Improving Resilience, Sustainability and Nutritional Properties of Specialty Crops Using Composted Spent Coffee Grounds

Non-composted, static pile composted and/or in-vessel composted spent coffee grounds were tested for phytotoxicity using germination and growth experiments. Germination and growth experiments were carried out at the Texas A&M Department of Horticultural Sciences, Horticulture Teaching Research and Education (Hort. T.R.E.C.) greenhouse facility. The spent coffee grounds used throughout this study came from a cold brew coffee company based in San Antonio, TX. Pro-Mix general-purpose potting mix was the peat-based media used in these experiments. Potting mixture treatments were made on a volume/volume basis and mixed by hand. Treatments included 10%, 25%, 50%, 75% and 90% SCG and potting mix in order to cover the full spectrum of the effect on the plant at high and low levels of SCG. The first eggplant experiment also included SCG and sand at the same ratios as above. These treatments were not repeated due to the difficulty and poor results of using sand in nursery pots.

Spent Coffee Ground Preparation and Compost 

The spent coffee grounds used throughout this study came from a cold brew coffee company based in San Antonio, TX. The SCG were delivered by dump truck loads to the Scotts Miracle-Gro Turf Science Center at Texas A&M University. They were dumped in a pile in an area at the turf center designated for large quantities of soil components.

Non-Composted SCG: The non-composted SCG used in these experiments were collected from the pile of SCG within a 24-hour period of delivery and dried in a 62 °C oven for 72 hours. The SCG were removed from the oven, cooled, and stored indoors in a plastic container. A sample was sent to Texas A&M AgriLife Extension Soil, Water and Forage Testing Laboratory for chemical analysis.

Static-Pile Composted SCG: The static-pile composted SCG used in these experiments were taken from the pile of SCG about nine months after composting began. Temperatures ranged from 38-60 °C depending on where in the pile the temperature was measured. Approximately 0.34 m³ of this nine-month composted SCG were taken from the pile and air dried for 48 hrs. After drying they were sieved to 2 mm, and stored indoors in a plastic container.

 In-Vessel Composted SCG: May 21, 2020 a new batch of spent coffee grounds were delivered to the Scotts Miracle Gro Turf Center. A small amount was taken to air dry in the greenhouses at the Hort. T. R. E. C. After they were dry, they were stored indoors in a plastic container. On August 17, 2020 SCG were taken from the pile and transported to the Poultry Science Center at Texas A&M University to put in their Ecodrum^® in-vessel composter. At this time temperatures of the pile of SCG ranged from 50-60°C which is considered thermophilic composting.  Four 200 kg barrels of SCG were added initially, then a barrel every other day for approximately 21 days (September 7, 2020).

            After 21 days a sample was taken from the composter and due to the lack of moisture water was added. Any SCG that had exited the composter were added back in but no new SCG were added. At this point, temperatures had reduced to a mesophilic stage of composting (>40°C). Turning was reduced to biweekly and temperatures continued to stay within mesophilic range, this continued for approximately 90 days. The composted SCG were removed from the composter and stored in plastic barrels indoors. Samples were sent to the Texas A&M AgriLife Extension Soil, Water and Forage Testing Laboratory for chemical analysis. To verify the compost maturity a Solvita CO₂ and NH₃⁺ co-variate test will be used.

Germination experiments

Each mixture was spread evenly into seedling trays (25 cm wide by 51 cm long, and 6.4 cm deep) with forty seeds per tray. Each tray was replicated three times. Seeds tested included Pisum sativum ‘Wando’ bush peas, Spinacia oleracea hybrid #7 spinach, and Raphanus sativus, Cherry Belle radish. Percent germination was calculated.

Growth

Solanum melongena ‘Patio Baby’ were planted into seed plug trays (2 cm by 4 cm cell size), one seed per plug, filled with seedling potting mix (Pro-Mix general purpose seedling mix). Eggplants were transplanted into 20 cm nursery pots after the first set of true leaves were fully developed. Each treatment had five replicates given a high fertilizer rate (15-5-25 at 0.4 g/L) and five replicates given a low fertilizer rate (15-5-25 at 0.1 g/L). Plants were watered daily using a dosatron set at a dilution rate of 1:100 The sand and potting mix treatments were sperate experiments, assigned to different benches with pots arranged in a Completely Randomized Design. Data collected on a weekly basis included: height, number of flowers and fruits. Nine weeks after transplanting the eggplants were harvested and fresh weight was recorded as well as fruit number, fruit weight, and fruit length. Eggplants were left on greenhouse benches to dry until a constant weight was achieved, and dry weight was recorded. This experiment was repeated but did not include the sand treatments because the sand did very bad in the pots. It also only included a high fertilizer rate because there were no differences between the high and low fertilizer treatments. 

Ocimum basilicum ‘Rutgers obsession’ seeds were started in 3.8 cm rockwool cubes and transplanted according to the methods described above. Treatments included potting mix and static-pile composted SCG, and potting mix and non-composted SCG with ratios the same as previously stated. A high fertilizer rate (15-5-25 at 0.4 g/L) was applied to all treatments, with five replicates per treatment and in a completely randomized design. Data collected on a weekly basis included: height, number of flowers and branches. Eight weeks after transplanting the basil was harvested and fresh weight was recorded. Basil plants were left on greenhouse benches to dry until a constant weight was achieved, and dry weight was recorded. 

