API: In-Soil Hatch Rates of Commercially Marketable Earthworm Cocoons August 30, 2005
(C) Table of Contents
(A) Cover Sheet (CSREES-667) 1
(B) Project Summary (CSREES-668) 2
(C) Table of Contents (not numbered)
(D) Response to Previous Review (not numbered)
(E) Technical Content 3
(1) Identification and Significance of the Problem or Opportunity 3
(2) Background and Rationale 3
(3) Relationships with Research and Development 4
(4) Technical Objectives 6
(5) Work Plan 6
(6) Related Research and Development 11
(7) References 14
(F) Key Personnel and Bibliography 17
John D. Reep, President Advanced Prairie, Inc. 18
William Kreitzer, Chief Technical Officer Advanced Prairie, Inc. 18
(G) Facilities and Equipment 19
Advanced Prairie, Inc 19
Soil Ecology Laboratory at The Ohio State University 19
(H) Outside Services 19
Professor Clive A. Edwards 20
Dr. Norman Q. Arancon 20
(I) Satisfying the Public Interest 21
(J) Potential Post Application 22
(K) Current and Pending Support 23
(L) Budget 24
Form CSREES-2004 24
Budget Narrative 25
(M) Documentation of Multiple Phase II Awards 26
(N) Certifications 26
(O) Assurance Statements [CSREES-2008] 27
(P) National Environmental Policy Act Exclusions Form [CSREES-2006] 28
Letters of Agreement from Consultants 29
Letter from Clive Edwards 29
Letter from Norman Arancon 30
Letter of Availability of OSU Facilities 31
(D) Response to Previous Review
We are grateful for the thoughtful and detailed comments from our previous proposal. Key, general differences from the previous Phase II proposal and this one appear below; other more specific recommendations have been incorporated throughout the text.
· The research plan for this proposal eliminates lifecycle studies, inoculation techniques, and effects of earthworms on soil and crop yield. As many reviewers noted, these types of studies exceed the timeframe of the SBIR projects, and the existing literature already has demonstrated that earthworms are invaluable elements in healthy soils. Instead, the proposed research has been simplified for Phase I, and the overarching questions for both Phase I and Phase II are 1) whether introducing earthworms into the soil increases the earthworm population and 2) what factors have the greatest impact on earthworm population.
· We have made sure to include no-treatment control groups in all of our experiments, and we provide greater detail about statistical techniques to be used.
· In response to concerns about breeding rate, cocoon harvesting, and deep burrowing (which presents problems for confined-space research) of Lumbricus terrestris, we have removed this species from the Phase I proposal. Many reviewers recognize the beneficial characteristics of this soil-turning, deep-burrowing species, however, so we plan to include it in the Phase II studies.
· We do not propose to find earthworm-free fields, but will select fields with low earthworm population as a result of previous tillage.
· We have responded to concern about being able to identify inoculated earthworms from populations that may already exist in the soil in three ways. First, the mesh cores we propose will prevent existing worms from interfering with initial hatch counts. Second, we will use species not normally found in Illinois soils but present in North America and non-injurious to the environment Third, the ability to distinguish recently introduced earthworms is not critical to the project’s success since we are comparing the total earthworm population of the test and control groups.
· As researchers we must be sensitive to the potential environmental impact of non-native species. We note that “non-native” is hard to define in this case since glaciation has left fields wormless, and most earthworm species in the central plains of the United States were subsequently introduced from Europe. For our work over more than a decade we have chosen species that are not common in Central Illinois but populate other areas of the continent without any known damaging effects to existing worm populations, to soil quality, or to crop yields.
· Market projections have been adjusted in two ways. First, our estimates for the agricultural market are lower. Second, we recognize, as did many reviewers, additional potential markets including forestry and animal waste treatment.
· We have duplicated the roles of the personnel from the Budget Narrative in the Key Personnel and Outside Services sections.
· We include more recent authorities to our bibliography and discussion of the state of the art.
· Regarding page count, we note to reviewers that not all of the pages of the proposal are included in the 25-page limit, only those pages through section L, Budget and Budget Narrative. The Table of Contents and this page are excluded from the page count.
