Schematic presentation of the Shikimate metabolic pathway indicating critical points where specific micronutrients are required as enzyme cofactors (Graham and Webb 1991).
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The creation of undesirable (soggy) sauerkraut resulted in the loss of $1,000,000 worth of organic sauerkraut in 2022, which prompted a multistep investigation of the cause and potential solution. The cause of this condition has been previously reported as unique fermentation conditions and the lack of key trace nutrients essential for cabbage (Brassica oleracea var. capitata) cell wall integrity. Because the condition was limited to organic sauerkraut in 2022, this investigation initially focused on differences in fermentation conditions between organic and conventional sauerkraut. No differences in fermentation conditions accounted for the condition; therefore, attention was focused on analyzing the mineral content of cabbage grown for sauerkraut production that pinpointed a deficiency in critical micronutrients such as iron, copper, manganese, boron, and zinc. This deficiency was traced to the use of poultry manure that was contaminated with glyphosate residue from conventionally fed turkeys and chickens that consumed genetically engineered (GE) feed and used as the fertilizer for organic cabbage production. The presence of glyphosate, a potent mineral chelator and antibiotic, was identified as a significant factor that impairs the absorption and physiological function of essential minerals in the shikimate metabolic pathway whereby cell walls and lignin are produced, thus compromising the structural quality of the sauerkraut. After this discovery, the study progressed to evaluate various remediation strategies aimed at eliminating glyphosate from the soil and restoring nutrient uptake. Corn grain and silage were selected as the test crops for this phase. Among the tested remediation solutions were raw sauerkraut juice containing Lactobacillus plantarum, which is reported to degrade glyphosate in the rumen of dairy cows and two patented proprietary microbial mixtures, PB027 and PB027SK, that degrade glyphosate by all three of the known metabolic pathways. These treatments were specifically formulated to degrade residual glyphosate in the soil. The results showed that these interventions could reduce soil glyphosate levels by 80% to 90% within 6 to 7 months to significantly enhance both the yield and quality of corn grain and silage. The increase in corn grain yield from glyphosate degradation on the Shiocton silt loam soil was 907.89 kg·ha−1 (13.5 bushels/acre). The increase in yield on the irrigated Kidder sandy loam soil was quantified at 726.31 kg·ha−1 (10.8 bushels/acre) for corn grain and 6.62 t·ha−1 (2.68 t/acre) for silage, with an additional improvement in silage feed quality beneficial for milk production. The findings underscore the importance of addressing both micronutrient sufficiency and glyphosate residue in soil to ensure the optimal growth of cabbage and the quality of sauerkraut produced. By successfully identifying manure as a subtle source of nutrient immobilization and implementing effective soil remediation techniques, this research highlights a clear path forward for improving crop yield and quality to ultimately enhance the structural integrity and consumer acceptance of sauerkraut. This study has broader applications for the nutritional content and crop yields of many organic crops that use conventional poultry and animal manures that may contain glyphosate in desiccated plant tissues or GE feeding operations.
Fermentation is an ancient method of preserving many vegetables to retain their nutrient value and wholesome characteristics (Pederson and Albury 1969). Fermented foods also are commonly used as probiotic sources to remediate gut dysbiosis and improve digestion in animals and humans. The fermentation process for cabbage/sauerkraut (Brassica oleracea var. capitata) is initiated rapidly by the Gram-positive microaerophylic bacterium Leuconostoc mesenteroides to establish a favorable anaerobic environment in sequence for lactic acid bacteria (primarily Lactobacillus brevis, L. plantarum, and Pediococcus cervisiae). Occasionally, the acidic anaerobic environment that is inhibitory to undesirable bacteria and their enzymes, which could otherwise soften vegetables, does not develop (Pederson and Albury 1969). During normal fermentation, L. plantarum, which is a high producer of acid, soon becomes the predominant fermenter. The fermentation temperature, salt concentration, mineral composition of cabbage, and initial presence of a sequence of lactic acid bacteria are the primary environmental factors that determine the rate and outcome of the fermentation process. Optimization of these factors generally results in a high-quality, crisp, white commercial product. Quality defects have occasionally been associated with an abnormality in the chemical composition resulting from low or high salt concentrations that affect surface fermentation caused by nonlactic acid bacteria (Pederson and Albury 1969). The potential for unfavorable effects of surface mold or yeast fermentation is reduced by covering tanks with plastic or wood covers to reduce surface aerobic exposure. Salt uniformity has been correlated with tenderness, whereas temperature is correlated with color. Consumers prefer a flavorful, crisp, and white to slightly opaque product.
Emphasis is placed on optimizing the fermentation environment for quality sauerkraut production; therefore, initially, the textural differences between the organically and conventionally grown cabbage in 2022 were attributed to differences in the source of salt used to break cell wall osmotic pressure pretreatment to fermentation. In this case, the conventional cabbage was treated with Superior TX-10 Salt (US Salt LLC, Watkins Glen, NY, USA) obtained from underground salt deposits by deep well solution mining and subsequent evaporation by the vacuum pan system. The organic cabbage was treated with Sea Salt Top 50 lb PA salt (Cargill, Minnetonka, MN, USA) that is manufactured using the natural evaporation of seawater from San Francisco Bay and refined by washing with clean saturated brine to remove surface impurities. Both salts are listed as food grade. An analysis of the two salts revealed that they were remarkably similar, with no significant differences apparent between them that would affect the textural quality of cabbage (Table 1). All other conditions of fermentation were the same for the conventional and organic sauerkraut.
