Updated November 30, 2021:
Development of Soy-formulate for organic ammonia production via hyper-ammonia-bacteria fermentation in a one-pot system
Ademola Hammed, Agricultural and Biosystems Engineering,
North Dakota State University
Objectives of the research
i. Development of a mild pretreatment for whole soybean
Completed work
i. Development of a mild alkali extraction of soybean protein
Preambles
Plant cell wall are rigid and prevent diffusion of protein needed during fermentation to produce organic ammonia. Therefore, we are developing chemical extraction processing to obtain protein from soybean. Since the targeted product is ammonia, we developed ammonia hydroxide extraction of soybean protein. Also, ammonia can be recovered and reuse making it an efficient extraction solvent.
Method
Whole soybean was grinded and sieved into different particle sizes >1, 0.425-1, 0.25 - 0.425 and <0.25 mm denoted, respectively as A, B, C, and D. Protein extraction was carried out on 5 g A, B, C, and D using 50 mL of different ammonium hydroxide concentrations (1, 2.5, 5, 10 and 15%). The mixtures were transferred into a shaker preset at 55 oC and 130 rpm. Extracts were collected at different time (0, 3, 6, 12, 24 and 48h). The extracts were centrifuged at 1000 rpm for 5 mins to separate the supernatant (extract) from the residue. The extract protein concentration was determined using the Bradford assay.
Results
Effect of particle size
The particle size affects extraction yield because increasing particle size reduces the solid surface area available for interactions with the solvent. Smaller particle size not only has high surface area that facilitates surface washing of extract but also has reduced diameter that aids extract leaching. However, reducing sample particle size is energy intensive, costly and prone to processing hazards including explosion. Therefore, we investigate the effect of particle size on the release of protein during protein extraction using ammonia hydroxide solution.
Figure 1 shows the result of protein yield at different particle sizes. Extraction yield increase across with reduction in particle size. At 15% solvent concentration and 48h, 50% protein extraction was achieved for sample A while for sample D, about 59% protein extraction was achieved. The results showed that particle size had a significant effect on extraction yield.
Figure 1: Effect of particle size at 48h and 15% solvent concentration
Effect of solvent concentration
Solvent polarity and pH affect extraction yield because high acidic/basic solvent disrupts plant cell wall and high OH- increase solvent polarity to dissolve polar solute. The degree of plant cell wall disruption and solute solubility could increase extraction yield. In order to determine the ammonium hydroxide concentration that favor soy bean protein extraction we used different ammonium hydroxide concentrations (0, 1, 2.5, 5, 10 and 15%) during processing. The result (Figure 2) shows that ammonium hydroxide solutions extract more protein than the control. Protein extraction increase rapidly (approximately 49%) with 1% solvent concentration. Further increase in concentration did not cause significant increase in protein.
Figure 2: Effect of solvent concentration at 48h
Analysis of variance
A two-way ANOVA was performed to analyze the effect of particle size, ammonium hydroxide concentration and time on protein extraction from soybeans. The ANOVA analysis in Table 1 shows that the processing conditions significantly affect the protein yield. The probability F is less than 0.0001 suggesting that the difference is highly significant.
Dependent variable: Protein Concentration
Source DF Sum of Squares Mean Square F Value Pr > F
Model 47 27650.59291 588.31049 50.43 <.0001
Error 72 839.89921 11.66527
Corrected Total 119 28490.49212
Extraction kinetics
In solvent extraction process, kinetic models are commonly used to simulate the extraction process. Kinetic modelling simplifies and facilitates optimization study design. Different kinetic models were used for the extraction and the best fit model (So and Macdonald’s model, Figure 3) was selected based on higher magnitude of linear correlation coefficient (R=0.94) and lower root mean square (RMS=0.24) value (Kitanovic, S et al., 2008).
Figure 3. Comparison of experimental (symbols) and fitted (line) yields for soybean pretreatment at 1% solvent concentration and 48h: The extraction kinetics was fitted by So and Mac Donald’s model.
Conclusion
The effect of extraction processing factors, ammonium hydroxide concentration, time and particle size on soybean protein yield have been investigated. Reducing particle, time and ammonium hydroxide concentration increases protein yield. The conditions for highest protein yield are 12 h, 1% ammonium hydroxide and < 0.425 mm particle size.
