Genetic Fingerprinting of Bacillus thuringiensis Isolates by Randomly Amplified Polymorphic DNA Polymerase Chain Reaction (RAPD-PCR)

Genetic Fingerprinting of Bacillus thuringiensis Isolates by Randomly Amplified Polymorphic DNA Polymerase Chain Reaction (RAPD-PCR)

Gyan Sundar Sahukhal^l*2, Upendra Thapa Shrestha¹, Binod Lekhak², Anjana Singh², Viswanath Prasad Agrawal¹

¹Research Laboratory for Biotechnology and Biochemistry (RLABB), Kathmandu, Nepal.

²Central Department of Microbiology, Tribhuvan University, Kirtipur, Nepal

Corresponding authors: gyan633413@gmail.com / upendrats@gmail.com

Abstract

Random Amplified Polymorphic DNA (RAPD) is a method of producing a genetic fingerprint of a particular species without its prior genetic information. Relationship between species may be determined by comparing their unique fingerprint information. B. thuringiensis was isolated from soil samples of Khumbu base camp of Everest region, Nepal. Crystal protein (delta endotoxin) producing strains (46 from Phereche and 40 from Sagarmatha national park) were tested against a series of 100 decamer RAPD primers (codes 201-300, obtained from University of British Columbia) by RAPD PCR. Primer 284 was found the best among the tested primers and the reaction condition for PCR was optimized with a PCR buffer containing 10mM Tris HCl, 50 mM KCl, 3 mM MgCl₂ with pH 8.3.; 200μm dNTPs each, 1U Taq polymerase , 40 pmol decamer primers, 20 ng template DNA and 1% DMSO as a final concentrations in 25μl reaction mixture. The thermal programme was programmed as initial denaturation temperature at 94^oC for 5 min followed by 35 cycles with denaturation at 94^oC for 1 min, annealing at 36^oC for 1 min and extension at 72^oC for 2 mins with final extension temperature at 72^oC for 10 min. Higher polymorphic fragments were found in the range between 700-900 bp. Next to it, the range of 400-700 and 1200-1600 bp were, too, highly polymorphic among the isolates. The discriminatory capacity (D) of the RAPD-PCR was found to be 0.9901. The isolates of cold tolerant B. thuringiensis from high altitude regions were found rich in genomic polymorphism.

Key words: RAPD-PCR, fingerprint, endotoxin, Bacillus thuringiensis Berliner, polymorphism, base pair (bp)

Introduction

Use of chemical pesticides has led to the emergence and spread of resistance in agricultural pests and vectors of human diseases and to the environmental degradation. The very properties that made these chemicals useful—long residual action and toxicity to a wide spectrum of organisms—have brought about serious problems. An urgent need has thus emerged for environment friendly pesticides to reduce contamination and the likelihood of insect resistance (Ben-Dov et al. 1997).

The soil bacterium Bacillus thuringiensis Berliner fulfills the requisites of a microbiological control agent against agricultural pests and vectors of diseases that lead to its widespread commercial application. It is a gram-positive, aerobic, endospore-forming saprophyte. All known subspecies of B. thuringiensis produce large quantities of insecticidal crystal proteins (Cry proteins) which are segregated in parasporal bodies (also known as δ-endotoxins). The genes coding for Cry proteins normally occur on large plasmids and direct the synthesis of a family of related Cry proteins classified as Cry1-28 and Cyt1-2 groups according to their degree of amino acid homology. Cry proteins have been used as biopesticide sprays on a significant scale for more than 30 years, and their safety has been demonstrated. The main target pest of B. thuringiensis include various lepidopterous (i.e. butterflies and moths), dipterous (i.e. flies and mosquitoes), and coleopterous (i.e. Beetles) species. Some strains have also been found to kill nematodes (Schenpf et al. 1998). Conventional B. thuringiensis preparations such as those registered in Germany and also found worldwide are mostly derived from the highly potent strain B. thuringiensis var. kurstaki HD1, which was isolated in the sixties ( Dulmage 1970).

Williams et al. (1990) used varieties of morphological and physiological characteristics to assign different bacterial strains into defined taxonomical clusters. However, most of the taxonomic methods are very time consuming and sometimes give ambiguous results. Genomic fingerprinting assays using RAPD have already been shown to be useful for differentiation of bacterial strains. This method is based on the amplification of distinct DNA sequences under low stringency conditions during annealing using an oligonucleotide of arbitrary sequence. The primer is not directed at any specific sequences within the template, making previous knowledge of the genome non-essential. The efficacy of the amplification procedure is primarily dependent on sufficient sequence similarity at the 3’ end of the oligonucleotide to allow adequate priming. The resulting pattern of amplification products of varying size can subsequently be used as a genetic fingerprinting of the organisms (Mehling et al. 1995) and can also be used to genetically link to a trait of interest for individual and pedigree identification, pathogenic diagnostics, and trait improvement in genetics and breeding programmes. Morphological , biochemical characterization and identification, isozyme analysis, restriction fragment length polymorphism (RFLP), minisatellites, microsatellites , randomly amplified polymorphic DNAs (RAPD) and fluorescence in situ hybridization (FISH) have been so far used to analyse genetic similarity and diversity for breeding research of animal/plant/microbes (Yoon & Kim 2001). In this study RAPD-PCR has been used for genetic and molecular studies as it is a simple and rapid method for determining genetic diversity and similarity in various organisms.