A mineral analysis of sauerkraut tissue showed that copper (Cu), manganese (Mn), and several other nutrients involved in plant cell wall development (Fig. 1) were lower in the soggy organic sauerkraut than in the higher-quality crisp conventional sauerkraut, thus indicating that the problem may have occurred during the production of cabbage. Environmental conditions that can affect sauerkraut quality during the growth of cabbage are not as readily identified; however, the effects of environmental conditions during the growth of cabbage on sauerkraut quality (texture, flavor, and color) have been reported (Pederson and Albury 1969). Drought or other conditions that limit the nutrient availability or sufficiency are influential. An example of these conditions is the difference in cabbage grown in upland soils rich in potassium (K) that yield solid, crisp heads of white cabbage compared with the loose-headed, green leaf cabbage grown in low-K marsh soils. Higher levels of K are recommended for high nitrogen (N) and peat-bog conditions (Hoffman and Latizko 1951). Molybdenum (Mo) deficiency prevents nitrate (NO3) reduction, resulting in high tissue N, and high N levels also can induce a Cu deficiency. A Cu deficiency impairs lignification of plant cell walls involved in crispness and texture (Fig. 1). The ratio of cell wall material to total dry matter is greatly decreased in Cu-deficient tissues. This decrease in cell wall formation and lignification occurs even with mild Cu deficiency and results in soft (soggy) tissues. In addition to Cu, Mn, iron (Fe), boron (B), and cobalt (Co) are other micronutrients that are required in the shikimate metabolic pathway for lignification and cell wall development (Graham and Webb 1991; Maeda and Dudareva 2012) (Fig. 1). Thus, the presence of anything that interferes with the shikimate pathway minerals and enzymes involved in cell wall composition, such as glyphosate [N-(phosphonomethyl) glycine]-based herbicides (Fig. 1), can damage the quality of the sauerkraut produced.
Citation: HortScience 59, 11; 10.21273/HORTSCI18041-24
Soil analyses performed during late 2021 (Table 2) for fields planted with conventional or organic cabbage in 2022 were similar. These analyses showed that several nutrients involved in plant cell wall formation and lignification (Graham and Webb 1991) were low to deficient in some irrigated sandy loam soils used for sauerkraut cabbage production. This suggests that the issue of soggy sauerkraut may originate during cabbage production rather than during the fermentation process, despite fertility programs being designed to ensure nutrient sufficiency for both conventional and organic systems. The soil mineral analysis of fields indicated that phosphorus (P), B, and Cu were low, but that most of the other mineral nutrients analyzed during Fall 2021 for the 2022 cabbage crop were moderate to high (Table 2). Additionally, previous cropping may influence mineral availability. For example, the Prince-2N field, where cabbage followed potato, had different mineral profiles compared with those of Prince-3 fields A and B, which followed green beans. These differences were noted with similar pH but lower organic matter (OM), K, calcium (Ca), and Mg and higher P levels (Table 2).
In addition to soil minerals, the OM content and soil pH significantly influence the availability of mineral nutrients for plant uptake. Soil analyses on 19 Aug 2021 of high and low OM portions of the Prince 3 organic field showed distinct differences in all soil minerals except K and B, despite similar pH levels (Table 2). The side with higher OM (Prince 3B) had higher Ca and Mg levels but less available P, Cu, Mn, and Zn. Because Cu, Mn, and zinc (Zn) are crucial for cell wall development and membrane permeability, deficiencies in these minerals could affect sauerkraut texture. The differences in mineral content and soil conditions between the two parts of the field suggest that they should be fertilized separately even though their pH levels are similar. These variations in mineral content could explain differences in organic sauerkraut quality unless corrected by a fertility program. Soil analyses for other conventional and organic fields planted with cabbage in 2022 are presented in Table 2.
In 2022, both conventional and organically grown cabbage arrived at the fermentation facility in good condition, with acceptable size, maturity, color, flavor, and texture. The cabbage was washed, cut, salted, and placed into fermentation tanks the same day. Fermentation proceeded normally, with good acid production and normal salt levels. The color and flavor were normal, and the sauerkraut from the conventional fields was rated as having a normal texture and as shelf stable.
However, the texture of the organic sauerkraut after fermentation varied widely, from normal, to below average, to mushy (apple sauce-like texture with very little to no solids remaining). This inconsistency resulted in the abandonment of several large vats of fermented cabbage and an estimated $1,000,000 loss to the sauerkraut company. The soft texture issue was more pronounced in 2022, when the consumer demand for organically grown cabbage was increasing compared to that for conventionally grown cabbage on similar soil types that produced good-quality sauerkraut.
Based on this information, research was initiated to identify and address soil and nutritional factors that occurred during commercial sauerkraut production that could negatively affect sauerkraut texture, even when fermentation conditions are optimized.
Cabbage was grown in fields of Shiocton silt loam soil near Shiocton, WI, USA, and in irrigated Kidder sandy loam soils near Colona, WI, USA, in 2022. All of the fields included organic cabbage that had been managed organically for at least 25 years. The cabbage cultivar grown and fermented in 2022 was Nixon. The cultivar was changed in 2023 to longer-maturing cultivars Passat and Typhoon, which have been historically used for storage situations.
