Patent Application
20170332644
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Feb. 25, 2016, is named Termites_SL.txt and is 1,672 bytes in size.
The present invention embraces a biological system that can be used as bio-repair, insecticide, termiticide and bio-additive. This invention provides a biomaterial based in a bacteria that produces cellulose from sugar derivate. The biological system increases the resistance and flexural strength and also has an insecticide effect.
Soil termites, also known as subterranean termites, are the most destructive termites in the United States. These insects, and other related insects can cause a lot of damage and should be controlled upon discovery.
Hundreds of thousands of termites in a colony well-organized among workers, soldiers and Queens tunnel 24 hours a day through soil and into the wooden frames of houses, fences and buildings providing new sources of cellulose for the entire colony.
If left untreated, termites can destroy the entire value of a home. According to the National Pest Management Association, termites are costing Americans more than $5 billion in damage each year. This is more than fire and floods combined. Destruction is boundless, because any home, regardless of design, can offer the ideal combination of heat, moisture and food for termites. In addition, many plans for housing are not covered by insurance for such damages. Without insurance protection, serious problems in selling a house may arise. Many lenders require a termite bond before lending money to homebuyers.
The present invention provides for the first time a biological system which provides the dual function of killing termites and other wood damaging insects while also producing a by-product substance having the capability of repairing damage by termites and other insects to wood and related cellulosic products.
In a particular embodiment of the present invention, a biological system, toxic to termites, is provided which produces a means by which damage caused by termites is repaired, said means comprising a by-product produced by a modification of the bacteria of the genus Gluconacetobacter. Preferably, the biological system is in the form of toxic bait.
In another embodiment of the present invention, a process is provided for killing termites and other wood damaging insects and for repairing damage to wood and related cellulosic products caused by termites comprising the steps of:
The by-product ooze is toxic to termites and other insects and non-toxic to humans.
A Gluconacetobacter bacterium from an apple was isolated. First, the apple was washed with distillated water and then it was crashed in 25 mL of sterile distillated water as well. The extract produced was incubated for 10 days at room temperature for the bacteria production. After this incubation, serial dilutions of the culture were done on LB agar plates and were incubated at 27-Celsius degrees for 2 days. The most diluted colonies corresponding to the white colored colonies were selected and analyzed by 16srRNA-PCR procedure using F8 forward primer (AGAGTTTGATCCTGGCTCAG) and R1492 reverse primer (GGTTACCTTGTTACGACTT) (Weisburg et al., 1991; Baker et al., 2003). The sequence obtained (
An evaluation of cellulose yield was done. G. malus was cultured in liquid mediums using different nutrient sources (glucose and sugar derivate) for 2 weeks at 27 Celsius-degrees without shaking (static culture) to produce cellulose. A cellulose yield of 128.8 g/L, 119 g/L, 111.9 g/L, 99.8 g/L and 94.9 g/L was produced by G. malus. From glucose, sugar beet derivates 1, 2, 3 and 4, respectively (shown in Table 1).
Biological System as Bio-Repair
To test the biological system as bio-repair, physical properties of these celluloses were assayed by doing a Dynamic Mechanic Analysis (DMA). Resistance and mechanical strength of cellulose are five times more in comparison with wood-cellulose.
Furthermore, electronic microphotographs shows how this biological system repairs and reconstitutes the damaged wood starting on the initial hours from its application to 8 days (
In USA there are 79.000.000 homes affected by termites. This biological product has a lot of advantages: is not toxic to the human, doesn't damage the environment and is a very effective as bio-repair product. It can be used as bio-repair on damaged wood's structures of homes caused by termites and other insects.
Biological System as Insecticide
To evaluate the insecticidal effect, an aliquot of the supernatant from bacterial cellulose cultures was settled on a plate with a coleopteran to emulate the natural environmental conditions. When the coleopteran reaches the supernatant, the insect dies. Contrary to when the insect eats the bacterial cellulose. These assays were performed using Brevipalpus chilensis (a mite that infects vine plants). The SN was added to the privet leaves, not directly to the insect. After 7 days of post-treatment, we measured the percentage of mortality. A 92% of mortality was shown using the SN of the bacterial cellulose culture (
Furthermore, a similar assay was performed using SN from bacterial cellulose cultures with different nutrient source. We determined that the different SNs were effective (
In the vinifera vine sprouting in early may cause tissue necrosis and death cause of outbreaks and also, dehydration rachis, pedicels and bronzing of leaves.
On the other hand, we test the insecticidal effect using 9 nematodes (Table 2). Nematodes-based termite s are phytoparasitic of a wide of vegetable cultivation like tomato and also vine plants. In this assay we use the SN (filtrated or not) of the liquid culture using Sugar Beet Derivate 1 as carbon source. All the insects die using the SN. Water added to the nematodes was used as negative control. The SN is effective against different types of insects.
