Formulation Of Moringa Oleifera Seed Powder And Ferrous Sulfate Heptahydrate Into A Matrix To Control Coliform Bacteria And Odor In Poultry Manure

Formulation Of Moringa Oleifera Seed Powder And Ferrous Sulfate Heptahydrate Into A Matrix To Control Coliform Bacteria And Odor In Poultry Manure

Joseph M. Manu*, Jeffrey T. Barminas, Buba A. Aliyu, Sunday A. Osemeahon Department of Chemistry, Modibbo Adama University of Technology,

`P. M. B 2076, Yola, Nigeria

A blend was formulated from a mixture of Moringa Oleifera seed powder ( MOSP) and ferrous sulfate heptahydrate crystals, each at blend ratios of 2%, 5% and 10% via factorial experiment in order to optimize efficacy of the blends for odor inhibition. It involved the two components as factors vize: factor A (Ferrous sulfate) at three levels each i.e a1, a2, a3 representing 2%, 5% and 10% and factor B (moringa oleifera) also at three levels each i.e b1,b2, b3 representing 2%, 5%, and 10%. This is a 2×3 factorial experiment from which a total of 9 treatment combinations were obtained. The products were coded F1 F9 and each applied to a 300 g fixed weight of poultry waste. Their odor reduction potential was evaluated on weekly basis up to 5 weeks incubation period using an odor panel. Results showed significant differences in their odor reduction potentials (p > 0.05). F2, F5 and F7 were particularly impressive and gave overall odor reductions of 53.6%, 53.2% and 52.8%% respectively. It is concluded that the formulations suppressed manure odors at both short and long term incubation periods in contrast to most commercial products. Therefore they show promise of pre-land application use.

Postal address: Department of Chemistry,

Modibbo Adama University of Technology,

P. M. B 2076, Yola, Nigeria

Over the years, there has been increased participation in rearing of livestock both in the rural and urban centres. The ultimate is mainly food and income and manure for crop cultivation.

Livestock production and the waste generated can pose a threat to soil, water and air quality and to human health. Some of the more serious problems with livestock waste include nutrient enrichment of soil and water, emission of offensive odors and green house gases, as well as presence and transmission of pathogenic microorganisms (Vincent, 2002; varel et al, 1999). Livestock farming have continued unregulated particularly in the poultry sector, with little or no regards to the wide range of the adverse effects of these facilities. This is because the legal and regulatory frameworks required to impose compliance are reported to be weak and in most cases uncertain on the statutory responsibilities and duties of the government with regard to environmental management and protection to guaranty clean air (Ogunba, 2004).

The offensive odor is the immediate impact of livestock production on the human population and is partly the result of incomplete anaerobic decomposition of plant fibre and protein in stored manure (Spoelstra, 1980; Hammond, 1989). Results from studies of different researchers have characterized these odorous substances. A list of 150 different compounds in swine manure was presented (Spoelstra, 1980). Another list of 168 volatile compounds related to animal production was reported (ONiell and Phillips, 1992). Schiffman et al, (2001) also gave a list of 331 volatile substances sampled from lagoons and air at swine production facilities. About 35 t0 73 decomposition products from diary manure were equally reported (Sunesson et al, 2001, Raband et al, 2003, Filipy et al, 2006). The products reported have far reaching environmental, health and socioeconomic concerns.

In recent years, many treatments such as biogas production, anaerobi or aerobic waste conversion and solids separation have been available to farmers for managing livestock odors and manure. Other modern abatement technologies and additives that reduce odors from animal units have equally been developed and categorized into digestive agents, oxidizers, adsorbents masking agents and disinfectants (McCrory and Hobbs, 2001., Mackie et al, 1998., Ritter et al, 1975., Warburton et al, 1980).

While some of these methods have proven effective, particularly for short term odor control, the results of many have been mixed and generally unsatisfactory especially for long

term odor reduction. Their use may equally be limited by cost and /or operative expertise and unavailability of products (Ritter et al, 1989, Eileen et al, 2011). Besides having variable effects, no single treatment or amendment has been successful for short or long term odor control. Odor abatement technologies are often targeted towards other livestock systems, but limited data exist for poultry waste odor management (Ulmans et al, 2004). Therefore nuisance odor generation from poultry farms has continued unabated and is a global concern. Due to the health and environmental issues associated with odorous emissions from the highly proliferated poultry farms and waste storage facilities, continued efforts and search for effective, cheap and available materials for its control is an urgent imperative.

This work is therefore a report of the synergistic effects of FeSO4.7H2O crystals and Morringa Oleifera seed powder used to inhibit microbial activities and reduce odors from poultry waste. The short term effect of FeSO4.7H2O as pH modifier in livestock waste has been mentioned (More et al, 1996). Morringa Oleifera, a natural biopolymer, is also well reported for its broad based antimcrobial activities (Raheela et al, 2008), but never tested for inhibition of odor causing microbial activities in poultry waste odor control. This is expected to provide both short and long term solution to odor problems in the vicinity of poultry houses in the developing countries and hence ensuring clean and healthy environment.