The disparity in the nutrient availability of Cu, Fe, and Zn for plant uptake in the organic cabbage indicated a possibility of glyphosate (a strong mineral chelator and antibiotic) contamination. Glyphosate is a patented herbicide that inhibits the shikimate metabolic pathway responsible for cell wall production and lignification in plants, and it reduces Fe and Mn uptake (Eker et al. 2006). Potential inadvertent sources of glyphosate contamination included well water used for irrigation and poultry manure organic fertilizer. Soil samples (eight random probes with a 0- to 30-mm depth per plot were bulked), water from the two irrigation wells, and poultry manure were analyzed by Health Research Institute Laboratories (Fairfield, IA, USA) using high-performance liquid chromatography with tandem mass spectrometry to determine the presence of glyphosate and aminomethylphosphonic acid (AMPA) (Jensen et al. 2016). The total effective glyphosate (TEG) was calculated according to the Food and Agriculture Organization (FAO) method, whereby the TEG residue was the sum of glyphosate plus 1.5 multiplied by the weight of its AMPA metabolite. Glyphosate is a powerful antibiotic against beneficial soil microbes and a strong mineral chelator that immobilizes essential micronutrients, such as Cu, Co, Fe, Mn, nickel (Ni), and Zn, that are essential for plant growth (Bernards and Thelen 2003; Huber 2021). An herbicidal dose of glyphosate is commonly 500 ng·g−1. Sauerkraut was not analyzed to determine the presence of glyphosate because Lactobacillus plantarum, the primary fermenting organism for sauerkraut, has been shown to fully degrade glyphosate (Gerlach et al. 2014).
Soil, manure, sauerkraut, and plant tissues were analyzed to determine the presence of plant essential minerals by Midwest Laboratories (Omaha, NE, USA). Analytical tests included Bray-1 and Bray-2 for P. Optimum nutrient reference standards for cabbage were provided by Midwest Laboratories and Bryson et al. (2014) for “mature cabbage.” The primary difference between the conventional and organically grown cabbage was the source of fertilizer amendments used to optimize nutrient sufficiency. The organic cabbage was fertilized with 8.65 t·ha−1 to 9.88 t·ha−1 (3.5–4 t/acre) of poultry manure (Table 3), and the conventional cabbage was fertilized with nutritionally comparable inorganic fertilizer based on the soil and tissue analyses of the field (Bryson et al. 2014). The significant increase in consumer demand for organic sauerkraut in 2022 necessitated the location of additional sources of poultry manure for the 2022 organic cabbage crop; therefore, manure from conventionally fed poultry (Table 3), which was approved for organic crop production, was included (Heckman et al. 2009).
Cabbage was processed by GLK Foods (Appleton, WI, USA). The only difference in the fermentation of the two cabbages, conventional and organic, was the source of salt: mined salt (US Salt) was used for conventional sauerkraut and San Francisco Bay sea salt was used for organic sauerkraut. Analytically, both salts were remarkably similar (Table 1), and fermentation conditions were the same for both cabbages. The sauerkraut texture was evaluated by the GLK Foods quality panel as “normal,” “below average,” or “soft and unusable.”
Desorption of residual glyphosate in soil after applying phosphate fertilizers has caused serious losses in nonherbicide crops. This occurs because glyphosate and phosphate compete for similar binding sites in soils, and phosphate can displace glyphosate, releasing a potent herbicide that is readily absorbed through plant roots. Glyphosate is difficult to degrade in many agricultural soils because many organisms lack the phosphite-lyase enzyme necessary for full degradation. The indiscriminate application of glyphosate-based herbicides during the last 50 years has resulted in the accumulation of highly toxic levels of residual glyphosate in many soils, water, food, and feed products (Gerlach et al. 2014; US Geological Survey 2014) and throughout the environment. While collecting a load of raw sauerkraut juice (RSKJ) in 2022 after reading the study by Gerlach et al. (2014) of RSKJ and its ability to degrade residual glyphosate in cattle feed, we became aware of serious concerns regarding soggy sauerkraut. When asked about possible causes, our initial response was that it was likely caused by a nutrient deficiency (probably Cu and or Mn) in the cabbage that was fermented. We offered to help identify a remediation procedure. It was fortuitous that our research of glyphosate degradation was established in common areas with cabbage production because we proceeded to identify causes of soggy sauerkraut, as reported in this work.
Although RSKJ contains only small amounts of plant-essential nutrients to compliment the basic fertility program (Table 4), it contains Lactobacillus plantarum and related microbial species that have been reported to degrade glyphosate. Gerlach et al. (2014) demonstrated that RSKJ fed to dairy cattle could degrade residual glyphosate in feed, thereby preventing chronic botulism caused by Clostridium botulinum. Two “satellite” studies were established to evaluate whether RSKJ, a new eight-organism biological cocktail called PB027, and the combination of PB027 with RSKJ (PB027SK) could degrade soil residual glyphosate and AMPA to lower TEG. A strip study of six replicates of alternating RSKJ-treated and non-RSKJ-treated 45.72-m-wide (150 feet) sprayer strips across the field were established on a Shiocton silt loam soil near Shiocton, WI, USA. For this study, 140.25 L·ha−1 (15 gal/acre) of RSKJ collected from the brine waste stream of a “conventional” sauerkraut fermentation vat was sprayed onto the soil surface previously analyzed to determine available nutrients, residual glyphosate, and AMPA on 2 Jun 2022. The materials were lightly incorporated with a harrow and then planted with cabbage. Severe inclement weather at harvest on 30 Oct 2022 prevented the collection of meaningful cabbage yield data. Soil samples (depth, 0–30 mm) were taken on 30 Oct 2022, 5 months after RSKJ application, for mineral availability and glyphosate analyses. Subsequently, corn hybrid LG44C27VT2PRO RIB was planted 22 May 2023 at a density of 77,838 seeds/ha following the 2022 cabbage crop. Corn grain yields from the plot areas were measured at 14% moisture during the 2023 harvest.