Carotenoids

After the plants were harvested eggplant fruits and basil leaves were placed in zip-loc baggies and stored in a deep freezer. Samples were freeze dried and mailed to UW Madison Department of Nutritional Science for analyses. Chemical analysis of eggplant fruit and basil leaves using HPLC and LC-MS were used to identify carotenoids (carotenes [beta-carotene, alpha-carotene], xanthophylls [lutein]).  Data will be compared to published nutritional value data for the same crops. A total of 80 samples were analyzed, 25 basil leaf samples and 55 eggplant fruits. Basil samples will be from 0%, 10%, and 25% non-composted and static-pile composted SCG treatments with five replicates from each treatment. Eggplant fruit samples will consist of 0%, 10%, 25%, 50%, 75% and 90% non-composted and static-pile composted SCG treatments with five replicates from each treatment. 

Supplies (per sample)

• 3 large test tubes/lids
• 3 15 mL screw top

test tubes/lids

• 3 HPLC vials / caps
• 6 disposable pipets/ bulbs

Set Up:

• *Start water bath and set sample(s) out to thaw
• Fill ice bucket and put in DI H2O to cool and internal standard to keep cool
• Label large test tubes with sample numbers
• Label disposable test tubes
• Prepare/label vials

PROCEDURE:

• Weigh first tube à zero scale
• Measure 03g – 0.035g / 0.15g – 0.155g of first sample into tube
• Repeat with rest of tubes/samples
  • Record each weight
• Label sample bag as analyzed and return to freezer
• Get 5mL pipet, set to 3 mL
• Add 6 mL (3 mL x 2) of EtOH + BHT in each tube
  • Cap each tube (don’t screw too tight, or may crack in hot water bath)
  • Vortex each tube ~30 seconds
  • Put in water bath for 5 min
• When 5 minutes is up, remove tubes from bath to rack
  • Add 500 uL KOH with 1 mL pipet set to 0.5
  • Vortex each tube again ~30 sec
• Add tubes back to hot water bath for 5 min
• When 5 min are up, vortex each tube and return back to bath for 5 more min
• When last 5 min are up, remove each tube from water bath and put into ice bucket
• Add 3 mL DI water (1 mL pipet x 3)
• Add 500 uL / 200 uL B-apo internal std
  • Vortex each tube ~15 seconds
• Add 4 mL Hexanes (5 mL pipet set to 4 mL) to each tube
  • Vortex ~30 sec and centrifuge ~ 2 min

     Extract:

• Take small tubes and pipets to hood
• Pipet off top layer of sample into corresponding 15 mL tubes USING DIFFERENT PIPET FOR EACH SAMPLE
• Add 3 mL hexanes to each tube
  • Vortex and centrifuge
• Repeat top layer extraction for each sample
• Add final 3 mL of hexanes to each tube
  • Vortex and centrifuge
• Repeat top layer extraction for each sample
  • **After all extraction steps, should have 10 mL of collected hexanes (4 mL, 3 mL, 3 mL)
• Put under nitrogen to dry

    Reconstitution:

• Once samples are dry, add 600 uL / 200 uL of 50:50 MeOH:DCE
• Vortex and centrifuge
• With remaining pipets, pipet samples into corresponding vials as well as a vial with B-apo
• Take to HPLC or refrigerate- inject 10 uL / 20 uL

Modified from previously published method: Howe, J. A., and S. A. Tanumihardjo. 2006. Evaluation of analytical methods for carotenoid extraction from biofortified maize (zea mays sp.). J. Agric. Food Chem. 54:7992–7997.

Mineralization Rate of SCG

A 70-day and 100-day laboratory incubation study was conducted at Texas A&M University in Fall 2019 and Spring 2021, respectively. The treatments included non-composted SCG (2.2-0.9-0.5), composted SCG [73-day (2.2-0.9-0.5), 100-day (3.9-0.1-0.7)], Milorganite (5-2-0), and Urea (46-0-0), and the control was soil with nothing added to it. Each treatment was mixed into 50 g of Booneville fine-sandy loam soil at a rate of 9.8 g N ^m2 for comparison. The soil was brought to field capacity (60% w/w) and a 9.8 g N ^m2 rate of each treatment was added to 50 g of dry-weight soil. Treatments were thoroughly mixed in 50 ml polypropylene beakers. Each beaker was placed inside a Mason jar with 5 ml of water in the bottom to create a microcosm. A CO² trap which consisted of a 50 ml polypropylene centrifuge tube, with no lid, filled with 20 ml of 1.0 M sodium hydroxide was also placed in each Mason jar. The jars were immediately sealed and placed in an incubator held at 25 °C in a completely randomized design with three replicates per treatment. Aerobic conditions were maintained by opening the jars every 7 days. The CO² (sodium hydroxide) trap was sampled every 10-days by adding barium chloride and back titrating with hydrochloric acid (HCl). The amount of HCl used in the titration was recorded and used to monitor CO² release throughout the incubation. The CO² flux was used as an index for net mineralizable nitrogen. Ammonium (NH₄⁺), and nitrate (NO₃^-) were extracted from destructive samples every 10-days by inductively coupled plasma spectrometry (ICP) following Keeney and Nelson (1982).