This biological product can be used as insecticide, mostly important as a termiticide to protect the wood structures from termites while this product is repairing the damaged wood as mentioned before. Also, can be used in the agriculture, mainly in the countries that are susceptible to insect damage by mites and other insects. This new biological compound shows a great potential to control the damage of Brevipalpus chilensis in our Vitis vinifera. The actually acaricides are not sufficient effective to control this mite.
Biological System as Bio-Additive
The biological compound can be used in the fabrication of added-resistance laminated and agglomerated wood panels. Plywood increases over 5 times its resistance to flexion.
In 2011, the International Agency for Research on Cancer (IARC) classifies the formaldehyde as carcinogenic agent, based on epidemiologic studies of cancer in animals and humans. The new biological compound can replace the formaldehyde to a polymer that catalyzes the dry and reduces the use of matchwood for the Eco-wood formulation, using materials that aren't toxic on humans.
Below, a report to determine the tensile strengths of wood veneers treated with the composition of the present invention and to analyze any possible influences treatment on tensile strength is included.
Report on the Tensile Strength of Wood Veneers Treated with Wood Eagleone
The following report by the Laboratory of Wood Technology (Laboratorio de Tecnologia de la Madera) of the Technical School of Rural Engineering (Escuela Técnica Superior de Ingenieros de Montes) at the Universidad Politécnica de Madrid.
Testing methods were created in compliance with the following regulations:
Wood specimens were prepared between Apr. 13, and 15, 2015, in the Laboratory of Wood Technology (Laboratorio de Tecnologia de la Madera) of the Technical School of Rural Engineering (Escuela Técnica Superior de Ingenieros [E.T.S.I.] de Montes). Specimens were prepared with pinewood veneer originating from the southeast United States. Overall, 470 specimens (150×23×0.65 mm) were prepared, of which, 110 (i.e. 100 specimens for assays and 10 as reserves) remained at the Laboratory of Wood Technology as a reference standard (treatment 0).
As requested by the client, the remaining samples (360 specimens) were sent on May 19th to the company Creative BioScience at Avda. del Valle Norte 857, Of 102, Ciudad Empresarial, Santiago, Chile. The specimens were divided into three groups for posterior testing with three distinct WOOD EAGLEONE treatments (100 specimens per assay and 20 reserve specimens for each treatment). To best homogenize variations in the wood, specimens were randomly distributed among the treatment groups.
The specimens sent to Creative BioScience were initially going to be subjected to three distinct WOOD EAGLEONE treatment assays, but five treatments were finally test. According to information provided by Mrs. Adda Mora Foppiano, the number of specimens used for each treatment assay was as follows:
On Sep. 24, 2015, the samples treated by Creative BioScience were received by the Laboratory of Wood Technology.
Before assays, the specimens were conditioned until reaching a constant mass in a climatic chamber at 20±2° C. and 65±5% relative humidity.
Prior to tensile strength tests, specimen densities (p) were calculated according to the following formula:
The tensile strengths of the veneer specimens were determined with a universal testing machine (Class 0.5 load cell, 5 kN capacity). (See
The tensile strength (k) of each specimen was calculated according to the following formula:
The mean and standard deviation were calculated for all results.
An analysis of variance (ANOVA) with a 95% confidence interval was used to detect possible significant differences between the applied treatments.
Prior to ANOVA assessments, data were tested for assumptions of normality, independence, and homoscedasticity against bias. Additionally, kurtosis was determined, and the Lavene's Test was applied.
The Grubbs' test was applied to all properties to detect outliers in the dataset. Each datum considered a statistical outlier was individually assessed to evaluate possible elimination from the dataset.
In cases where statistical differences were detected, confidence intervals were assessed to establish between which treatments such differences existed. These assessments were carried out using the Fisher's Least Significant Difference (LSD), Scheffé, and Bonferroni tests, thereby minimizing the effect of sample size.
Fisher's LSD test is the method that detects the most significant differences and is highly efficient in detecting real differences between means. However, this method is not very conservative and is not adequate for evaluating a high number of treatments, particularly since type I errors (i.e. rejection of the hypothesis when there are no significant differences) increase with the number of treatments.
In turn, the Scheffé test does not require an equilibrated design; in other words, this test does not need the same quantity of samples in each treatment, as in the present case. Furthermore, the Scheffé analysis is one of the most robust mean comparison tests and presents fewer type 1 errors.
Finally, the Bonferroni test is an adequate method when the number of comparisons is not very high. This method is more conservative than the Scheffé test and is better than Fisher's LSD test at controlling for type I errors.
For additional data confirmation, bootstrapping was used to obtain the confidence intervals for the tensile strength ratios of each treatment. This robust numerical method does not assume previous conditions of normality and homoscedasticity, which are required for the other performed analyses. The inclusion of the unit within the indicated confidence interval indicates that significant differences do not exist between the assessed strengths.
Descriptive statistical analyses, ANOVA, and confidence interval assessments were performed using the STATGRAPHICS Centurion XVI v.16.2.04 program. To obtain the confidence intervals of the tensile strength ratios, the MATLAB R2013b program was used. All statistical analyses were performed considering a 95% confidence interval.