Materials and Methods

Bacterial isolates

B. thuringiensis strains were isolated by acetate selection method from the soil samples collected from Khumbu base camp of Everest region. The isolates were identified by standard microbiological techniques including colonial, morphological and biochemical characteristics according to Bergey’s manual of systematic bacteriology (Claus & Berkeley 1986).

Preparation of template DNA

Templates were prepared from 16 to 18hr cultures in Luria-Bertani medium as described by Ben-dov et al. (1997). Aliquots of 3 to 4.5 ml were harvested by centrifugation and washed once in TES (10 mM Tris-HCl of pH 8.0, 1 mM EDTA, 100 mM NaCl), and the pellets were resuspended in 100 ml of lysis buffer (25% sucrose, 25 mM Tris-HCl [pH 8.0], 10 mM EDTA, 4 mg of lysozyme per ml). The cell suspension was incubated for 1 hr at 37°C. Further, DNA extraction was performed as described by Sambrook et al. (1989). Extracted DNAs were quantitated by spectrofluorometer and diluted up to 20ng DNA/ul in order to feed on the PCR reaction mixture.

RAPD reaction

One hundred RAPD primers (10-mers) of arbitrary sequence obtained from University of British Columbia, Canada, were screened for the ability to produce discriminatory polymorphisms. RAPD-PCR mixture was set up that contained 20-50 ng of genomic DNA, 40 pmol of primer, 1 U of Taq polymerase (Bangalore GENEI.), 200 uM (each) deoxynucleoside triphosphate, 10 mM Tris-Cl (pH 8.3), 50 mM KCl, 3 mM MgCl2 and 1% DMSO. Each reaction mixture was overlaid with 25 ul of mineral oil and amplified with a Perkin-Elmer Cetus DNA Thermal Cycler model TC-1 as follows: (i) Initial denaturation, 1 cycle consisting of 5 min at 94^oC and (ii) 35 cycles, with 1 cycle consisting of 1 min at 94^oC, 2 min at 36^oC, and 3 min at 72^oC, followed by a final extension step at 72^oC for 10 min.

RAPD products were separated by agarose gel electrophoresis (1 %) with 1X TAE buffer for 2 hrs. Molecular size standards (l DNA HindIII digest and f X 174 phage DNA type II digest) were also included in each gel, photographed by the Fototdyne camera using polaroid film (Porplan 667). The RAPD fingerprints were analyzed visually and the molecular size of each band migrated was calculated by plotting standard curve (log of molecular weight vs distance traveled) of the standard DNA ladders.

Results

Ninety one soil samples collected from the Khumbu base camp of Everest region, Nepal were processed at Research Laboratory for Biotechnology and Biochemistry (RLABB). A total 109 B. thuringiensis isolates were obtained from the soil samples of Phereche (P) and Sagamatha National Park (SNP). From 52 Phereche soil samples, 63 isolates were obtained and from 39 soil samples from SNP, 46 isolates were obtained but only 86 isolates were found to produce crystal protein which were preceded for RAPD-PCR (Shrestha et al. 2006)

Identification of discriminatory primer(s) for RAPD analysis

Nine primers (Table 1) with GC content - 50-80%, were found to amplify genomic DNA fragments with reproducible polymorphisms suitable for strain differentiation of the B. thuringiensis isolates (Fig 1). Primer 284 with GC content 70% was found to produce more polymorphic bands than other primers and was used to obtain RAPD profiles of some selected B. thuringiensis isolates (Fig 2 and 3).

RAPD fingerprinting of the B. thuringiensis isolates

Of 108 isolates, 86, the crystal protein producers, were typed by RAPD-PCR to study the polymorphism patterns. The RAPD typing results are summarized in Table 2, table 3 and table 4.

