TO: Lanham Frazier, Big S 

FROM: Drs. Evan Siemann & Meenakshi Bhattacharjee, Rice University 

DATE: 3-11-2025 

RE: Untargeted metabolomics analyses of EARTI ™  microgreens 

We analyzed the metabolites in arugula (Eruca vesicaria) microgreens grown in the EARTI ™  chamber and arugula microgreens bought at local grocery stores. Untargeted metabolomics simultaneously measures all detectable compounds in a sample (hundreds to thousands of compounds), determines the relative concentrations of each compound among the samples via differences in peak areas, and identifies each compound (if possible). However, it does not tell the absolute concentrations of compounds so it is not possible to determine the relative concentrations of different compounds, even in the same sample.  But, it gives a comprehensive chemical profile that can be used to compare among samples.  

Arugula was grown for seven days post planting in the EARTI ™  chamber. Then the arugula microgreens were cut at the base, ground in liquid nitrogen with a mortar and pestle, and stored at -80°C. Another set of microgreens were harvested two days later and processed the same way. Later, another set of seven day microgreens were grown and processed. Arugula microgreens were bought from HEB and Sprouts and processed the same way as the EARTI ™  samples. All the samples were shipped in a container of dry ice to Creative Proteonomics in Shirley, NY. Details of their chemical analysis methods are at the end of this report.  

The data were analyzed at Rice University. First, the untargeted metabolomics data were processed to remove peaks that could not be assigned to a specific compound. Second, Principle Component Analysis (PCA) was used to examine the overall chemical composition of samples and to determine which samples had similar vs. distinct chemical compositions. This was done separately for data generated using positive vs. negative detector modes. Third, each chemical compound was tested to determine whether it differed in concentration between the EARTI ™  vs. commercial samples using ANOVAs. P-values were adjusted using sequential Bonferroni correction (alpha=0.05) to control for false discovery with the large number of tests being performed. Fourth, the ratios of concentrations of each compound in EARTI ™  vs. 

commercial samples were examined to identify compounds that occurred in much higher concentrations in EARTI ™  vs. commercial samples. Finally, a literature search was conducted to determine effects on human health for compounds that were present in significantly higher concentrations in EARTI ™  vs. commercial samples. 

Key findings were: 1) Arugula microgreens grown in EARTI ™  had chemical compositions that were distinct from those of arugula microgreens available at local grocery stores in Houston, TX. 2) A shorter time to harvest in EARTI ™  produced arugula microgreens that had consistent chemical composition. 3) Seventeen compounds were significantly elevated in EARTI ™  arugula microgreens vs. those bought at local grocery stores and ten were significantly lower in EARTI ™  samples. 4) Another three compounds were not significantly different between EARTI ™  samples vs. those bought in local grocery stores but were present at more than 100 times the concentration in EARTI ™  samples. 5) More chemical compounds were elevated in arugula microgreens grown in EARTI ™  than in those bought at local grocery stores. 6) Dipeptides were commonly elevated in EARTI ™  samples.  

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PCA: The overall chemical composition of the samples fell into three distinct groups (Figure 1). In these graphs, points closer to each other have more similar chemical compositions and points more distant from each other have more distinct chemical compositions. In these graphs, positive vs. negative on the axes is arbitrary. The key finding was that, the same three groups are found in both positive and negative detector mode data. One group is the commercial samples from grocery stores (“C1” – H2Organics bought from HEB, “C2” – BrightFresh bought from Sprouts). Another was the short time to harvest samples from 

EARTI ™  (“T1” – planted 10/2/24 and harvested on 10/9/24; “T2” – planted 10/19/24 and harvested on 

10/26/24). The longer time to harvest EARTI ™  sample was separate from the others (“T3” – planted 10/2/24 and harvested on 10/11/24). At harvest, the T3 sample seemed to have some microbial contamination based on appearance and odor. It is important to note that it is possible that different arugula varieties were grown in EARTI ™  vs. those for commercial samples which could contribute to the differences in their chemical compositions. 

Figure 1 Chemical composition of arugula microgreen samples grown in EARTI ™  for 7 days (T1, T2) or 9 days (T3) along with commercial arugula microgreen samples (C1, C2). Numbers indicate the percent of variation explained by each axis.   

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ANOVAs: For examining which compounds contribute to these distinct chemical compositions, we restricted statistical analyses to only the shortterm EARTI ™  vs. commercial samples. After correcting for examining a large number of compounds (~1000 in total) with a sequential Bonferroni procedure, there were 27 compounds that differed significantly between EARTI ™  vs. commercial samples (Table 1). If T:C >1 (i.e., log[T/C]>0) then there is a higher concentration in EARTI ™  samples vs. commercial samples [rows in green] while T:C<1 means greater concentrations in commercial samples [rows in red].  

Table 1. The relative concentrations of samples that differed significantly between arugula microgreens grown in EARTI ™  vs. those bought at local grocery stores. Mode = detector mode. CODE indicates the peak number. RT = retention time in minutes. MW = molecular weight. T:C is the ratio of the compound’s concentration in EARTI ™  vs. commercial samples.   

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Relative concentrations by sample: Although they are not significantly different between EARTI ™  and commercial samples, three compounds were present at more than 100 times the concentration in EARTI ™  vs. commercial samples. They were N-Butylbenzenesulfonamide (sulfonamide) which was ~200 times higher in EARTI ™  samples along with p-methoxybenzylisothiocyanate (isothiocyanate) and 2,3Dihydroxypropyl nonadecanoate (fatty acid ester) which were ~100 times higher in EARTI ™  samples.  

Figure 2. The relative concentrations compounds (log transformed) in arugula microgreens grown in EARTI ™  [7 days] vs. those bought at local grocery stores.   

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