Final Report for SW09-053
Project Information
Agriculture is highly dependent on fertilizer made through energy-intensive industrial nitrogen (N) fixation. As energy prices increase, so does the price of fertilizer. Biological N fixation using cyanobacteria has the potential to supply N to crops while reducing input costs and increasing energy-efficiency. The goal of this project was to develop and test an on-farm biological N fixation system. The primary scientific components included development and optimization of cyanobacteria-based N fixation, on-farm testing through participatory research and economic analysis. A manual, newsletter article, presentations and social media were used to raise awareness and educate producers and professionals regarding this new technique.
Our objectives are to:
1) Evaluate cyanobacterial species and growth conditions necessary for biofertilizer production,
2) Evaluate the economic and social feasibility of on-farm or community-scale fertilizer production using a biological N-fixation system,
3) Optimize the harvesting, processing and application of cyanobacteria-fixed N through on-farm testing,
4) Inform the bioreactor design and utilization of biofertilizers through consultations with farmers, and
5) Develop educational materials providing information on the production and utilization of cyanobacteria-based N fertilizer and disseminate that information to farmers and agricultural professionals through a variety of means.
Nitrogen is the limiting factor for agricultural yields in most cropping systems. Nitrogen gas (N2) makes up approximately 78 percent of the Earth’s atmosphere, but in order to be useful for most microorganisms, plants and animals, N must be in a “fixed” form such as ammonium or nitrate. The discovery of the industrial N fixation process in the 20th century, now called the Haber-Bosch process, ushered in the era of industrial fertilizer by enabling the large-scale production of ammonia and its derivatives. In the Haber-Bosch process, N2 is combined with hydrogen gas (H2) in the presence of a catalyst, at high temperature (several hundred degrees C) and high pressure (several hundred atmospheres) to form ammonia gas, from which all other nitrogenous fertilizers are derived (Havlin et al., 2005). Methane, also known as natural gas (CH4), is typically used as both a hydrogen source and an energy source to maintain the high temperature and pressure required. The process is energy-intensive, and the use of natural gas as a feedstock further ties N fertilizer production to this non-renewable and costly resource. Natural gas prices have been volatile in recent years, but until the recent economic crisis, they had increased two- to three-fold since 2000 (US EIA, 2012). Though natural gas prices have fallen from their highs prior to the economic crisis, N fertilizer prices have recently returned to levels at or near record highs (USDA ERS, 2012).
Biological N fixation, by contrast, takes place at ambient temperature and pressure. It is catalyzed by the nitrogenase family of enzymes, which are found in a variety of symbiotic and free-living microorganisms that have nearly ubiquitous distribution on Earth. Unlike industrial N fixation, which uses chemical energy in the form of fossil fuels, biological N fixation obtains the required energy by capturing it from the sun through the process of photosynthesis. Naturally occurring N-fixing organisms have directly supported life on Earth for billions of years. Leguminous plants in association with Rhizobium bacteria, in particular, have played an important part in maintaining the N fertility of agricultural lands.
Cyanobacteria are photosynthetic bacteria, many of which are capable of producing nitrogenase enzymes and thus fixing atmospheric N. All cyanobacteria can exist as free-living cells but some are also capable of forming symbiotic relationships with plants. The symbiotic relationships of cyanobacteria with plants have been studied extensively (e.g., symbiosis between cyanobacteria and Azolla ferns has been exploited for rice production in India). Free-living forms have been subject to much less scrutiny but can be effective in flooded environments such as rice paddies. Boussiba (1991) noted the potential of cyanobacteria as a bio-fertilizer “inoculum” in rice paddies, and Kannaiyan et al. (1997) showed cyanobacteria to be effective in this environment. Hashem (2001) also investigated various cyanobacterial strains for their potential as bio-fertilizer inocula for rice.
Research on other crops is limited, but Banerjee et al. (1997) applied naturally occurring colonies of cyanobacteria to rice as well as mustard crops. The benefits of cyanobacterial addition to soils have also been reported for the cultivation of several grains and vegetables (Dadhich et al. 1969, Patterson 1996). Silva and Silva (2003) and Silva et al. (2007) have investigated preservation methods for cyanobacterial inoculum to be tilled into the soil in land-based agriculture. To date, nearly all work on bio-fertilizers focuses on inoculating fields. The use of intensively-cultivated, non-inoculum-based, locally-produced cyanobacterial bio-fertilizer in environments other than rice paddies should be possible. However, no such products are currently commercially available in the U.S. This project builds on previous research by testing cyanobacteria as a fertilizer rather than as a microbial inoculum, with the cyanobacteria being applied at N fertilizer rates. It also develops a process for on-farm cyanobacterial bio-fertilizer production, which is unprecedented.
On-farm methods for production and application of cyanobacterial cultures remain a major obstacle (Hashem, 2001). Mishra and Pabbi (2004) reported success in growing bio-fertilizer in open-air ponds from environmental cultures. They provided little qualitative or quantitative data but did note that controlled growth conditions could increase production. As asserted by Postgate (1997) and Kant et al. (2006), the potential of cyanobacteria has not been fully exploited. The intensive culture of cyanobacteria for production of a N bio-fertilizer remains largely unexplored and could hold the key to ambient temperature, energy-efficient N fertilizer production.
Cooperators
Research
Objective 1: Evaluate cyanobacterial species and growth conditions necessary for biofertilizer production
Milestone 1.1. Culture N-fixing cyanobacteria and determine baseline growth and N-fixation
Soil, water and sediment samples were collected from 24 locations around the Front Range and Western Slope of Colorado, including wetlands, lakes, rivers and farmers’ fields. Nitrogen-fixing cyanobacteria were initially cultured using two methods: (1) construction of Winogradsky columns or (2) selective sub-culturing with Allen and Arnon media. Cultures were initially grown in Erlenmeyer flasks under 12 hour light (2500 lux):12 hour dark cycles, ambient temperature, and aerated by means of a low speed (100 rpm) orbital shaker.