Work to be completed
i. Optimization of NH4OHaq aided extraction of soybean protein.
ii. Biochemical hydrolysis of protein extract.
iii. Inclusion substrate essential elements
References
Kitanovic, S., Milenovic, D., & Veljkovic, V. B. (2008). Empirical kinetic models for the resinoid extraction from aerial parts of St. John's wort (Hypericum perforatum L.). Biochemical Engineering Journal, 41(1), 1-11.
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Updated July 2, 2022:
Statements of goals/objectives
Our goal is to develop an alternative manufacturing method that could eliminate the CO2 footprint which can be employed by local farmers, thus, bringing ammonia close to where it is needed to reduce the emissions associated with both the production and distribution.
• Development of soy-based substrate for the production of green ammonia
Deliverables
Ammonia is very critical to the manufacturing of fertilizers. The industrial production of most of the world’s fertilizer using the Haber-Bosch process require huge amounts of energy to generate the high temperatures and pressures needed to combine nitrogen and hydrogen into ammonia. The high cost of transporting fertilizer from large manufacturing facilities to remote rural areas, such as sub-Saharan Africa is another herculean task, making it hard to be obtained in such areas. It is, therefore, necessary to develop a smaller-scale alternative that could be used locally to produce fertilizer for farmers or a small community of farmers.
The hydrogen gas used for the Haber-Bosch process is usually obtained from methane derived from natural gas or other fossil fuels. High temperatures (500 degrees Celsius) and pressures (200 atmospheres) are required to make the very unreactive nitrogen react with hydrogen to form ammonia. The process is not only very expensive but also emits a great deal of carbon dioxide, thus, making ammonia the largest contributor to greenhouse gas emissions, among all chemicals produced in large volumes.
Milestones and key performance indicators with
• Hydrolysis of soybeans
• Effect of different factors affecting soybean conversion to ammonia
Brief summaries progress, accomplishments and deliverables
Effect of substrate composition on microbial growth and ammonia production
The composition of substrate is among the major factors that affect fermentation process. Both protein and carbohydrate can provide carbon source for microbial proliferation. On one hand, bacteria generally prefer carbohydrates, primarily glucose for their growth. On the other hand, protein contain nitrogen that provide the amino base for ammonia production. To assess whether HAB can produce ammonia in cultures containing sugars (glucose or starch) only and sugars with a nitrogen source (casamino acid), it was observed that sugars alone could not support the growth of HAB; both starch and glucose give relatively poor growth of the bacteria (Figure 2). A similar result was seen in ammonia production when only sugars were used as the substrates. Nearly zero ammonia was produced in glucose only and starch only cultures (Figure 3). When the carbohydrate-containing cultures were supplemented with proteins, growth of the HAB surged (Figure 2), but not with concomitant high production of ammonia (Figure 4). This result showed that sugar inhibits ammonia production even when nitrogen-containing substrates are present. This is further evident as soybean flour which contains about 15% percent of sugars and more than 40% of protein among other components yielded low ammonia relative to peptide and casamino acid alone when each was used as substrates in different cultures (Figure 5).
Protein isolation and enzymatic hydrolysis enhance ammonia synthesis
Having discover that sugar inhibit ammonia synthesis and peptide/amino acids support early ammonia synthesis, we conducted the effect of protein isolation and enzymatic hydrolysis on ammonia production during HAB fermentation. According to Figure 6, a reduced ammonia concentration was observed in cultures containing soybean flour. But when soybean protein isolate (SPI) was used as the substrates, a relatively high concentration of ammonia was obtained in the culture. The rate of ammonia production was much higher in hydrolyzed protein (HP) cultures, but both SPI and HP cultures performed better vis-à-vis ammonia production relative to soybean flour, reaching a concentration of 1200 mg/L (Figure 6). Soybean flour showed a delay in ammonia production. This is expected due to the presence of non-protein components such as sugars, oil, and flavones, amongst others. Some bacteria are known to digest large biological molecules into useful products without prior pretreatment or degradation of such large polymers.
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