A second location was studied to determine changes in the mineral content after remediation of residual glyphosate (conducted by Dr. Tim Maloney, Agri-Tech Consulting and GLK Foods). The location was the Findlay Farm located at Whitewater, WI, USA, which has a fertile, well-managed, irrigated Kidder sandy loam soil (pH 6.7). The previous crop was wheat. The stubble was chisel-plowed in the fall, and the field was cultivated in the spring to incorporate fertilizer and level the field for planting. Two adjacent study areas were established, one for grain yield and the other for silage. Each experimental design was a one-factor, randomized, complete block with six treatments (Table 5) and four replications. In-house studies by the company that produces PBO27 and PB027SK (Paleobiotica Inc., San Luis Obispo, CA, USA) demonstrated that these products can facilitate the degradation of glyphosate. PB027 is an eight-organism, patent-pending, biological cocktail that degrades glyphosate by all three known metabolic pathways (Mohanty and Das 2022). Catawater (Catawater, Plano, TX, USA) is a proprietary organic biocatalyst technology product that also has been shown to enhance glyphosate degradation (McNeill, unpublished data 2022). Based on their results, evaluations of the activity of Catawater and the PaleoBiotica biological cocktail product (PB027) in a water carrier and in combination with RSKJ (CatawaterSK and PB027SK, respectively) were performed during the glyphosate degradation study in cooperation with Agri-Tech Consulting near Whitewater, WI, USA.
The four 76.2-cm (30-inch) row plots were 3.05 m × 15.24 m (10 × 50 feet) and planted 19 May 2022 to DS4018AMXT genetically engineered (GE) hybrid at 86,485 seeds/ha (35,000 seeds/acre). Glyphosate herbicide was applied for weed control. The two center rows [6.10 m (20 feet)] of the 15.24-m-long (50 feet) silage plots were harvested 14 Sep 2022, and all four rows [13.72 m (45 feet)] of the 15.24-m (50 feet) grain plots were harvested 11 Nov 2022. Gross weights were adjusted to 65% moisture for silage and 15% moisture for grain. Treatments (Table 5) were applied preplanting and broadcast on 18 May 2022, followed by light incorporation. Corn plant emergence, density, and stand were recorded 27, 29, and 31 May 2022 and 3 Jun 2022.
Corn leaf tissue (leaf above the ear) was analyzed to determine the mineral sufficiency at silking (26 Jul 2022) and grain fill (14 Sep 2022) of the grain plots and at harvest (14 Sep 2022) for the silage study. Silage quality was evaluated to determine crude protein, neutral detergent fiber, total tract neutral detergent fiber digestibility, acid detergent fiber, crude ash, crude fat, and indexed milk production (Shaver 2007) by Rock River Laboratory (Watertown, WI, USA). Corn grain was harvested 11 Nov 2022, and the percent moisture, weight (adjusted for 15% moisture), and test weight data were recorded.
A statistical analysis of glyphosate levels and their impact on mineral nutrients was conducted using a two-tailed Student’s t test assuming unequal variances. This test was chosen to compare the means of two independent groups (glyphosate treatment vs. the control) while accounting for the possibility of unequal variances between the groups. The level of significance was set at P = 0.05, meaning that P < 0.05 was considered statistically significant. The same statistical approach was used to analyze conventional crop yield and other parameters. A two-tailed Student’s t test assuming unequal variances was conducted to compare the means of the treatment and control groups for each parameter. P < 0.05 was considered statistically significant (R Core Team 2023; Wickham 2016; Wickham 2019).
Soil analyses (Midwest Laboratories, Omaha, NE, USA) performed in 2021 (Table 2) of fields before planting cabbage in 2022 indicated that soil P, K, Ca, Mg, and Zn should be sufficient for cabbage; however, the B, Cu, Fe, and Mn levels, which are essential elements for cell wall production, were low to severely deficient for cabbage and should be increased through fertilizer amendment. Several of the nutrients critical for sauerkraut texture (Mn, Cu, B) are often also severely deficient in many of the conventional fields planted to cabbage but still produced a quality sauerkraut (Tables 2, 6, 8, and 9). In contrast, tissue analyses of organic cabbage grown on these soils indicated that the expected availability of certain micronutrients were not always taken up by the plants. Although B had low to deficient levels according to the soil tests performed in 2022 and 2023 (Tables 2 and 6), it was excessive in plant tissues from some fields (Table 8). The soil OM content and management practices influenced the availability of macronutrients Ca, Mg, and P and micronutrients Cu, Fe, Mn, and Zn, with locations with higher OM showing less available micronutrients (Table 2).
Textural differences between the conventional and organic sauerkrauts were significant. The sauerkraut texture of conventionally grown cabbage in 2022 was rated as good quality, with a crisp texture and shelf-stable status. In contrast, some of the organically produced cabbage was severely deficient in Cu, Fe, and Zn, high in N, B, and S, and generally sufficient in the other essential nutrients. This nutrient imbalance caused significant variability in the quality of the organic sauerkraut, with some batches producing soft and mushy sauerkraut that was not marketable, resulting in a loss to the company of $1,000,000.
A soil analysis of irrigated, sandy loam fields that were planted to cabbage in 2023 indicated that they should have had sufficient P, K, Ca, Mg, and Fe for cabbage; however, B, Cu, Mn and Zn were low to severely deficient in many fields before fertilization (Table 6). Thus, one or more of the nutrients critical for sauerkraut texture (Fig. 1) were often severely deficient in many of these fields.