Once cultures were actively growing (by observing the appearance of green planktonic or aggregate cultures), baseline growth and N-fixation measurements were recorded. Growth was monitored by determining the absorbance [optical density (OD)] of cultures at 655 nm. Absorbance was found to be well correlated with dry weight biomass. N-fixation was estimated by measuring the total Kjeldahl N (TKN) of samples at the beginning and end of 14-day growth periods.
Once cultures were growing consistently in liquid media, frozen culture stocks were made (in replicate) and placed in a -80oF freezer for long term storage. After a few weeks, one tube of culture stock was revived to verify the success of the method and ensure long-term viability of our culture collection.
Milestone 1.2. Evaluate performance of cyanobacteria grown with and without aeration
Based on preliminary experiments, aeration is key to enhancing nitrogen fixation in filamentous, heterocystous strains of cyanobacteria. To simulate cultivation of cyanobacteria in an open pond system, four test cultures were inoculated into three gallon aquariums containing Allen and Arnon media. Each culture was grown with or without aeration. A sample from each tank was analyzed for total nitrogen analysis using the Kjeldahl method.
Milestone 1.3. Determine performance of cyanobacteria grown in different water sources
We developed a laboratory method for evaluation of various growth parameters. We named this the light box technique (Fig.1) and utilized it to evaluate the effect of water source on cyanobacterial growth and N fixation, as well as in subsequent studies. Three water sources were compared: distilled water, tap water and river water (a common source of irrigation water). We generally use distilled water in all of our laboratory work. However, since distilled water is not practical for on-farm conditions, it was important to evaluate the impact of other water sources on cyanobacterial growth and N fixation.
Milestone 1.4. Determine the tolerance of cyanobacteria cultures to a range of temperatures
Two cultures (H1 and H4) were grown at four temperatures to evaluate the influence of temperature on the growth and N fixation of the cultures.
Milestone 1.5. Determine the optimum nutrient media for growing cyanobacteria
The composition of the nutrient media is another critical component that is currently being evaluated. Cyanobacteria fix their own carbon and nitrogen from the carbon dioxide and nitrogen in the atmosphere by photosynthesis and nitrogen fixation, respectively. The most critical nutrients for cyanobacteria are the trace metals required by the enzymes involved in these processes.
The micronutrient solution in Allen and Arnon (AA) is more complete than that of BG-11 in that it contains vanadium; however, BG-11 has higher concentrations of some micronutrients. Four environmental cultures were grown side-by-side in AA and BG-11 media. Biomass and total N fixed were determined after 14 days of growth.
Milestone 1.6. Design a nutrient media that can be used in certified organic food production
AA media contains several chemicals prohibited in organic food production. Since we expect a major market to be organically-certified farmers, we have begun to develop a growth media for N-fixing cyanobacteria that can be organically-certified as well. The substitutions shown in Table 1 were made to develop a nutrient solution (OAA1) for cyanobacterial production using entirely certified organic ingredients. This media was then compared to the control treatment (AA media) and another media formulation (AA-V) that was identical to AA except that Vanadium was eliminated because there is no organic substitute. The media were analyzed in two ways: first, they were analyzed chemically to determine concentrations of the various macro and micronutrients. Secondly, a replicated growth experiment was conducted using the light box technique in which H4 culture was inoculated into each of the three media treatments to determine differences between growth and N-fixation of the cultures.
Objective 2: Evaluate the economic and social feasibility of on-farm or community-scale fertilizer production using a biological N-fixation system
Milestone 2.1. Interview farmers regarding their current practices and opinions regarding feasibility of cyanobacterial bio-fertilizer production and utilization
Potential adopters were examined for on-farm adoption of production and were categorized as small or large organic farms. Characteristics of each are described below.
Small Organic Farms: 2-15 acres of land upon which varied and integrated crops are grown, including vegetables, fruits and often flowers. These farms represent a growing trend in the U.S. for Community Supported Agriculture (CSA) providing produce to local buyers. All those interviewed classify themselves as “organic” or “natural” and are intensely concerned with sustainable practices; however, most are not USDA-certified organic. They rigorously avoid all synthetic inputs and chemicals. Currently, N is sourced through integrated on-farm soil management and growing methods such as biodynamics, composting and manure management, and cover cropping. Few, if any, purchased soil amendments are used.
Large Organic Farms: own and/or manage more than 1,000 acres. These growers have found success by specializing primarily in vegetable crops for regional, national and even export markets, increasing the value proposition with the USDA Organic label. Due to large areas of land, there is significant need for inputs that would facilitate growth while simultaneously keeping with organic requirements. Growers interviewed exhibit a personal belief that organic farming practices are best; however, business logic and acumen is what has driven their success to this scale. Currently N is sourced through a combination of purchased compost, fish emulsion, manure, guano and cover cropping.
Objective 3: Optimize the harvesting, processing, and application of cyanobacteria-fixed N through on-farm testing
Milestone 3.1. Evaluate the practicality and efficacy of settling, filtration and centrifugation as harvesting methods
Some strains of cyanobacteria possess gas vacuoles, allowing them to float. Other strains naturally settle. Thus cells are naturally able to concentrate themselves either at the surface or at the base of a pond or reactor, greatly facilitating harvesting. Four criteria were considered in the evaluation of harvesting techniques: (1) efficiency of recovery, (2) settling/flotation time, (3) energy requirements, and (4) cost of required harvesting equipment. The settling behavior of different cyanobacterial strains was evaluated in order to estimate how well they would settle in a larger system.