Poultry fecal waste was collected from caged broilers grown on a modern farming skill acquisition center in Yola, Adamawa state of Nigeria, after being excreted over night. A clean 8 x 6ft nylon sheet was placed underneath the cages to avoid contamination of manure from the floor. This was carefully wrapped and transferred to the laboratory for further processing.

The biopolymer based matrix was formulated from a mixture of MOSP and ferrous sulfate heptahydrate crystals by physical blending. The materials were first oven dried according to standard methods, ground to fine powder and sieved using 200 mm mesh size. A factorial design

approach was used to optimize efficacy of the formulation of the biopolymer based matrix for odor inhibition. The strategy is to study the effects of ferrous sulfate hepta-hydrate and MOSP at different ratios as variables on odor inhibition. It involved the two components as factors vize: factor A (Ferrous sulfate) at three levels each i.e a1, a2, a3 representing 2%, 5% and 10% and factor B (MOSP) also at three levels each i.e b1,b2, b3 representing 2%, 5%, and 10% (Table 1). This is a 2×3 factorial experiment from which a total of 9 treatment combinations were obtained and were coded F1 F9.

Factors levels Treatment combinations

A a1 (2%) a1b1 a1b1 a2 (5%) a1 a1b2 a1b2

a3 (10%) a1b3 a1b3

a2b1 a1b1

a2 a2b2 a2b2

a2b3 a2b3

B b1 (2%) a3b1 a3b1 b 2 (5%) a3 a3b2 a3b2

b3 (10%) a3b3 a3b3

Quantity of waste used = 300 g

The two components and the various formulations obtained were added to about 300 g each of the poultry waste and thoroughly mixed together using a motorized mixer. This was transferred into 1-L capacity wide mouth glass jars each and sealed with covers underneath which was cotton swatches, as odor absorbents. A controlled jar with no addition was included. The jars were painted black o minimize the visual impact of manure during sampling and analysis. The jars were transferred into the sniffing room and incubated at room temperature. The contents of the flask were sampled at weeks 1, 2, 3, 4, and 5 for analysis.

The microbial load in the samples was estimated using method outlined by El-Jalil et al, 2008. Ten (10) grammes of each sample was blended in 90 cm3 of saline water (0.9 % W/V NaCl ) with a Warring blender to prepare the initial dilution. Colony forming units (CFU) were determined by standard pour plate methodology. Decimal dilution for total viable counts was made in (0.85% W/V saline solution) and 1 cm3 was placed in duplicate on standard plate count agar – Enterobacteria were enumerated on MacConkey Agar (Leininger, 1976). The plates were incubated at 30°C for 24 h.

Analysis of odor was performed according to the procedures authored by Sobel, (1972); Sweteen and Akanbi, (1994). A panel of 10 members was presented with the odor samples who rated the samples on a given scale of 0 5 over a 5 week period. The 0 represents no odor while 5 represents maximum odor levels. Observations were taken by lifting up the lids of the jars with a gloved hand and smelling the treated and controlled samples at a distance of 6 8 inches and odor levels were rated accordingly.

All treatments were in duplicate and data in figures are expressed as means. Statistically significant differences at p< 0.05 probability level were obtained from ANOVA tests.

Fecal coliforms were enumerated from the controlled and treated samples. The no addition treatment initially contained 4.65 × 103 CFU ml-1, which increased to 5.33×103 CFU ml-1 in 7 days before declining to a level below the initial concentration at the end of 35 days (0.81×102 CFU ml-1). The steady decline in the concentration of coliform bacteria may be due to increase in

toxic secretions and degradative acidic products which became lethal to a select population of organisms in the control. When compared to the no addition treatment (control), the FeSO4.7H2O and MOSP treatments significantly reduced the number of viable coliform bacteria as shown in figure 1a & b (p < 0.05).

From Fig.1a, the 2%, 5% and 10% concentrations of ferrous sulfate hepta hydrate all effectively suppressed the populations of the coliforms in the waste. The 10% FeSO4.7H2O treatment was seen to be more effective as it accelerated the decline of the viable populations from 2.85 × 103 CFU ml-1 at the start of the incubation to 0.29 × 101 CFU ml-1 after 35 days period. This effect may be due to the hostile nature of the highly acidic FeSO4.7H2O. The MOSP treatment was however more effective at the lowest concentration of 2% (Fig.2). The population of viable coliforms decreased from 3.05 × 103 CFU ml-1 initially to 0.22 × 101 at the end of the 35 days incubation. MOSP seed is a natural biopolymer and the carbohydrate content at the higher concentrations of 5% and 10% may increase nutrient for microbial growth rather than inhibition, thereby limiting the antimicrobial strength evidenced by the low values.