The nutrient content of the poultry manure used as the primary nutrient source for the organically produced organic cabbage is presented in Table 3. In addition to the mineral fertilizer value of the poultry manure, it also contained a significant amount of the herbicide glyphosate from contaminated feed (Table 7). A subsequent analysis of glyphosate found that some of the poultry litter (turkey and chicken litter) applied late in 2021 and 2022 contained as much as 371 ng·g−1 total equivalent glyphosate (Table 7). As shown in Table 7, the levels present in manures used in 2021 and 2022 were considerably higher than those in manures available in 2023 and 2024. Other alternative by-products of agriculture that are used in organic production can also contain significant levels of glyphosate (Table 7), and processing may concentrate it because it is fairly intransigent in the environment.
Analyses of cabbage destined for sauerkraut production in 2022 and 2023 that were performed to determine the mineral content, identified Fe, Cu, Mn, and Zn (Tables 7 and 8) as the most common micronutrient deficiencies, although differences in macronutrients were also obvious in cabbage from certain fields. A tissue analysis of cabbage from the 2023 cabbage crop compared samplings from the outer leaves (older leaves) and inner leaves (younger) (Table 9). This comparison indicated the nutrient availability throughout the growing season; marginally sufficient soils may be adequate for early growth but may not be able to supply, or the plants may not be able to retranslocate, mobile nutrients from older to younger more actively growing tissues. This could also be an indication of environmental and previous crop effects on different fields. The lower concentration of Mn, a relatively immobile element in younger tissues compared to that in older tissues, indicated an inadequate sufficiency of this essential nutrient available in the soil to meet optimum plant needs during head formation (Table 9).
Tissue analyses of the various fields for the 2022 organic crop generally showed low tissue N, P, K, Ca, and Mg levels but very low to deficient Fe, Mn, Cu, and Zn levels, with a few exceptions for N, Ca, Mg, Mn, and B. The sulfur (S) and Mo levels were sufficient to high. The concentration of P, a mobile nutrient in plants, increased in younger tissues, whereas K, Ca, Mg, and Mn decreased in younger tissues compared with those in older tissues. Additionally, Fe, Mn, B, Cu, and Zn in the 2023 cabbage crop were severely deficient and generally similar to those of the 2022 crop. A foliar application of deficient nutrients that affect enzymes of the shikimate pathway for cell wall formation and turgor was recommended because some preplant applications were not performed. Levels of Fe, Mn, Cu, B, Zn, and Mo during late spring are almost universally deficient in the cabbage plants in 2023, whether grown under conventional or organic management. Excess N was often, but not always, associated with low Mo because levels of N in the poultry manure also varied. Although nutrients come as a “package” with the manure used as a nutrient source in organic production, there is some flexibility to adjust individual nutrient levels that are deficient based on soil or tissue analysis results, and individual nutrient needs can be addressed with individual inorganic fertilizer sources if approved by certifying agencies (Heckman et al. 2009).
Almost 83% of the initial 207 ng·g−1 TEG was degraded to AMPA during the 5-month incubation period, along with further degradation of 88% of the AMPA within 5 months after treatment with RSKJ to an average of 31.59 ng·g−1. Only 13% of the initial TEG remained after the 5-month incubation period (Table 10). The available levels of B, Fe, Mn, and Zn were significantly increased with the decreased level of residual soil glyphosate (Table 11). Subsequent cropping of these RSKJ-treated and nontreated strips with corn hybrid LG44C27VT2PRO RIB planted at 86,485 seeds/ha (35,000 seeds/acre) in 2023 resulted in a significantly higher (907 kg·ha−1, 13.5 bushels/acre) grain yield from the RSKJ-treated areas (15.40 t·ha−1, 229 bushels/acre) compared with that of the untreated areas (14.49 t·ha−1, 215.5 bushels/acre).
Treating the soil with 140.25 L·ha−1 (15 gal/acre) of RSKJ increased the availability of B, Fe, Mn, and Zn at the Shiocton silt loam location (Table 11). Other nutrients remained unchanged or the differences were too small to be detected during a general soil analysis. This location has a pH that is slightly alkaline, very low in soil B, Cu, Mn, and Zn, and high in Ca, Mg, Fe and S. Only the first four of the six replicates were analyzed to determine glyphosate and AMPA. Although Fe, Mn, Cu, and Zn in the soil were initially low, all of them were slightly more available 6 months after treatment with 140.25 L·ha−1 (15 gal/acre) RSKJ from the fermentation waste stream (Table 11).
Most of the essential nutrients were at or above the target optimum levels during the two corn studies of remediating residual soil glyphosate at the Agri-Tech Consulting location near Whitewater, WI, USA (Kidder sandy loam soil). The only exception was the low Mn level. All treatments that received 140.25 L·ha−1 (15 gal/acre) RSKJ had increased vigor on 3 Jun, 23 Jun, 25 Jul, and 20 Aug (Table 12). There was no evidence of phytotoxicity caused by the 140.25 L·ha−1 (15 gal/acre) rate of RSKJ used during these studies.