Several fabrics were tested to determine if fabric filtration could provide a low-cost option for harvesting cyanobacteria from ponds. Silk blends, cotton blends, synthetic fabric, as well as varying weights and weaves of 100% cotton were tested to determine the efficacy (speed and % recovery) of filtration of cyanobacteria. The OD of a sample of 14-day old liquid cyanobacteria was measured. 250-mL aliquots of the remaining culture were filtered through the fabric treatments. The OD of the filtrates was then measured and compared to the initial OD.
In addition, using large-scale (625-gallon) ponds at Thin Air Nitrogen Solutions’ headquarters, several methods of harvesting were evaluated. Settling was evaluated by turning off the paddlewheel and allowing settling to occur for 24-48 hours and then pumping off the liquid while leaving the settled cyanobacteria to air-dry. Filtration was tested using recommendations based on the laboratory evaluations described above. Centrifugation was assessed by modifying a separator originally designed to separate milk from cream.
Milestone 3.2. Compare rates of N mineralization of cyanobacterial fertilizer to other organic fertilizers
Organic farmers require organic sources of N fertilizer. Cyanobacteria can be grown on site and may be an effective N fertilizer, but N mineralization potentials of this material need evaluation. The N mineralization potential of cyanobacteria (in liquid and solid form) was compared with commonly-used organic fertilizers (fish emulsion and composted manure) in two soils of contrasting textures in a laboratory incubation study. Incubation occurred in the lab at constant temperature (25oC) and moisture content (60% water-filled pore space). The experimental units were arranged in a Randomized Complete Block Design. Organic fertilizers were applied based on the field application rate of 50 kg N ha-1. Soils were destructively sampled over the course of 140 days and analyzed for NH4+-N and NO3--N. Nitrogen mineralization percentage, total C, total N, soil organic C and soil microbial biomass C were analyzed at the end of the incubation study.
Milestone 3.3. Determine how cyanobacterial fertilizer application affects yield and quality of lettuce compared to other organic fertilizer applications
A greenhouse study was conducted to determine the N mineralization potential of soil-applied and foliar-applied organic fertilizers in comparison between cyanobacterial and commonly-used organic fertilizers applied to clayey and sandy soils. The pot experiment was carried out from May to August 2012 in a greenhouse at Colorado State University. The study was conducted for 63 days using foliar application (liquid cyanobacteria and fish emulsion), fertigation (liquid cyanobacteria and fish emulsion) and soil application (composted manure and dried cyanobacteria) methods. Two N rates (50 and 100 lbs N acre-1) were evaluated. The experiment was arranged in a Randomized Complete Block Design (RCBD). At the end of the study, soil inorganic N (ammonium and nitrate), plant height, leaf chlorophyll content, fresh yield, leaf area, shoot and root biomass, zinc, iron and Vitamin A content in leaf tissue were measured.
Objective 4: Inform the bioreactor design and utilization of biofertilizers through consultations with farmers
Milestone 4.1. Consult with farmers to solicit input to guide prototype design
We had annual meetings with our Farmer Advisory Group to update them on our achievements in the previous year and to receive their feedback and advice for the following year. In addition, we had booths at the Colorado Big & Small Conference (Brighton, CO) and the New Mexico Organic Conference (Albuquerque, NM) in 2011 to solicit farmer input into our bio-fertilizer production and utilization system. A broad survey of farmers was carried out at these conferences using a one-page survey.
Milestone 4.2. Construct and test an open pond system at Happy Heart Farm (Summer 2011)
Following the farmer workshop in January 2011, we designed the ponds and aeration system for installation at Happy Heart Farm in summer 2011. Rectangular (4 foot by 6 foot) 100-gallon lined ponds were constructed. Hoop houses are known to maintain higher temperatures and allow for some filtration of harmful UV rays. Therefore, two of the ponds were placed outside, and two were placed in a hoop house (Fig. 2) to evaluate the potential impact of these different climates on cyanobacterial growth and N fixation. The nutrient solution was continuously dripped into the pond at a rate just enough to replace evaporation, and fish tank air pumps were used to add air in each of the four corners. Optical density (OD), dissolved oxygen (DO), pH and electrical conductivity (EC) were measured daily. Cyanobacteria were grown in two 2-week batch studies performed twice: once in June and once in July.
In both studies, the cyanobacteria in the outdoor raceways died within 24-36 hours. After evaluating alternative explanations, it was concluded that the high ultraviolet light intensity resulted in the death of the cultures grown outdoors. Therefore, hoop house cover materials were evaluated in summer 2012. In our 100-gallon prototypes in the hoop house at Happy Heart Farm, pH levels rose to above 10, and growth was limited. Elevated pH can indicate a CO2 deficiency. Therefore the addition of carbonate was tested in field studies during summer 2012.
Milestone 4.3. Construct and operate pilot-scale raceways at CSU’s Horticultural Research Farm (Summer 2012)
Initial prototype testing done on-farm during summer 2011 indicated some problems with our design that needed to be evaluated, including possible CO2 deficiency and photo-inhibition from intense UV light outdoors. The parameters of depth, carbonate addition and hoop house covers were evaluated in replicated batch studies carried out in 50-gallon pilot-scale raceways. A 20 foot x 48 foot hoop house on Colorado State University’s Horticultural Research Farm, just NE of Fort Collins, was used for the pilot tests. Fifteen 50-gallon pilot-scale raceways were constructed using sheep troughs and materials available from a hardware store (Fig. 3).
