The NC State Soil Microbiome Extension program conducted a microbial biofertilizer butterhead lettuce greenhouse trial in 2025. Biofertilizers are a class of biostimulants that are comprised of living microbes (e.g., bacteria, fungi, or algae) that can enhance plant growth by improving nutrient availability and/or nutrient use efficiency. The objective of this study was to evaluate the impact of a biofertilizer comprised of Xanthobacter autotrophicus on butterhead lettuce production and to consider environmental sustainability impacts by quantifying greenhouse gas emissions. The biofertilizer used in this study was a non-commercial, prototype formulation developed by Kula Bio.

Kula Bio offers commercial biostimulant products comprised of a single Xanthobacter autotrophicus strain, which is a naturally occurring, free-living, nitrogen-fixing bacteria. This microbe colonizes plant root zones where it fixes atmospheric nitrogen that can be directly taken up by plants. Xanthobacter autotrophicus may increase soil fertility by replacing or supplementing synthetic nitrogen fertilizer and can be used in a variety of specialty and row crops (including strawberries, lettuce, and tomato). This product is delivered through fertigation and must penetrate the soil and contact the root system. As of May 2026, products are commercially available for both conventional (Kula-N) and organic production systems (Kula-Next) and in different formulations (liquid, concentrated liquid, and shelf-stable powder) (Table 1). Application rate for Kula-Next is 2-8 oz of Kula Bio product per acre per lb of nitrogen desired, but recommendations vary across formulations and production systems.

The experiment was conducted at the North Carolina State University Phytotron greenhouse facility. Butterhead lettuce Lactuca sativa (Johnny's Selected Seeds Product ID 2358; Adriana variety) was grown in a standard potting mix containing 33% Sun Gro Propagation Growing Mix (50-65% Canadian Sphagnum peat moss, vermiculite, dolomitic lime, 0.0001% Silicon dioxide) and 66% pea gravel. Lettuce was grown for 28 days under ideal environmental conditions (26 ˚C during the day and 22 ˚C at night), and pots were well-watered.

We fertilized plants using a standard nutrient solution with nitrate as the nitrogen (N) source, and adjusted total N as needed. The fertilizer solution and Xanthobacter autotrophicus test product were applied weekly (4 total applications) dispensed as a drench at the base of the plant. Fertilization rates were GSP equivalent to 120 lbs N/acre (100% Nitrogen or 100N) or reduced fertilization equivalent to 96 lbs N/acre (80% Nitrogen or 80N). X. autotrophicus test product was applied at a rate equivalent to 8 oz/acre, and nitrogen replacement and boosting/supplemental effects were both evaluated. Treatments were installed using a randomized block design (with 6 replicate pots) and included:

1. 100N Control (100% Nitrogen fertilizer control without X. autotrophicus)
2. 80N Control (80% Nitrogen fertilizer control without X. autotrophicus)
3. 100N + X. autotrophicus test product (100% Nitrogen fertilizer plus microbe; boosting effect)
4. 80N + X. autotrophicus test product (80% Nitrogen fertilizer plus microbe; replacement effect)

We harvested butterhead lettuce after 28 days of growth (Figure 1). We determined yield by measuring lettuce head biomass (total harvest weight without roots). Nitrogen application rate determined lettuce yield, but X. autotrophicus application did not impact biomass production. Lettuce grown with 100% Nitrogen (120 lbs/acre) had greater biomass compared to lettuce grown with 80% Nitrogen (96/lbs/acre) (110.1 g/head or 3.9 oz mean vs. 79.5 g/head or 2.8 oz mean; P-value < 0.001). Lettuce yield of the 100% N fertilizer control was approximately 5 oz per head including roots which is at the lower end of market weight, indicating that growth conditions did not reflect commercial operations. We did not observe a significant nitrogen replacement effect (80N Control vs. 80N X. autotrophicus) or boosting effect (100N Control vs.100N X. autotrophicus) for either of the microbial treatments (Figure 2).

Nitrogen management practices that minimize nitrogen loss support environmental sustainability. In agricultural systems, nitrogen loss pathways include nitrate leaching and nitrous oxide (N2O) emissions, a potent greenhouse gas, via a microbial process called denitrification. We measured N2O emissions across the course of the greenhouse experiment, and cumulative N2O emissions did not differ between fertilization rates or X. autotrophicus biofertilizer treatment (Figure 3). We also did not detect differences in N2O emissions between lettuce pots and soil-only controls (not pictured). In summary, under the study conditions microbial denitrification did not represent a significant nitrogen loss pathway. We cannot account for potential differences in nitrate loss between treatments as we did not measure nitrate leaching in this study.

• Nitrogen fertilization rate determined butterhead lettuce yield under the study’s greenhouse conditions. Lettuce head biomass was greater for plants grown at 120 lbs N/acre (100% Nitrogen or GSP) versus plants grown at 96 lbs N/acre (80% Nitrogen or reduced nitrogen).
• The non-commercial biofertilizer formulation comprising Xanthobacter autotrophicus had no impact on butterhead lettuce yield in this study. We did not observe a nitrogen replacement effect (no difference in biomass between 80% N Control and 80% N + X. autotrophicus) or a boosting effect (no difference in biomass between 100% N Control and 100% N + X. autotrophicus).
• We did not detect differences in nitrous oxide (N2O) emissions, a potent greenhouse gas, between treatments. This indicates that under these study conditions, microbial denitrification did not represent a significant nitrogen loss pathway. In real-world agronomic conditions, nitrogen loss pathways are critical considerations for environmental sustainability and economic profitability.
• This applied research project evaluates the impact of Xanthobacter autotrophicus on butterhead lettuce production under the study conditions and does not reflect other usage scenarios. Crop response to fertilization and Xanthobacter autotrophicus application may differ in other systems, including different field or greenhouse environments, product formulation, crop variety, and fertilizer composition (nitrogen source).

Kula Bio, Inc. (Natick, MA USA) provided funding and research materials for the project. Madaris Serrano Perez (Soil Microbiome Extension Program Research Technician) installed the greenhouse experiment, maintained the trial, collected data, and contributed to data analysis and visualization. Isabella Borrero (NC State PhD student) contributed to data collection and analysis. Akieliah Robinson and Matthew Clayton (NC State undergraduate students) contributed to study setup and data collection.

The use of brand names and/or any mention or listing of specific commercial products or services herein is solely for educational purposes and does not imply endorsement by North Carolina State University or our partners, nor discrimination against similar products or services not mentioned.