Raw sauerkraut treatment alone and with PB027SK and CatawaterSK increased the silage yield 5.2% to 9.3% compared with that of the untreated control (Tables 13 and 14). The RSKJ plus PB027SK was the highest-yielding treatment, with an increase of 9.3% (6.62 t·ha−1, 2.68 t/acre) increased silage/acre compared with that of the untreated control. PB027 alone increased the silage yield by 1.28 t·ha−1 (0.52 t/acre). The mixture of 224.17 g·ha−1 (3.2 oz/acre) of Catawater in 140.25 L·ha−1 (15 gal/acre) RSKJ (CatawaterSK) increased the silage yield by 4.32 t·ha−1 (1.75 t/acre) compared to that of the untreated control. No improvement in silage yield was noted with Catawater in water alone treatment. Although the tissue contents of K, Ca, and Fe were less than the optimum levels, and although Mn was deficient at the silking stage of growth, other essential nutrients were sufficient (S, P, B, and Zn) or excessive (N, Mg, and Mo) (Tables 15 and 16). Additionally, the level of K was slightly less than optimum at silking; however, it was at full sufficiency at grain fill (Table 17). However, Mn was very low throughout, even after treatment with all of the remediation treatments. All other minerals were nearly optimal or slightly higher than the optimum at the grain-fill growth stage (silage harvest). Similar to the Shiocton study, there was no evidence of phytotoxicity caused by RSKJ during any of these studies.
The average corn grain yield was 14.80 t·ha−1 (220 bushels/acre) in 2023 following cabbage in 2022 on the silt loam soil; the corn yield was 907.89 kg·ha−1 (13.5 bushels/acre) higher in the RSKJ-treated areas compared to that of the nontreated control. The combination of RSKJ and a biological cocktail consisting of eight bacterial species that degrade glyphosate by all three known metabolic pathways (PBO27SK) increased corn grain yields by 523.89 kg·ha−1 (7.79 bushels/acre) to 729 kg·ha−1 (10.84 bushels/acre) and silage by 6.62 t·ha−1 (2.68 t/acre). The reduction of residual soil glyphosate also resulted in silage with higher feed quality for milk production (Tables 13 and 14). Similar to the Shiocton study, there was no evidence of phytotoxicity associated with the RSKJ with any of these treatments.
All treatments containing RSKJ had higher corn grain yield (382.66 kg·ha−1, 5.69 bushels/acre) that was as high as 732.36 kg·ha−1 (10.84 bushels/acre), with the combination of RSKJ plus PBO27 (PBO27SK) yielding significantly more grain compared with that of the untreated control (Tables 18 and 19). The combination of RSKJ and PB027 (PB027SK) treatment showed the greatest increase in yield. All five treatments significantly degraded glyphosate in the soil, with 84% or more of the residual soil glyphosate degraded after 6 months of incubation (Table 20). The combinations of Catawater and PBO27 with RSKJ caused the greatest percent reduction of glyphosate compared with that of the control. An analysis of glyphosate levels in some midwestern US dairy cow rations (Table 21) indicated a serious need for attention to glyphosate contamination of feedstuff and the manure resulting from it.
The plant nutritional status on the corn grain plot was similar to that in the adjacent corn silage plot. Additionally, levels of N, Mg, and Mo in tissue of the corn grown for grain were high, K, Ca, and Fe were low, P, S, B, Cu, and Zn were sufficient, and Mn was deficient at tasseling for all treatments (Table 22).
This research points out the frequent nutrient deficiencies present in some Wisconsin soils. These deficiencies not only reduce yield and production efficiency but also can lower product quality and consumer acceptance. The overall mineral effects of glyphosate magnify the impact of the already deficient soils. The low level of Mo in the 2023 cabbage could explain the high N level and the further increase in N with time that was observed because Mo is required for nitrate-N utilization (Marschner 2011). Greater attention to remediation of these deficiencies through crop tissue analyses and subsequent fertilization to compensate for their absence or limited availability in soil is recommended. Residual glyphosate in soils from the direct application of glyphosate-based herbicides used for weed control or, more subtly, as identified in the current situation, from glyphosate contamination of organic manures used as nutrient sources from feed fed to animals, may exacerbate the extensive soil micronutrient deficiencies observed because it could immobilize essential mineral nutrients through chelation or reduce nutrient availability by acting as an antibiotic against soil microorganisms that make nutrients more available for plant uptake (Huber 2021). The high concentration of glyphosate, AMPA, and TEG in fresh poultry manures available in 2022, compared with those available in 2023 (Table 7), and AMPA was the principal factor that contributed to the costly loss of sauerkraut in 2022 compared with that in 2023.
The indiscriminate application of glyphosate-based herbicides during the last 50 years has resulted in high levels of residual glyphosate in many soils, water, food, and feed products (Gerlach et al. 2015; US Geological Survey 2014) (Table 21) and throughout the environment. As a water-soluble and persistent chemical compound, there are many subtle ways it can contaminate a product besides through contaminated feed products. It is immobilized and bound to soil particles; however, glyphosate and phosphate have similar binding sites in soil; therefore, the application of phosphate fertilizers to plants or soil can desorb residual soil glyphosate to create extensive damage to crop production and decrease crop and environmental quality (Bott et al. 2011; Gomes et al. 2015). Direct and indirect (chronic) health effects of agricultural chemicals, especially glyphosate, on humans, animals, and the environment are of great concern (Mason 2013; Swanson et al. 2014; Van Bruggen et al. 2018; Wilson and Huber 2021). Because of these interactions, it is important to degrade residual soil glyphosate rather than just immobilizing it in soil by binding to soil particles and through chelation that also immobilizes plant essential mineral elements.