Milestone 4.4. Determine if culture depth affects cyanobacterial productivity
Large-scale productivity of microalgae is usually determined by surface area rather than volume. Thus, water depth was evaluated as a parameter to determine if cyanobacteria could be grown more efficiently using less water. Three replicates of two treatments (8 inch depth, 10 inch depth) were set-up in a randomized complete block design. The six raceways were filled to the appropriate depth with AA media and then inoculated with H4 culture at a 1:10 dilution ratio. Raceways were continuously mixed with a submersible laminar water pump (1500 gph) which pushed the water around a center divider in order to improve mixing as compared to the rectangular ponds in 2011. Air was also bubbled into the tank using an aquarium aerator fitted with a stone diffuser. Water temperature, pH, dissolved oxygen (DO) and electrical conductivity (EC) were measured three times per day. Optical density (OD) of the cultures, which was used to assess culture growth, was measured once per day. Chlorophyll was extracted from the cultures to determine the correlation between culture OD and chlorophyll content. Cultures were examined microscopically daily. Twice during the study, pH, temperature, EC and DO were measured hourly from sunrise to sunset in order to better understand daily fluctuations in these parameters. At the end of the study, triplicate samples from each raceway were analyzed for total Kjeldahl N (TKN) and total solids (TS).
Milestone 4.5. Evaluate carbonate addition as a means to improve growth
Elevated pH and reduced growth of the prototype ponds located in the hoop house at Happy Heart Farm in 2011 were concerns as we began field testing in 2012. Since CO2 is frequently added to improve productivity of microalgal cultures in other industries, we wanted to test the effect the addition of CO2 might have on the buffering capacity, cyanobacterial growth and N fixation. Bubbling CO2 into the ponds is not likely to be affordable or practical for an on-farm system. Therefore, potassium bicarbonate (KHCO3) was added at different rates to test a range of CO2 levels. The treatments were as follows: control (no KHCO3 addition), low dose (0.19 mM KHCO3) and high dose (1.9 mM KHCO3). Carbonate dosages were determined from the range of CO3 in various freshwater cyanobacterial growth media (Andersen, 2005). Triplicates of the three treatments were set-up in a Randomized Complete Block Design using the 50-gallon pilot-scale raceways. The nine raceways were filled with AA to a depth of 8 inches and then inoculated with H4 culture at a 1:10 dilution ratio and grown for 14 days. Cultures were mixed and aerated as described above. Water temperature, pH, DO and EC were measured three times per day. OD measurements and microscopic observations were done daily. The above analyses were carried out daily for the first five days and then reduced to every other day until the end of the study (14 days total). Chlorophyll extraction was done three times over the course of the experiment. Twice during the study, pH, temperature, EC and DO were measured hourly from sunrise to sunset. At the end of the study, triplicate samples from each raceway were analyzed for total Kjeldahl N (TKN) and total solids (TS).
Milestone 4.6. Evaluate hoop house covers as a means to filter out harmful UV rays and prevent photo-inhibition
Based on the hypothesis that intense UV light led to the death of the outdoor cultures during the 2011 study at Happy Heart, we evaluated the efficacy of various hoop house covers on the growth and N-fixation of cyanobacteria grown in 50-gallon pilot-scale raceways. Five treatments were evaluated: no cover, standard 6 mil hoop house plastic (SuperDura from AT Films, Edmonton), transparent 6 mil construction plastic, LUMINANCE® High Tunnel Film (AT Films, Edmonton), and Dura-Film® ThermaxTM (AT Films, Edmonton). The raceways were arranged outdoors next to the hoop house in a Randomized Complete Block Design. Cover treatments were applied with mini hoop houses that were designed to fit over each pilot-scale raceway. Raceways were filled to a depth of 8 inches, and H4 culture was inoculated at a 1:10 dilution. The study was run for 14 days and measurements were taken as described in the carbonate study.
Milestone 4.7. Construct and operate prototype raceways at Thin Air Nitrogen Solutions’ headquarters (Summer 2012)
Two 625-gallon raceways (6 foot x 18 foot) were built above-ground. They were framed with wood, corners were rounded with flexible metal sheeting, and they were lined with 6-mil transparent plastic. Details are described and illustrated in our manual entitled, “Building a Raceway for Cyanobacterial Bio-fertilizer Production.” An A-frame hoop house was built over the two raceways and covered with 6-mil transparent plastic sheeting. Paddlewheels were built using PVC blades, pulleys to anchor the blades and an axel (Fig. 4). AA media was added to a depth of 10 inches, and H4 culture was added to the raceway in a 1:20 dilution. The AA media was dripped into the raceways at a rate to replace evaporative loss. The raceway was continuously mixed and aerated via the paddlewheel for two 14 day production cycles. In the third production cycle, the paddlewheel was only run during daylight hours when the cyanobacteria were actively photosynthesizing. OD and pH were measured daily, and samples were routinely examined under the microscope as well. A final sample was analyzed for nitrogen content.
Milestone 4.8. Estimate the land and water use requirements for cyanobacterial bio-fertilizer production as compared to cover crops
Water and AA media additions were carefully monitored in the 625-gallon raceways so that a comparison could be made of how much water was needed for each unit of N fixed in the raceways as compared to cover crops. Consultation with our Farmer Advisory Group provided us with figures on how much irrigation water was needed to grow various cover crops, and N fixation by those crops was collected from scientific literature.
Objective 5: Develop educational materials providing information on the production and utilization of cyanobacteria-based N fertilizer and disseminate that information to farmers and agricultural professionals through a variety of means
Milestone 5.1. Develop educational materials on the production and utilization of cyanobacteria-based N fertilizer
We developed a manual for raceway construction entitled, “Building a Raceway for Cyanobacterial Bio-fertilizer Production,” attached to this report. In addition, an article was published about our work in the Colorado State University College of Agricultural Sciences magazine (http://www.agsci.colostate.edu/news/Newsletter_1212/FoodforThought-1212.pdf).
Milestone 5.2. Disseminate information to farmers and agricultural professionals through a variety of means
We held annual Farmer Advisory Group meetings and began reaching out to both the agricultural and farming communities through presentations, the internet and social media. More detail is provided below in the Results and Discussion section.