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The efficacy of the combination of the two additives at the various concentrations on fecal coliforms was also investigated (Fig.2). All the formulations exhibited improved performance in comparison with the single component treatments. Although the odor control formulations did not completely eliminate the microbial mass in the treated samples, they

however accelerated the death of the coliforms.

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F2 F3 F4 F5 F6 F7 F8 F9

The performance of these treatments compared well with, and in some cases, proved better than a number of commercial products that claim to eliminate coliform bacteria from manure. Varel et, al (2004) reported a total CFU of 0.02 x 105 ml-1 after 14 days of treating cattle waste with thymol, a plant derived oil. In another study, N- (n-butyl) thiophosphoric triamide (NBPT) at 20 and 80 mg/kg-1 gave 0.4 x 105 and 0.13 x 105 CFU ml-1 respectively after 42 days of waste treatment (Varel, et al 2006). Their actions is predicated on the capacity to reduce or eliminate microbial pathogens, the odor causing agents in manure. Clearly, reducing the concentrations of potentially pathogenic bacteria in manure is a key to reducing manure odors and is an attractive feature of odor controlling agents (Della et al, 2002).

Consequently, an odor assessment of the treated samples was conducted to ascertain the odor reducing potential of the formulations throughout the treatment period. The odor control effectiveness of the formulations on weekly basis is shown in Figure 3.

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Formulations

From Figure 3, the odor levels generally decreased with time. From the first week, all the formulations indicated positive effect on the odor. F6 gave minimum reduction of 22% and F7 showed the highest value of 42% reduction. By the end of the treatment period of 5 weeks, F7, F5, F2 gave impressive results of 70%, 76% and 82% respectively.

The overall effectiveness of the formulations over the entire period of treatment is depicted in Figure 4. The results showed that F6 was the least with odor levels reduced by 36%, while F2 was the best with odor levels of 53.6% reduction.

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From the nine formulations, only F2, F5 and F7 reduced the overall odor levels by more than 50%. F6 seemed to be the least effective with odor reduction of 36%. The level of effectiveness of the remaining formulations was in-between 36% and 50%. F2, F5, and F7 were particularly impressive and can therefore be used to effectively inhibit malodor generation from poultry waste.

The performances compared well with the efficacy of some commercial odor reducing products reported in the literature. Amon et al., (1995) used saponin at 6.2g/day for 60 days to treat hog waste and reported 26% odor reduction. A pH modifier, AMGUARD (an organic acid), applied to waste for 8 months, gave 42% reduction in odor levels (Hendriks et al., 1997). In another study, a digestive agent used over 22 months period inhibited odor generation only by 30% (Hendriks et al., 1997). Wheeler et al (2011) investigated the efficacy of other 22 commercial manure amendments covering five classes (microbial, chemical, adsorbent, masking and disinfectant) for both short and long term periods. Three products showed significant short term odor control. A microbial digest/ enzyme (MAF) showed a 33% reduction in odor, another, a disinfectant (H2O2) with a 45% reduction and the third a masking agent (Hyssops officinalis) essential oil with a 27% reduction.

These products were reported to offer potential for use prior to the transition from storage to land application by providing short term odor reduction. Reductions of odor emission by 22 or 17% respectively were also reported in manure treated with peppermint oil and basil oil. The efficacy of zeolite was also tested and was found to reduce odors by only 11%. Odor emission rates were reduced by 48% following the addition of digestive / facultative MBR and chemical only as a frequent re-application to diary manure. However, some of these compounds tested were found to influence odor generation instead. Manure treated with odorless glycerol, a product of biodiesel production, increased odor emission by 27% (Wheeler et al, 2011). In contrast to its performance at 3 days, H2O2 increased odor by 46% after 30 days incubation period, Zeolite increased by 177% after 30 days.

On the whole, they concluded that none of the 22 manure amendments significantly reduced odor emission from animal manure for both 3 and 30 days incubation periods. This agrees with the report that a large percentage of the commercial odor reducing compounds are not effective, besides being costly and scarce (Ritter et al, 1989; Wheeler et al, 2011). This is an indication that

short, medium and long term odor control from poultry houses remains challenging. Comparatively, the effects of the formulations reported in this work showed significant coliform reduction and consequent odor inhibition at both short (7 days) and long term (35 days) incubation periods. However, none of the products was able to totally eliminate odors.

Results suggest that both the single components and their formulations were generally effective at both short and long term durations, therefore may require no repeated additions and can be used to inhibit malodor generation from poultry waste. However, studies under field conditions are expected to verify claims of efficacy at the optimized concentrations.

The authors wish to thank the manager of the farming skill acquisition centre, Yola where the sample waste was obtained. The assistance of Mr. Adeogoke of Microbiology department in the microbial analysis is also acknowledged. Finally, we thank the final year students of Chemical Engineering department, who were part of our panel members.

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