Levels of glyphosate and its early degradation product, AMPA, in manures will often fluctuate depending on the level in feed that is fed to the animals (Dupmeier 2021). The amount of TEG in the poultry manure applied to the soil in late 2021 and 2022 for the 2022 cabbage crop was equivalent to 60% of the herbicidal rate of glyphosate-based weed killers (500 ng·g−1). As a strong cationic mineral chelator, spray drift (one-fortieth of the herbicidal rate of glyphosate: 28.7 g·ha−1, 12.5 g/acre) can inhibit plant uptake of Fe, Mn, and Zn and reduce translocation of Fe in the plant by 85%, Mn by 90%, and Zn by 34% (Eker et al. 2006) to immobilize and greatly reduce the availability and functional activity of the cationic essential minerals Ca, Co, Cu, Fe, K, Mg, Mn, Ni, and Zn. Conventionally managed soils also differ in residual glyphosate depending on management practices to accommodate GE crops in the rotation (Table 7) or use as a harvest aid (crop desiccation). The influence of the previous crop in the rotation could have a carry-over effect on specific nutrients and reflect differences in soil management or soil microbiological activity that influences nutrient availability.
The mineral content of cabbage that was fermented for sauerkraut was influenced by available soil nutrients, soil type, soil OM content, and the presence of glyphosate residues. Deficiencies of micronutrients involved in cell wall development and lignification (B, Fe, Mn, Cu, and Zn) were common in both conventional and organically grown cabbage; however, low tissue Cu, Mn, and Zn were most commonly associated with soggy (soft) sauerkraut from organic production fertilized with glyphosate-contaminated poultry manures. Glyphosate in irrigation well water generally ranged from nondetectable or trace to 0.11 ng·g−1, with a high level of 0.70 ng·g−1, and could provide a minor secondary source of glyphosate contamination. Residual soil glyphosate was significantly reduced by spraying 140.25 L·ha−1 (15 gal/acre) RSKJ from the cabbage fermentation process or RSKJ with the CatawaterSK or PB027 biological cocktail. Using RSKJ from the fermentation waste stream as a “carrier” for the PB027 (PB027SK) further increased its effectiveness in degrading residual soil glyphosate and significantly increased the yield and quality benefits. In addition to the increased yield and vigor of silage corn, increased milk production and other quality factors may be realized by degrading much of the residual glyphosate in the soil to increase the availability of immobilized nutrients (Shaver et al. 2007) (Tables 15 and 16). This could be especially important for situations in which animal feedstuff is contaminated with glyphosate (Table 21) (Dunham 2021; Dupmeier 2021). Available B, Fe, Mn, and Zn levels significantly increased in soil at 6 to 7 months after applying RSKJ to degrade residual glyphosate in soil. Like the silage area, the corn grain study included a high-fertility situation, with most nutrients at or above optimum levels. Furthermore, Mn was the only essential mineral that was deficient, at approximately half of the optimum value. Considering the importance of Mn for photosynthesis, hormones, cell wall development, and stress resistance and the interaction with chelation immobilization by glyphosate, increasing access to Mn could be especially important in this soil because residual soil glyphosate could be a major factor that reduces Mn availability in the soil and the plant (Table 22).
Soil (Tables 2 and 6) and plant tissue samples of cabbage cultivar Nixon from production fields (Tables 8 and 9) analyzed to determine the mineral composition were low to deficient in K, Mn, Cu, and Zn. Soils were generally very low in Cu. Glyphosate and AMPA are strong mineral chelators (Bernards et al. 2003) that immobilize cationic minerals; therefore, they are not available for physiologic functions in plants (Jolley et al. 2004; Popov 2001). Glyphosate and AMPA are also powerful antibiotics against numerous beneficial organisms involved in soil mineralization that increase the solubility and function of mineral nutrients (Kremer 2021). Biodegradation of residual glyphosate in soil may manifest as reduced toxicity to plants (increased yield, vigor) or a beneficial effect on specific components of the soil microbiota involved in mineralization or nutrient availability (Dunham 2021) that could be observed as improved growth with or without increased nutrient density. Glyphosate is toxic to N-fixing and Mn- and Fe-reducing microbes (Huber 2021; Kremer 2021); therefore, removal of this microbiological toxicity could increase the availability and uptake of these essential minerals. The increases in Mn, Zn, Fe, and Cu (Tables 15 and 16) could also occur through direct release from glyphosate chelation–immobilization, with previously chelated minerals becoming available for plant uptake, as observed during tissue analyses. After treatment with RSKJ plus PB027 (PB027SK), N, B, Mn, Zn, Fe, and Cu levels increased in soil with reduced glyphosate (Table 11); therefore, both mechanisms could be indicated. Only a small quantity of these nutrients would be added through the 140.25 L·ha−1 (15 gal/acre) RSKJ used as a carrier for the CatawaterSK or PB027 biological cocktail applied. Although a glyphosate-tolerant GE corn hybrid was used in the sandy loam degradation study, there is nothing in the GE plant that affects the mineral chelation or soil antibiotic activity of glyphosate. Thus, the yield or quality of the GE corn hybrid used in the Agri-Tech Consulting study could have been compromised by the residual soil or applied glyphosate (Zobiole 2010a, 2010b).
Glyphosate is a compound that is difficult to degrade because most soil organisms lack the carbon-phosphite lyase enzyme required for full degradation. As a consequence, in soils with a low pH or high clay content, significant herbicidal levels of glyphosate can accumulate (Fahrenhorst et al. 2009; US Geological Survey 2014). Glyphosate and P have similar binding sites in soil; therefore, P fertilizer applications performed later can desorb residual soil glyphosate and damage successive crops (Bott 2011; Gomes et al. 2015), including GE types (Zobiole 2010a, 2010b).