Objective 1: Evaluate cyanobacterial species and growth conditions necessary for bio-fertilizer production
Milestone 1.1. Culture N-fixing cyanobacteria and determine baseline growth and N-fixation
Selective sub-culturing proved to be the most effective method of culturing nitrogen-fixing cyanobacteria and was used as the standard method for subsequent soil samples that were collected. Filamentous heterocystous cyanobacteria are the dominant type of cyanobacteria selected for under these conditions (Fig 5). Based on morphology, we have identified cyanobacteria from at least four genera, including Nostoc, Anabaena, Calothrix and Cylindrospermium in our environmental cultures, with strains from Nostoc and Anabaena being the most dominant.
This task was completed in year 1, but our culture collection is constantly being expanded. We have collected and cultured N-fixing cyanobacteria soil/water samples from Nebraska, Iowa, Illinois, Michigan, Pennsylvania, New York and Maine, as well as Ethiopia, Costa Rica, Nicaragua, Taiwan and Cambodia.
After being diluted at a ratio of 1:10, cultures generally take two weeks to go through the lag, exponental, stationary and decline phases before requiring another transfer. This has held true both in the laboratory and in outdoor pilot-scale and prototype tests. On average, cultures harvested after two weeks contain about 6-7% N (on a dry weight basis).
Milestone 1.2. Evaluate performance of cyanobacteria grown with and without aeration
Based on preliminary experiments, aeration is key to enhancing nitrogen fixation in filamentous, heterocystous strains of cyanobacteria. The aerated cultures fixed over 2.5 times more total nitrogen (p=0.006) than those that were grown under static conditions (Fig. 6).
Milestone 1.3. Determine performance of cyanobacteria grown in different water sources
Although growth was highest in the distilled water, there was no significant difference in growth between the tap and river waters (Fig. 7a). On the other hand, N fixation rates were the same in the distilled and tap waters but lower in the river water (Fig. 7b). Selection of water sources on-farm (river water, ground water, city water) will be an important factor in optimizing productivity.
Milestone 1.4. Determine tolerance of cyanobacteria to a range of temperatures
The H1 culture, although it grew well previously in the laboratory, did not perform well at any of the temperatures evaluated. However, the H4 culture grew optimally in the 27-29oC temperature range (Fig. 8a), fixed the most nitrogen at 27 oC (Fig. 8b) and was generally more vigorous at all temperatures than the H1 culture.
We compared two of our most promising cyanobacterial cultures (H1 and H4) at different temperatures and in different water sources in order to select the most-resilient culture for up-scaling under field conditions. In all cases, the H4 culture has outperformed H1; therefore, H4 was utilized in on-farm trials in summer 2011 and thereafter.
Milestone 1.5. Determine the optimum nutrient media for growing cyanobacteria
When four environmental cultures were grown side-by-side in AA and BG-11 media, three of the cultures showed greater biomass production with AA media (Fig. 9). However, there was no significant difference in the TKN of the final cultures between those grown in AA and those grown in BG-11 media.
Milestone 1.6. Design a nutrient media that can be used in certified organic food production
The organic formulation of AA (OAA1) was lower in P and Fe than the control AA media due to solubility and pH issues (Table 2). AA-V did not have levels of vanadium that were significantly different from AA, so it was excluded from further analysis. When cyanobacteria growth in AA and OAA1 were compared, cyanobacteria grown in OAA1 had slightly lower growth rates and total N fixed than the AA media (Fig. 10). Initial attempts to increase the concentration of P were unsuccessful. However, by using alternative sources of iron, either ferric citrate or ferric tartrate, the percentage of dissolved iron could be significantly increased compared to using ferric sulfate as the organic source of iron (Fig. 11). The use of ferric citrate may also enhance P dissolution since it could serve as a chelator of the excess calcium supplied by bone meal, which was believed to be another factor decreasing the dissolution of P. Once we have developed an organic nutrient solution in which cyanobacteria can achieve equivalent levels of growth and N-fixation as in the control AA media, we will begin the process of optimizing nutrient content to reduce cost and getting the new formula certified through the Organic Materials Review Institute (OMRI).
Objective 2: Evaluate the economic and social feasibility of on-farm or community-scale fertilizer production using a biological N-fixation system
Milestone 2.1. Interview farmers regarding their current practices and opinions regarding feasibility of cyanobacterial bio-fertilizer production and utilization
Direct interviews with representatives from both farm categories (described above in the Materials and Methods) were used to determine the level of potential interest in adoption of a cyanobacterial bio-fertilizer and the economic decision-making framework for each category. Varying levels of interest for on-farm production of N fertilizer exist across both categories of organic farmers. Small-scale organic farmers typically were committed to producing their own inputs and were initially not interested in the cyanobacterial fertilizer both because of economic constraints but also due to their commitment to an existing philosophical approach (such as closed-loop or bio-dynamics). However, interest was piqued when we explained that they could possibly grow their own. The larger-scale organic growers expressed keen interest in on-farm production stating they had the necessary land, water and production capacity. Ultimately, both categories of growers were interested in on-farm production of cyanobacterial bio-fertilizer. However, for the large organic growers, other factors were identified that may drive the preferred method of production away from a fully distributed model (of on-farm production) towards a somewhat more centralized model (such as at nearby cooperatives with larger scale production facilities to service a given region). These factors include the productivity of the cyanobacterial cultivation technology itself, economies of scale in that technology, capital requirements for building production facilities and risk sharing.