API: In-Soil Hatch Rates of Commercially Marketable Earthworm Cocoons August 30, 2005
(E) Technical Content
(1) Identification and Significance of the Problem or Opportunity
Earthworms improve soil quality, reduce costs, and increase yields
There is an abundant literature attesting to the beneficial effects of earthworms in the soil [Lavelle and Martin, 1992; Villenave et al., 1999; Brown et. al, 2000; Pashanasi et al., 1992]. Earthworms improve overall soil structure, form water-stable aggregates, and provide channels for root growth and improve aeration, porosity, drainage and soil moisture retention both of which minimize surface water erosion. In no-till soils, which now represent a large proportion (over 50% of soybeans) of agricultural crop soils, earthworms perform the essential function of taking crop residues down into the soil, breaking them down and recycling the nutrients they contain [Desjardins et al., 2003; Caravaca et al., 2005]. In addition they release valuable nutrients into the soil [Edwards, 2004] thereby reducing the need for chemical supplements. Furthermore, they are required for optimal soil microbial activity [Schindler-Wessels et al., 1997; Bohlen et al., 2002, Scheu, 1992; Daniel and Anderson, 1992]. Most of these activities are by lumbricid earthworms that were introduced into the U.S. from Europe and elsewhere. However, not all agricultural soils in the U.S. have healthy populations of these earthworms, which spread only slowly between sites and regions by natural mechanisms. Extensive tillage has effectively annihilated earthworm populations in many soils. Introduction of earthworms into sites that are currently lacking or deficient in earthworms has enormous potential for increasing the germination growth and yields of crops.
Better techniques can bring earthworms to agriculture and other industries
Advanced Prairie, Inc (API, until recently Advanced Biotechnology, Inc.) is developing commercially available methods of introducing earthworms into agricultural and other sites. One technique, encapsulation, involves breeding earthworms in organic matter, such as peat or separated animal manures or soils; separating the earthworm cocoons from the breeding medium; and encapsulating them with a patented-by-API, durable, but water-soluble coating. The encapsulated cocoons can be produced in similar sizes to corn or soybean seeds. This provides the commercial potential of introducing them into farm lands using a seed planter or standard soil-sampling equipment. Encapsulated cocoons are relatively easy to count and handle, thus facilitating shipping and other inoculation protocols including hand-introduction and introduction during soil sampling. We have demonstrated in a previous SBIR Phase I proposal that encapsulating has no negative effect on hatch rate in laboratory conditions, and they have a shelf life of at least five months. API is also beginning to experiment with liquid storage, shipping, and delivery of earthworm cocoons. This approach would open the door to earthworm inoculation during applications of liquid additives to the soil.
Over time we will develop a system for soil and residue management using earthworms and taking into account various factors such as soil type, moisture, temperature, additives, residue, intended use of the land, and past and future farming practices. The marketing could be on a large-scale and sold to farmers across the country, eventually from sub-production stations. The proposed research will pave the way to an improved system of soil management that supports improved health and conservation of the soil (e.g., no-till practices) in a manner that is economically advantageous to the farmer. A wide variety of additional industries also stand to benefit from improve knowledge and handling of earthworms. These additional industries include home gardening, land reclamation, forestry, waste recycling, and more.
(2) Background and Rationale
Farming practices have a great influence on earthworm populations. Extensive tillage alters the soil structure and interrupts lifecycle of the earthworm and population dynamics between earthworm species [Shuster et al., 2003; Springett et al., 1992; Lavelle et al., 1994]. Other research on both farmland and vermicomposting points to additional key factors, especially moisture content of soil, temperature, soil type, organic matter in the soil [Shaw and Pawluk 1986; Shuster et al., 2001 & 2003] and species of earthworm [Shuster et al., 2003, Butt, 1999]. We therefore propose to study the effects of these factors on earthworm populations. There are many additional factors to consider as well, including inoculation techniques, pH, surface texture, soil structure attributes such as surface texture and drainage class, type of crop being grown, interaction of various earthworm species, and more. In Phase I we will focus on the basic factors of moisture content, species, organic matter in soil, and whether or not cocoons have been encapsulated. In Phase II we will expand the experiments and address additional factors.
Our previous Phase I research allowed us to demonstrate the viability (greater than 70% hatch rate for both encapsulated and unencapsulated cocoons) and shelf life (at least 5 months) of our patented cocoon encapsulations. This proposal emphasizes the hatch rate and survival factors of earthworms in soil conditions using three experiments. The overall project (Phase I and Phase II combined) addresses two basic questions. Simply put:
1) Does the introduction of earthworm cocoons into the soil result in an increase in earthworm population?
2) What factors have the greatest impact on earthworm population?