The fertility level in the silage and grain plots was near optimum for the corn hybrid and environmental conditions of the study area near Whitewater, WI, USA. Exceptions were observed, however; K and Mg levels were slightly less than optimum and Mn was low to deficient, with only approximately half the optimum level, in the corn plants at both silking and grain fill growth stages (Tables 15, 17, and 22). Furthermore, Mn is critical for splitting water in photosynthesis and important in various other enzymatic reactions for cell wall, hormone, and stress defense reactions of the shikimate metabolic pathway.
Glyphosate is a common contaminant of animal manures and urine from animals fed glyphosate-contaminated feed (Table 21), especially herbicide-tolerant (GE) or herbicide-desiccated crops (Dunham 2021; Dupmeier 2021). Fermentation is a successive process involving Leuconostoc-producing metabolites that are key for the next stage of fermentation. Antibiotic activity against Leuconostoc could also interfere with the fermentation process to produce soft (soggy) sauerkraut; however, this may not occur because L. plantarum, the principal organism present during fermentation of cabbage, was shown to degrade glyphosate. Although actual levels of nutrition varied, cabbage grown in central Wisconsin was commonly deficient in Fe, Mn, Cu, and Zn. Additionally, Ca, K, and Mg were sometimes low or deficient. It appears that greater attention to the micronutrients, especially those required for cell wall development and membrane integrity, is important. Because most commercial cabbage production is contracted through a third party, closer involvement of the processing program with cabbage production could avoid costly quality deficiencies.
To assure the quality of sauerkraut, nutrient programs should ensure the sufficiency of the essential micronutrients to optimize cabbage yield and quality. Soft (soggy) sauerkraut may result from nonoptimal fermentation conditions, nutrient deficiencies during cabbage growth, or, as shown here, by glyphosate contamination of soil through manure used as a nutrient fertilizer. Soggy sauerkraut is associated with deficiencies of Fe, Cu, Mn, B, or Zn, which are essential nutrients for cell wall formation and membrane permeability through the plant’s shikimate metabolic pathway. In addition to the lack of one or more specific nutrients in the growth medium, nutrient deficiencies may be induced by factors that prevent nutrient uptake or the physiological function in the growing cabbage plants (Graham and Webb 1991; Maeda and Dudareva 2012). Minerals that were especially impacted in cabbage were the micronutrients B, Cu, Fe, Mn, and Zn, which are critical for cell walls and lignification through the shikimate metabolic pathway that is inhibited by glyphosate-based herbicides. Treatment of soils destined for cabbage production with 140.25 L·ha−1 (15 gal/acre) of RSKJ or a biological cocktail (PB027) of microbes shown to degrade residual glyphosate in soil, decreased soil residual glyphosate in both silt loam and sandy loam field soils by 80% to 90% within 6 to 7 months and significantly increased corn grain and silage yield. Although many of the fields selected for cabbage were low to deficient in one or more of the micronutrients involved in cell wall formation or cell membrane integrity, the presence of residual glyphosate in the fertilizer manure used for organic production exacerbated the existing mineral deficiencies and was the primary factor that caused the soggy fermented product produced in 2022 and extensive economic loss.
Residual soil glyphosate may come from a multitude of independent factors and reduce the yield and quality of subsequent crops, as demonstrated in this study. In this study, the origin of glyphosate was contaminated poultry feed that carried through to the manure used as a fertilizer source for the organic cabbage. Additionally, RSKJ, a biological cocktail of glyphosate-degrading microorganisms, or their combination, significantly reduced the level of this contaminating mineral-immobilizing phytotoxin in soil and significantly increased crop yield and quality. A soil application of 140.25 L·ha−1 (15 gal/acre) of RSKJ and/or PB027, along with balanced fertilizer applications, can significantly increase production quantity and product quality.
This study offers groundbreaking insights regarding effective bioremediation strategies that significantly mitigate the adverse effects of glyphosate and has broader application to nutritional content and crop yields when conventional manures that may contain glyphosate from feed and environmental contamination are used (Dunham 2021; Dupmeier 2021; Gerlach et al. 2014).
Schematic presentation of the Shikimate metabolic pathway indicating critical points where specific micronutrients are required as enzyme cofactors (Graham and Webb 1991).
Contributor Notes
Funding for analytical costs and field research was provided by GLK Sauerkraut, 400 Clark Street, Bear Creek, WI 54922, USA.
We thank Tim Malony, PhD Coordinator, Agri-Tech Consulting, White Water, WI, USA, for research coordination and implementation of corn grain and silage field research with the RSKJ and biological cocktails to evaluate the impact of degradation of soil residual glyphosate on mineral uptake. We thank Larry Van Straten, Shiocton, WI, USA, for field evaluation of raw sauerkraut juice to degrade residual soil glyphosate. We thank Adam Flyte, Flyte Farms LLC, Coloma, WI, USA, for field research and mineral analysis of organic and conventional grown cabbage. We thank John Fagan, Health Research Institute Laboratories, Fairfield, IA, USA, for guidance regarding the glyphosate analysis.
The first author oversaw and coordinated all the research, obtained funding, and helped perform sampling for the analyses. The second and third authors provided professional expertise regarding planning and implementing the research, data collection, and evaluation of data. The fourth author analyzed texture, coordinated with growers, obtained funding, and participated in the evaluation of data. The fifth and sixth authors developed and provided biological cocktails for evaluation of glyphosate biodegradation and performed statistical analyses.
D.M.H. is the corresponding author. E-mail: huberd@purdue.edu.
Schematic presentation of the Shikimate metabolic pathway indicating critical points where specific micronutrients are required as enzyme cofactors (Graham and Webb 1991).