Objective 3: Optimize the harvesting, processing, and application of cyanobacteria-fixed N through on-farm testing
Milestone 3.1. Evaluate the practicality and efficacy of settling, filtration and centrifugation as harvesting methods
The settling behavior of different cyanobacterial strains was evaluated in order to estimate how well they would settle in a larger system. Theoretical recoveries ranged from 60-90% within seven hours of settling time (Fig. 12). The strain we selected for further technology development, H4, exhibited the highest recovery (90%).
We evaluated harvesting methods from the 625-gallon prototype ponds. In the first run, we turned off the paddlewheel after two weeks of growth and allowed the cyanobacteria to settle for 24 hours prior to pumping off the supernatant into another pond. We were able to pump-off more than half of the supernatant before the settled cyanobacteria began to be stirred up. Then we waited another 24 hours and pumped off the supernatant again, achieving removal of 75% of the liquid and leaving less than two inches in the first pond. After that we used a shop-vac to pump the remaining cyanobacteria into a large conical tank and allowed further settling. This helped, but it became clear that to harvest a dry cyanobacterial bio-fertilizer, we would probably have to add an additional filtration step.
Initial testing of different filtering fabrics showed that natural woven fabrics were capable of greater recovery of cyanobacteria than synthetic or knit fabrics (Fig. 13). In subsequent testing, we determined that four layers of heavy-weight 100% cotton would allow for the best recovery; however, when tested in the field, the filtration process was too slow for it to be a viable harvesting option.
Next, we tried centrifugation using a hand-crank cream separator. It did not work well, so we sped up the centrifugation by hooking it up to a motor. That resulted in a clear liquid stream and a thick green slime. However, the separator became filled with the cyanobacterial slime and had to be stopped and taken apart every few minutes to clean it out. This was clearly not a practical approach!
When we harvested the second prototype pond, we allowed a longer settling time of 36 hours and were able to pump off more than 60% of the liquid. Then we allowed another 24 hours of settling and were able to pump off all but one inch of the remainder. The rest went to the conical tank for further settling followed by filtering through a single-layer of cloth. The cloth clogged repeatedly and had to be scraped to collect the bio-fertilizer.
When we harvested the third prototype pond, we used 36 hours of settling followed by pumping down to one inch, and then allowed the cyanobacteria to air-dry in place (Fig. 14). We hooked up a fan to speed the air-drying process, and in about 48 hours, we were able to sweep up the cyanobacterial bio-fertilizer from the pond liner. This procedure was the most successful and will be utilized in future prototype evaluations.
Settling by turning off the paddlewheel and pumping off the nutrient solution into another pond for re-use, followed by a few days of air-drying, is the best system for harvesting the cyanobacteria in solid form. Alternatively, the cyanobacteria can be utilized directly in liquid form through fertigation, thus simplifying the harvesting process.
Milestone 3.2. Compare rates of N mineralization of cyanobacteria fertilizer to other organic fertilizers
Nitrogen mineralization was significantly greater from fish emulsion than from liquid cyanobacteria, and was significantly greater from solid cyanobacteria than from composted manure (Fig. 15). Overall, the cyanobacterial bio-fertilizers provide an option that is intermediate between the quickly available fish emulsion and slow release compost and could be an important tool in managing soil fertility on organic farms.
Milestone 3.3. Determine how cyanobacterial fertilizer application affects yield and quality of lettuce compared to other organic fertilizer applications
For fresh yield of lettuce, soil-applied fish emulsion recorded significantly higher yield (117g) compared to control and the rest of the treatments when applied at 50 lb N acre-1 (Fig. 16). At 100 lb N acre-1, soil-applied fish emulsion recorded significantly higher yield (146 g) compared to soil applied liquid cyanobacteria (111 g) and soil-applied solid cyanobacteria (59 g) on clayey soil. Overall, the solid cyanobacteria yielded the same as the compost applied at the same N rate, and the liquid cyanobacteria yielded a little less than the fish emulsion but more than the compost or solid cyanobacteria applications (Fig. 17). Therefore, not only is the liquid cyanobacterial bio-fertilizer easier to harvest, but it also results in a better plant response compared to the solid cyanobacteria.
Objective 4: Inform the bioreactor design and utilization of bio-fertilizers through consultations with farmers
Milestone 4.1. Consult with farmers to solicit input to guide prototype design
To target our research to stakeholder needs, and, therefore, be in a better position to motivate on-the-ground producer adoption, we advanced our understanding of producer needs and opinions beyond our Farmer Advisory Group by surveying a broader base of farmers. Attendees at both the Colorado Big and Small Conference and New Mexico Organic Conference in 2011 were surveyed regarding their soil fertility practices. Fifty-three farmers were surveyed representing CO (20), NM (28) and AZ (5). Farm size ranged from less than one acre to 1,900 acres. Thirteen farms were certified organic, 26 considered themselves to be organic but were not certified, 10 were conventional, one was biodynamic and three gave no answer. All but one of the farms were irrigated and could use a portion of their water to both grow, and potentially deliver, a new nitrogen fertilizer to their fields. In addition, respondents use a variety of N fertilizers: 43% use compost, 40% manure, 26% cover crops/green manures, 19% fish emulsion, 11% kelp, 9% blood meal and 4% worm castings. On average, farmers spent $110/acre on fertilizers; however, the range was quite broad, from zero to $1,000/acre. Prices per pound of N are variable, with bat guano as high as $50/lb N, and seabird guano and blood and feather meals at <$10/lb N. We will use this information to ensure that cyanobacterial bio-fertilizer is both effective and affordable for farmers.
Farmers also described the problems they face in meeting the nutrient needs of their crops with soil amendments and fertilizers. Responses included: inadequate nutrient contents, too expensive, inconsistent quality, high application costs (equipment and labor), soil salinization concerns, inadequate organic matter content, bulkiness of low nutrient materials (e.g., manures), dispersal of weed seed in manure, odor, sustainability (e.g., fish emulsion) and inadequate composting space. On a scale of 1-10, farmers were moderately satisfied with their soil fertility practices (6.8) and interested in new approaches (7.8). Surveyed farmers (91%) strongly preferred producing their own fertilizers over purchasing them. These results directly influenced the research team in the development of an on-farm production system for cyanobacterial bio-fertilizer.
