol production using yeast strain. Known volume of filter sterilized hydrolysate thus obtained was inoculated to the sterile basal medium (Mukhopadhya et al 2008).Two ml of Saccharomyces cerevisiae inoculum was added to the media. For inocula preparation, a loopful of S.cerevisiae culture (obtained from Microbial Type culture collection- MTCC ,Chandigurh ,India) was inoculated into sabouraud dextrose broth. Inoculated broth was incubated at 35°C for 48 hours under shaking condition. After the incubation period, the fermented broth was filtered,collected filtrate was analysed for total ethanol content (AOAC 1990).
In this study, methylene blue was selected for the adsorption efficacy of WHB under batch condition. Methylene blue analytical grade (Qualigens) was prepared as required concentration (100,125,150,175 and 200 ppm) in 100 ml of distilled water followed by the addition of 0.1 and 1.0 % dosage of WHB separately. Seeded flasks were incubated under orbital shaker (100 rpm) at ambient temperature. At the respective time periods, the samples withdrawn from the flasks and the amount of residual dye was m0easured at 668 nm using Shimadzu-1800 spectrophotometer.Effect of WHB on the chemical oxygen demand (COD) of the methylene blue solution was carried out.
Water treatment efficacy
Effect of WHB on the waste water treatment efficacy was studied by batch studies under controlled condition. Brewery industry effluent with the following parameters pH 9.4,1860 mg/L TDS,980 mg/L BOD,1875 mg /L COD, 45.3 mg/L TOC,32.4 mg/L TN,36.4 mg/L TP ) that collected from Empee brewery industry, Padappai, Kanchipuram, Tamil Nadu was used in this study to check the water treatment efficacy. Known quantity of collected sample (1L) was transferred to 2L of conical flask followed by the addition of 1.0 % WHB. Seeded flasks were incubated at ambient temperature under shaking condition (150 RPM). To determine the WHB mediated waste water treatment efficacy, changes in the various chemical parameters (pH ,total dissolved solids (TDS),chemical oxygen demand (COD),biological oxygen demand (BOD),total organic carbom, total nitrogen, total phosphorous ) was done by using standard methods of APHA. Biofilm inhibition efficacy was also investigated by modified method of Toole & Kolter, 1998). In this method, known volume of collected water sample (5ml) was transferred to the sterile screw cap vial under aseptic condition followed by incubation without shaking at 37°C for three days. After the incubation period, adherent biofilm was stained by crystal violet followed by solubilisation with ethanol. Biofilm forming efficacy was calculated by measuring the optical density of the ethanol solubilized biofilm at 570 nm. Morphological changes of the biofilm after exposure to the WHB treatment was studied by scanning electron microscopy analysis (Namasivayam et al 2019). Adherent biofilm was isolated from the inoculated vial, fixed with glutaraldehyde and observed for morphological changes.
Evaluation of reusability of WHB treated water and dye
Reusability of WHB treated brewery effluent and methylene blue was done by phytotoxicity assessment. Phytotoxicity assessment was studied by determination of germination index (seedlings emergence percentage).
Seeds collection and treatment
Healthy seeds of Vigna mungo were collected from seeds collection centre of Chengalpattu Agriculture Department, Washing of the seeds with the sterile distilled water was done before seed treatment. Washed seeds were grouped into control and treatment followed by soaking in the suspension derived from the respective treatment at room temperature for 1 hour. After the incubation period, treated seeds from the respective treatment groups were transferred to the sterile petri plate, incubated at ambient condition. From the data of seedlings emergence in the respective group, germination index was determined.
Zebra fish developmental toxicity
Non target toxic effect was studied by determination of developmental parameters of zebra fish as described in our recent study (Namasivayam et al 2018).
Evaluation of enhanced growth stimulation efficacy (GSE)
Enhancement of growth stimulation efficacy (GSE) of biomass and its synergistic impact with different biofertilizers Rhizobium sp, Azospirillum sp and Phosphobacterium sp on V.mungo was examined on green gram (Vigna mongo ) (under pot culture condition. Particular biofertilizer was utilized as powder definition. One gram of powder detailing of individual biofertilizer was blended well in with one gram of WHB independently and moved to the mud pot pre-loaded up with soil (2 Kg/pot).Three replications and control was kept up for every treatment. Healthy seeds of V.mungo were moved to the pots and permitted to develop Water was applied every day. Plant growth advancing impact was studied by estimating different parameters of tested plants after 30 and 60 days treatment.s
Influence of WHB media on biomass production of fungal biopesticidal agent Beauveria bassiana
Assessment of WHB on the biomass and spore production of potential fungal biopesticide Beauveria bassiana was done under in vitro condition and the pesticidal activity of biomass in this manner acquired from WHB medium was screened against castor caterpillar Achaea janata.
Isolation of B.bassiana
The tested fungal strain was isolated from groundnut nearby field soil tests embracing soil dilution technique as portrayed by our recent reports (Namasivayam et al 2018). Well homogenized collected soil sample was serially diluted and the aliquots was spread plated on selective media and the isolated fungal culture was distinguished dependent on the morphological and cultural characteristics embracing standard strategies. Isolated pure culture was maintained on agar slant. Inoculum of the fungal strain for mass production was derived from agar slant.
Evaluation of biomass and spore production of fungal biopsticidal agent Beauveria bassiana
WHB based liquor medium was prepared by suspending different concentration of homogenized WHB (0.1%, 0.25%, 0.5%, 1%, 2%, 2.5%, 5% ) in 100 mL of distilled water, sterilized by autoclaving. After sterilization, 0.1 ml of the inoculum was inoculated into the WHB medium. Seeded flasks were incubated under static condition at 28ºC for 5 days. After the incubation time, the medium was filtered and the dry weight of collected biomass was recorded. For the determination of spore count, collected biomass was washed with sterile distilled water,centrifuged at 2,500 rpm for 10 minutes. Collected supernatant was used as the source of inoculum.
The impact of water hyacinth media on spore germination was additionally contemplated utilizing cavity slide method. Spore suspension thus obtained was transferred to the sterile cavity slide kept in the petriplates fixed with sterile filter papers moistened with sterile distilled water, incubated at 28ºC. Spore germination was observed under microscope and the information got was utilized to record germination index ( %)..
Effect of fungal spores on survival parameters of Achaea janata
Assessment of the pesticidal action of spores got from the fungal biomass was tried against significant castor semi-looper Achaea janata. The pesticidal action was confirmed by examining total survival rate of respective treatment groups
Effect of fungal spores on cumulative mortality against Achaea janata
Various larval instars of Achaea janata (1-1V) were used to determine the insecticidal activity of B.bassiana. Spore suspension was gotten from the fungal strain was utilized as the wellspring of inoculum for the pesticidal activity Spore concentration of the suspension was previously determined and aliquots were set up from the stock utilized for additional investigations. In this study, respective larval instar collected from the laboratory stock culture were sprayed with various aliquots of spore suspension using ultra-low volume sprayer. Triplicates and control was maintained. Everyday observation on larval mortality was recorded for a time of 4 days. Total mortality was recorded in the individual measurements of treatment which was used to predict lethal concentration 50 (Finney, 1962).