Protocol Improvement & Product Development of Liquid Organic Fertilizers from Fermented Plant Extract
This research project evaluated the microbial and chemical properties of different botanical concoctions to help the farmers standardized their own biological extracts. Biological extract preparations commonly known as fermented plant extract/juices (FPE/FPJ), indigenous microorganisms (IMO) and fermented fruit extract (FFE) were prepared according to current farmer’s practice. Additional preparations with modifications on the current mixing protocol and use of inoculation were also employed as part of the improvement and standardization of the biological extract.
The fermented plant extract (FPE) was prepared using ground leaves of madre de cacao added with 25% brown sugar and 25% water for untreated set-up. Inoculated treatment contained an additional 10% FPE solution obtained from previous set-up. For FFE, grounded banana peelings was used at 50% , brown sugar, 25% water, 25% for untreated set-up. For inoculated set-up, 1% inoculum was added using FPE or IMO. For IMO, it was conducted using 1kg cooked rice that was inoculated with white fungus. After 3 days, molasses of different concentrations (100%, 20%, and 10%) were mixed to the cooked rice. Sampling was conducted every 3 days and parameters like pH, microbial community structure, population dynamics, microbial identities and their attributes, and chemical analysis were noted at different fermentation periods.
The microbial population dynamics was analyzed using a culture-independent approach like denaturing gradient gel electrophoresis (DGGE). In addition, culture-dependent technique was also employed like microbial isolation and purification. Identification of the isolates of interest was conducted using VITEK 2 and VITEK MS identification systems which uses analytical profiling index based on biochemical utilization/reaction. Finally, some isolates were also identified by 16S rDNA sequence analysis.
FPE DGGE band pattern obtained at days 0, 15, and 30, were not significantly different between the uninoculated and inoculated samples. However, differences on the band thickness or density were significantly different indicating a marked change in the population level of the microorganisms involved. Among the inoculated samples, there were no significant change in the band pattern from days 0 to 30 which seemingly indicate a more stable community. The microbial community based on the DGGE profile was composed of Weissella sp., Chryseobacterium sp., Lactococcus sp., Lactobacillus, Pseudomonas and an uncultured bacterium. At earlier stages, the non-lactic acid bacteria seemed to predominate while the lactic acid bacteria group predominated towards the end of the fermentation period in general, such observation was the same through in both inoculated and uninoculated set-ups.
In the fermentation of IMO, the microbial community involved were of the bacillus group, Sphingomonas sp, and lactic acid bacteria group. The use of 100% molasses and 20% molasses seemed to affect the component of the microbial community. Using a 100% molasses, the most predominant microbe was of the bacillus group while reducing the molasses concentration to 20% favoured the growth of the lactics. In the production of IMO, farmer’s usual practice baits their fungal inoculum using cooked rice. Accordingly, a fungi characterized merely by a white mycelium that grew on the cooked rice was the proper starter inocula. Based on DGGE results, the predominant fungal species were identified as Kazachstania naganishi, Monascus purpureus and uncultured Candida.
Culture dependent or culturable microbes showed that; in general, the FPE, FFE, or IMO microbial counts using some selective medium showed that the bacterial group predominated in all trials, followed by the yeast, lactic acid bacteria and finally the molds group. Modifying the current mixing protocol by dissolving the sugar first in water before addition to the madre de cacao leaves relatively resulted in higher microbial counts in inoculated set-up as compared to the uninoculated ones. Likewise, the chemical analysis also showed positive improvements on most of the microelements but did not much have significant effect on N-P-K content.
Further, culturable microorganisms were isolated, characterized and identified. Most of the isolates were identified as Bacillus sp., Staphylococcus sp., Enterococcus sp., and Stenotrophomonas sp., Pantoea sp., Enterobacter sp., Vibrio sp., Streptococcus sp., and Serratia sp., using the VITEK II compact or VITEK MS Systems. All of the isolates were preserved by Liquid-drying and were accessioned at the Philippine National Collection of Microorganisms (PNCM).
A total of 191 isolates were obtained during the fermentation of different biological extracts and analyzed for their functional attributes. Among the FPE isolates, 62.1% were nitrogen fixers (NF), 17.2% were potassium solubilizers (PS), and no phosphorus solubilizer (KS) was detected. In FFE isolates, the 54.5% were NF%, 1.8% were PS and 19% were KS. In IMO isolates, the NF were 82% no PS, and KS were 8.7%. A high percentage of the isolates also exhibited IAA equivalents in FPE, FFE, and IMO as follows, 77.6%, 73.6% and 65.2% respectively: Moreover, IAA equivalent noted on the biological extracts were as follows, uninoculated FPE: 2.22 ug/ml; Inoculated FPE: 2.62 ug/ml; uninoculated FFE: 2.85 ug/ml; FFE with FPE inoculum: 3.22 ug/ml; FFe with IMO inoculum: 2.08 ug/ml; IMO (10% molasses): 6.54 ug/ml; and IMO (20% molasses): 7.21 ug/ml.
Some of the strains obtained from the improved protocol include the following microbes, Bacillus megaterium, Bacillus circulans, Stenotrophomonas maltophilia Pseudomonas geniculata, Bacillus tequilensis, Bacillus altitudinis, Sphingomonas sp., Staphylococcus sp., and Lysinibacillus sp., which seemingly can be used as a potential component of the fermenting biological extracts. Using 16S rDNA sequencing, fungal isolates were identified as Rhizopus oryzae, aspergillus fumigatus and Aspergillus niger.
Applying the modified mixing protocols and the use of microbial inoculation proved to be effective in the above results. Likewise, the results are seemingly useful in standardizing the preparation of the botanical extracts and further improve the current practice that would result to a more uniform and quality biological extract. It is also recommended that a phase two of the research project be implemented to focus the studies on production and efficacy testing of the product.