Milestone 4.2. Construct and test an open pond system at Happy Heart Farm (Summer 2011)
The initial on-farm testing in 2011 was very important in guiding our next steps. We learned that outdoor production ponds could be challenging due to the high light intensity in Colorado. This finding resulted in the construction of both pilot-scale (50-gallon) and prototype-scale (625-gallon) ponds under plastic fabrics to reduce light intensity.
Secondly, we determined that our aeration of the Happy Heart ponds was inadequate, and that there was a potential CO2 deficiency (demonstrated by pH levels above 10) due in part to that aeration system (and also due to high photosynthesis rates when the cyanobacteria are growing well). Therefore, in both the pilot and prototype scale raceways used in 2012, we added a center divider and more rigorous aeration and mixing processes (pumps in the 50-gallon raceways and paddlewheels in the 625-gallon raceways).
Milestone 4.3. Construct and operate pilot-scale raceways at CSU’s Horticultural Research Farm (Summer 2012)
Fifteen 50-gallon raceways were built as described above and placed inside of the hoop house at CSU’s Horticulture Farm. The purpose of the pilot-scale raceways was to enable replicated research on various production parameters which would not be possible with the large 625-gallon prototype raceways.
Milestone 4.4. Determine if culture depth affects on cyanobacterial productivity
Both the optical density (growth) and the net N fixed by the cultures grown at the two depths were significantly higher in the 8 inch depth as compared to the 10 inch depth (Fig. 18). This means that the cyanobacteria are more productive when grown with fewer resources (water and nutrients).
Milestone 4.5. Evaluate carbonate addition as a means to improve cyanobacterial growth
The net N produced by the three treatments was significantly influenced by treatment. The high dosage of carbonate had a negative effect on both growth and net N fixed (Fig 19). The low dosage of carbonate did not offer a statistically significant advantage in terms of growth or N-fixation. Further study is needed to determine if the addition of carbonate, perhaps as bubbled CO2, would be more beneficial to cyanobacteria than a chemical source.
Milestone 4.6. Evaluate hoop house covers as a means to filter out harmful UV rays and prevent photo-inhibition
Preliminary results indicate that some type of greenhouse film enhances growth and N-fixation (Fig 20). However, data and statistical analysis are still on-going. We believe that the size of the raceway and the method of aeration can affect the rate of photosynthesis, growth and N-fixation; therefore in future studies, we plan to conduct more tests on the role of covers on large-scale raceways.
Milestone 4.7. Construct and operate prototype raceways at Thin Air Nitrogen Solutions’ headquarters (Summer 2012)
We successfully produced cyanobacterial bio-fertilizer in 625-gallon raceways (Fig. 21). We experimented with the length of time from seeding a pond to harvesting the bio-fertilizer and concluded that 12 to 14 days is optimum. In one cycle, we went longer (17 days), but the culture declined during that time. It is interesting that this optimum harvest time is the same whether in laboratory flasks or prototype raceways.
We also experimented with different paddlewheel designs and concluded that a six-paddle design achieved the necessary flow rate and reduced splash and energy requirements compared to an eight-paddle design. In addition, we learned that it was not necessary or efficacious to run the paddlewheel at night when cyanobacteria are not photosynthesizing; this finding reduces the power requirements in half, an important finding to achieve economical and practical on-farm fertilizer production.
Milestone 4.8. Estimate the land and water use requirements for cyanobacterial bio-fertilizer production as compared to cover crops
Our goal is that less than 0.1 acre of ponds would be required to produce 50 lb N fertilizer annually, an average amount of N needed for one acre of vegetable production. However, to date, our production system requires 0.2 acre of ponds per acre of vegetables. Therefore, we are continuing to work on improving cyanobacterial growth and productivity. When leguminous cover crops are used to provide N, land is required in a 1:1 ratio.
We have calculated the N production in lb N per 100,000 gal of water use for cover crops and for solid cyanobacteria (Table 3). Cyanobacteria are more water efficient than most commonly-grown cover crops. If cyanobacterial bio-fertilizer is applied in liquid form, the water is being used for irrigation, and hence there is no additional water consumption except for a minor amount of evaporative loss.
Objective 5: Develop educational materials providing information on the production and utilization of cyanobacteria-based N fertilizer and disseminate that information to farmers and agricultural professional through a variety of means
Milestone 5.1. Develop educational materials on the production and utilization of cyanobacteria-based N fertilizer
An instruction manual for the construction of raceways has been finalized and is attached to this report (entitled “Building a Raceway for Cyanobacterial Bio-fertilizer Production”). In addition, an article was published about our work in the Colorado State University College of Agricultural Sciences magazine (http://www.agsci.colostate.edu/news/Newsletter_1212/FoodforThought-1212.pdf). Although we have made a lot of progress in developing a protocol for cyanobacterial bio-fertilizer production and optimizing the best ways to use the bio-fertilizer, we felt that it was premature to develop educational materials on production and utilization. We look forward to developing those after completing some additional research.
Milestone 5.2. Disseminate information to farmers and agricultural professionals through a variety of means
In addition to the many scientific presentations given at the yearly American Society of Agronomy meetings (See PUBLICATIONS AND OUTREACH), we began reaching out to the broader agricultural community. Our progress and research has been shared regularly through our website (www.thinairnitrogen.com) and the popular social media outlets of facebook (www.facebook.com/ThinAirNitrogen) and twitter (www.twitter.com/thinairnitrogen).