All bias and kurtosis values were within the interval (−2, 2), corresponding to normal distribution. Therefore, the hypothesis that property data originate from a normal distribution cannot be discarded.
Each statistically determined outlier was individually assessed, and none presented technical reasons warranting elimination from the global dataset.
Since the p-value was greater than 0.05, no significant differences existed between the detected variances (95% confidence interval).
Since the p-value was less than 0.05, significant differences existed between the distinct sample treatments (95% confidence interval).
Considering a 95% confidence interval, significant differences did not exist between treatments 0, 1, and 2 or between treatments 3 and 4. However, significant differences did exist between treatment 5, treatment group [0, 1, and 2], and treatment group [3 and 4].
Significant differences existed between treatment 5 and the remaining treatments, as well as between treatments 0 and 3. However, no significant differences existed between the treatment group [1, 2, and 4] and treatment 0 or treatment 3.
Significant differences existed between treatment 5 and the remaining treatments, as well as between treatment 0 and treatment group [3 and 4]. However, significant differences did not exist between treatments 0, 1, and 2; between treatments 2, 3, and 4; or between treatments 1, 2, and 4.
The following table provides the tensile strength ratios obtained following each of the distinct treatments.
(0.87-0.96)
(0.88-0.96)
(0.80-0.87)
(0.88-0.96)
(0.89-0.96)
(0.80-0.87)
(0.90-0.99)
(0.90-0.99)
(8.82-0.90)
(1.04-1.14)
(1.04-1.14)
(1.01-1.11)
(0.86-0.95)
(1.05-1.13)
(1.04-1.13)
(1.01-1.11)
(0.87-0.94)
(1.15-1.26)
(1.15-1.26)
(1.11-1.23)
(1.05-1.16)
(1.06-1.16)
Significant differences were detected between treatment 5 and the remaining treatments, as well as between treatment group [0, 1, and 2] and treatment group [3 and 4]. However, no significant differences were detected between treatments 0, 1, and 2 or between treatments 3 and 4.
The obtain tensile strength data met assumptions of normality and equality of variance. Consequently, ANOVA-based methods were acceptable for studying the influence of treatments on the tensile strength of samples.
A precursory evaluation of the data would indicate the existence of three groups within the treatments. The first group would be comprised by treatments 0 (standard), 1, and 2. The second group would be comprised by treatments 3 and 4, and the third group would be exclusively comprised of treatment 5.
Nevertheless, extended evaluation reveals that such group delineations are not so clear. What can be confirmed is the existence of significant differences between treatments 0, 3, and 5, with all analyses (i.e. Fisher's LSD, Scheffé, Bonferroni, and tensile strength ratios) arriving to this same conclusion. These differences indicate increased tensile strengths of 9.47% between treatments 0 and 3, of 20.42% between treatments 0 and 5, and of 10.00% between treatments 3 and 5.
Similarly, significant differences were confirmed by all analyses (i.e. Fisher's LSD, Scheffé, Bonferroni, and tensile strength ratios) between treatments [1 and 5], [2 and 5], and [4 and 5]—The determined increases in tensile strength for these treatments were 19.80%, 16.86%, and 10.46%, respectively.
While differences existed between treatments 1 and 3, indicating the existence of distinct groups, these results must be interpreted with caution. Although the intervals for Fisher's LSD, Bonferroni, and tensile strength ratio tests showed significant differences, this result was not obtained by Scheffé interval analysis. In particular, the Scheffé test indicated that the upper confidence interval limit of treatment 1 (88.84 N/mm2) overlaps with the lower confidence interval limit of treatment 3 (88.23 N/mm3).
This same situation, albeit more pronounced, occurred between treatments 2 and 3, where significant differences were found by the Fisher's LSD and tensile strength ratio analyses but not by the confidence intervals obtained in the Bonferroni and Scheffé tests.
Regarding treatment 4, although significant differences were found with treatments 3 and 5, the same cannot be definitively said for the remaining treatments. On the one hand, confidence interval analysis for the Fisher's LSD and tensile strength ratio tests revealed significant differences with treatments 0, 1, 2, and 5. On the other hand, analysis of the Bonferroni confidence intervals only resulted in significant differences with treatments 0 and 5. Similarly, the Scheffé confidence intervals only showed significant differences with treatment 5.
The uncertainty caused by analysis-dependent variations in confidence interval overlapping, or lack thereof, between treatments could be due to a lack of data normality or an insufficient number of data points. As previously mentioned, the coefficients of skewness and kurtosis were within the interval expected for normal distribution. Considering this, it is more likely that above mentioned uncertainties are the result of insufficient data points, an issue that could have been aggravated by the lack of an equilibrated assay design (i.e. the number of assessed specimens varied between treatments).
This Continuation-In-Part application is based on U.S. Nonprovisional patent application Ser. No. 14/120,777, filed on Jun. 26, 2014, which is incorporated herein in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 14120777 | Jun 2014 | US |
| Child | 15288703 | US |