Figure 1. Basic Research Questions, Phase I and Phase II.
The proposed Phase I effort consists of three experiments design to address these questions.
The proposed Outdoor Hatch Rate Experiment will assess hatch rates in natural, uncontrolled, real-life field conditions. We will place earthworm cocoons into a removable double-mesh core, which we will then place in an operating farm field and remove for inspection periodically during the growing season to determine hatch rate. We expect to observe a hatch rate of 50% from the mesh core observations.
Determining the effects of various factors on earthworm population requires the ability to control those factors. The proposed Indoor Hatch Rate Experiment, therefore, consists of a large number of plastic bins containing the soil from the outdoor plots, but placed indoors in controlled conditions. Each bin receives a specific treatment of moisture (high or low), organic matter (3% or 10%), earthworm species (Lumbricus rubellus or Dendrobaena veneta), and encapsulation (yes or no). The controlled conditions will allow us to assess not only worm count but size and maturity as well. We expect to observe a hatch rate of 60% for optimal treatments in these controlled conditions.
The Outdoor Population-Over-Time Experiment will provide baseline data regarding long-term earthworm population in the soil. Results of this experiment will have limited import in the 8 months of Phase I, but will yield much more significant results if continued as planned into Phase II.
(3) Relationships with Research and Development
Previous Phase I results: viability and shelf-life of encapsulations
Our previous Phase I work serves as a foundation for the proposed effort in having demonstrated the shipping, storage, and planting viability of encapsulated earthworm cocoons. Here is a brief summary of the previous Phase I results:
1) In controlled situations, unencapsulated cocoons had a hatching rate of 73%, which is acceptable for commercial development.
2) Average hatching rate was 70% to 76% after encapsulation; therefore encapsulation does not reduce hatching rate.
3) There were no significant differences in hatching rates among 5, 9, 13, 17, or 21-week storage periods.
4) 79% of cocoons (all encapsulated) recovered from soil inoculations showed evidence of hatching.
5) Encapsulated cocoons survived vacuum-type sowing equipment but not mechanical sowing equipment.
Result 4) revealed the likelihood of cocoon hatching in real-life field conditions, but an improved research design (i.e., the one we are now proposing) is needed for more conclusive results. Result 5) brought to light problems with mechanical planting of encapsulated cocoons, which has encouraged us to consider alternative inoculation methods.
Proposed Phase I foundation for Phase II
Hatch rates in soil. In the relatively short time span of Phase I it is impossible to determine population growth in a real-life field environment. Therefore, we have designed the Phase I proposal to assess the first stage in increased population, that is, hatch rates in both real-life (outdoor) and controlled (indoor) conditions. Only if hatch rates are promising is it worth investigating population growth in greater detail.
Factors affecting worm populations. We have structured the Phase I research to consider a few of the most basic factors: moisture, organic matter content, earthworm species, and encapsulation. For each of these variables, we will have only two values. The results will influence the direction of research in Phase II. If, as we expect, there is no difference between encapsulated and unencapsulated treatments, we may not investigate that area further in Phase II. If, however, low moisture kills the entire population while the high moisture group does well, we may expand that aspect of the research to include a variety of moistures so that we may determine the optimal moisture for effective earthworm propagation.
Furthermore, depending on Phase I results and the state of research at the time, we may be able to include additional factors in our Phase II research. These may include temperature, inoculation protocols and densities, pH, surface texture, soil structure attributes such as surface texture and drainage class, type of crop being grown, or others.
Population rates in soil. Also, realizing the need for longer-term experimentation, we will initiate an experiment in Phase I that is intended to continue through Phase II if funded, at which point more meaningful results will be possible. In Phase II, we plan to initiate a second, similar, longer-range outdoor experiment based on our experience and knowledge from Phase I.
(a) Technical, economic, social, and other benefits to the nation
Earthworms improve soil quality and encourage farm management practices that build up rather than erode the soil. By providing an economically advantageous way for farmers to increase their earthworm populations, we will be helping build one the country’s most fundamental real assets: the very land itself. The Conservation Security Program (CSP) recognizes the importance of promoting such practices by offering conservation payments ranging from $15.90 per acre to $42.40 per acre [NRCS, 2004]. The alarming rise in gas prices is also driving farmers toward practices that use less fuel. With improved means of earthworm inoculation, farmers can rely more on nature’s tillers and less on petroleum-consuming machines for cultivation.