Modern agriculture is highly dependent on fertilizer made through fossil energy-intensive industrial N fixation. As energy prices and input costs increase, so does the price of N fertilizer. In recent years, high demand has led to fertilizer shortages, yet even more N fertilizer is needed to meet the demands of our crowded and hungry world.
Biological nitrogen fixation by cyanobacteria has the potential to supply N to crops while reducing input costs and increasing the fossil energy efficiency of N fixation. Cyanobacteria use light energy to fix carbon and N from the atmosphere and can, therefore, produce biologically-available N without other energy inputs such as natural gas. A distributed, on-farm or local, community-scale N fixation system will provide additional benefits including reduced transport, decreased emissions, lower input costs, diversified income for farmers and employment opportunities in rural communities. This type of system would initially lend itself to small- and mid-size farms growing specialized, high-value crops. Cyanobacterial bio-fertilizer also has potential uses in certified organic production, which is growing rapidly, driven by strong consumer demand. This segment of the market is expected to continue to grow and represents a unique potential application for cyanobacterial bio-fertilizer.
In the short-term of this three-yearproject, we have developed an increased understanding and awareness of production and utilization of cyanobacterial bio-fertilizer as an alternative. In addition, we have developed the skill set necessary to produce and utilize cyanobacterial bio-fertilizer in both scientific and farming communities.
In the medium-term, we have begun working with organic and conventional fertilizer certifiers to achieve regulatory clearance for the use of cyanobacterial bio-fertilizer in organic and conventional farming systems. We are also developing improved decision-making abilities regarding cyanobacterial bio-fertilizer production and utilization.
In the long-term in the years to come, we aim for reduced fossil fuel use and greenhouse gas emissions from fertilizer manufacturing and the expansion of rural jobs, a reduced-cost on-farm fertilizer source and alternative income opportunities. A great opportunity exists to provide multiple benefits to small producers, rural communities, and United States agriculture by commercializing an on-farm biological nitrogen fixation system and optimizing bio-fertilizer utilization methods.
Research Outcomes
Education and Outreach
Participation Summary:
Several oral and poster presentations have been made at the American Society of Agronomy Meetings over the past three years. The references and links to these publications are as follows:
Barminski, R., H. Storteboom, and Y. Yang, S. Dominick, K. Athey, H. Wang, M. Stromberger, and J.G. Davis. Development of an organically certified growth-medium for cyanobacteria. American Society of Agronomy Annual Meeting, October 2012.
http://scisoc.confex.com/scisoc/2012am/webprogram/Paper72444.html
Davis, J.G., H. Storteboom, Y. Yang, R. Barminski, A. Sukor, H. Wang, F. Stonaker and M.S. Massey. Developing an on-farm bio-fertilizer production system using cyanobacteria. American Society of Agronomy Annual Meeting, October 2012.
http://scisoc.confex.com/scisoc/2012am/webprogram/Paper71281.html
Sukor, A., H. Storteboom, M.E. Stromberger, and J.G. Davis. Nitrogen mineralization potential of cyanobacterial fertilizers compared to traditional organic fertilizers applied to clayey and sandy soils. American Society of Agronomy Annual Meeting, October 2012. http://scisoc.confex.com/scisoc/2012am/webprogram/Paper72219.html
Wolde-meskel, E., H. Storteboom, S. Yigrem, Y. Yang, R. Barminski, S. Dominick, M.S. Massey and J.G. Davis. 2011. On-Farm cyanobacterial nitrogen fertilizer production in Ethiopia. American Society of Agronomy Annual Meeting, October 2011. http://scisoc.confex.com/scisoc/2011am/webprogram/Paper65955.html
Storteboom, H., R. Barminski, E. Wolde-meskel, M. Massey and J.G. Davis. 2010. Exploring the potential for cyanobacterial nitrogen fertilizer to improve soil fertility in Ethiopia. American Society of Agronomy Annual Meeting, October 2010. http://scisoc.confex.com/scisoc/2010am/webprogram/Paper58501.html
In addition to these presentations, two publications have resulted from this project to date:
Olsen, N. 2012. Nitrogen from thin air. Food for Thought. Fall 2012. Colorado State University College of Agricultural Sciences. pp. 6-7. http://www.agsci.colostate.edu/news/Newsletter_1212/FoodforThought-1212.pdf
Swanson, L.L., and J.G. Davis. 2012. Building a Raceway for Cyanobacterial Bio-fertilizer Production. 15 pp.
Additionally, Arina Sukor recently defended her M.S. thesis, entitled “Nitrogen Mineralization and Nitrogen Use Efficiency: Effects of Cyanobacterial Fertilizers Compared to Traditional Organic Fertilizers on Growth and Yield of Lettuce (Lactuca sativa).” This thesis is expected to result in two publications: one reporting the mineralization data of the soil incubation study and the other describing the greenhouse fertilizer trial. These two manuscripts will be prepared for submission in early 2013 to Biology and Fertility of Soils and Plant and Soil, respectively.
Other outreach activities are described under Objective 5 in previous sections of this report.
Education and Outreach Outcomes
Areas needing additional study
In the future, we plan to further optimize the yield and efficiency of the on-farm bio-fertilizer production system, keying in on the parameters of CO2 addition via bubbling and aeration strategies. We intend to focus on large-scale on-farm production in future studies and, specifically, on cost minimization strategies. In addition, we plan to expand the cyanobacterial bio-fertilizer utilization studies from the greenhouse to on-farm irrigated vegetable systems in order to optimize bio-fertilizer utilization within typical cropping systems in the region. It is critical that we quantify the costs and benefits of on-farm production and utilization of bio-fertilizer in order to optimize economic returns for farmers. In addition, we are very interested in determining the carbon footprint of cyanobacterial bio-fertilizer compared to other methods of fertilization, in both organic and conventional production systems.