Table 1. RAPD primers producing reproducible polymorphisms with B. thuringiensis

S.No	Primer	Sequences of primers (5’ to 3’)	GC% of primers	No. of bands produced	Molecular size of the bands (bp)
1	208	ACG GCC GAC C	80	8	2821, 1842, 1473, 1282, 747, 639, 502, 357
2	254	CGC CCC CAT T	70	6	4228, 3102, 2357, 1125, 789, 639
3	256	TGC AGT CGA A	50	2	1125, 372
4	268	AGG CCG CTT A	60	6	2575, 1583, 1282, 936, 789, 404
5	275	CCG GGC AAG C	80	8	4228, 2575, 1282, 993, 789, 672, 526, 372.
6	276	AGG ATC AAG C	50	7	2357, 1473, 1282, 1056, 936, 747, 639
7	284	CAG GCG CAC A	70	10	3425, 1995, 1583, 1373, 1200, 936, 834, 747, 639, 480
8	292	AAA CAG CCC G	60	3	1373, 993, 708
9	299	TGT CAG CGG T	60	2	1373, 993

Table 2. RAPD types of the Phereche isolates

S. No	Codes of the isolates	No. of RAPD bands produced	Number of the base pairs (bp)
1	P1	1	2061
2	P2	1	812
3	P3	6	1843, 1563, 1213, 962, 690, 495.
4	P5	4	1563, 1053, 811, 690
5	P6	2	1274, 811
6	P7	7	1378, 788, 738, 692, 611, 512
7	P9a	10	2061, 1436, 1270, 1173, 1046, 937,815, 738, 692, 630
8	P10	1	1563
9	P12a	6	2139, 1944, 1629, 1501, 1337, 1200
10	P12b	1	2497
11	P13	6	1648, 1449, 1187, 1023, 889, 775
12	P14	1	1499
13	P15	5	3148, 2638, 1944, 1389, 927
14	P17b, P25, P27, P28,P30	2	1944, 1501
15	P18	6	2368, 2139, 1775, 1629, 1501, 1200
16	P21	1	1629
17	P22	6	3353, 2638, 2139, 1050, 1289, 1121
18	P24	2	2139,1700
19	P26	2	2249, 1944
20	P29	3	2638, 1944, 1501
21	P31	3	1086, 670, 527
22	P32	1	1501
23	P33	4	1156, 921, 811, 551.
24	P34	5	1553, 1326, 763, 567, 375
25	P35	10	1499, 1378, 1128, 972, 873, 788, 738, 692, 542, 512
26	P37	7	2006, 1553, 1229, 991, 717, 508, 413
27	P38	3	1142, 717, 567
28	P39	6	1553, 1229, 991, 763, 600, 375
29	P40	10	2804, 2393, 1874, 1635, 1322, 1128, 937, 844, 738, 670
30	P41	5	1483, 1274, 962, 718, 571
31	P42	6	1837, 1433, 991, 867, 717, 567
32	P43	5	1229, 926, 812, 717, 636
33	P45	5	1378, 1173, 844, 738, 512
34	P46	4	1229, 867, 717, 567
35	P47	4	2638, 2368, 2139, 1775
36	P48a	5	1229, 926, 812, 675, 567
37	P44,P49,P50, P51b, P52	5	1229, 991, 867, 763, 636
38	P53	4	2368, 2038, 1775, 1443

Table 3. RAPD types of the SNP isolates

S No	Codes of the isolates	No. of RAPD bands produced	Number of the base pairs (bp)
1	S1	5	2291, 1811, 1409, 787, 465
2	S2a	5	2079, 1733, 1355, 787, 465
3	S2b	3	1409, 1087, 477
4	S3	8	1255, 1074, 962, 807, 706, 663, 520, 417
5	S4	6	1723, 1512, 1283, 1187, 953, 755
6	S5	8	1792, 1656, 1534, 1235, 1012, 893, 817, 771
7	S6, S7	5	1361, 1074, 754, 464, 375
8	S8	7	1764, 1419, 754, 684, 586, 395, 339
9	S10	10	3793, 2181, 1733, 1466, 1256, 1087, 950, 867, 701, 538,
10	S13	4	1733, 1466, 891, 787
11	S14	5	950, 787, 701, 628, 512
12	S15a	11	1983, 1591, 1355, 1167, 982, 891, 837, 787, 701, 663, 448
13	S15b	5	1304, 1087, 891, 742, 663
14	S16	6	2291, 1894, 1466, 1256, 787, 701
15	S20	4	807, 586, 339, 294
16	S21	4	706, 586, 440, 280
17	S23	4	1594, 1431, 601, 473
18	S24	3	930, 726, 647
19	S25	5	2277, 1170, 819, 755, 522
20	S26	5	2776, 1154, 980, 794, 519
21	S27a	7	1170, 930, 819, 560, 505, 443, 320
22	S27b	5	1784, 1431, 1291, 891, 392
23	S28a	8	2990, 2009, 1784, 1431, 1291, 972, 891, 647
24	S28b	8	1594, 1359, 1229, 1017, 930, 726, 624, 458
25	S30	8	1866, 1235, 1080, 893, 794, 709, 603, 545
26	S31	10	2028, 1593, 1325, 1235, 1080, 980, 841, 709, 653, 519
27	S32	8	1288, 1049, 866, 791, 694, 638, 588, 433
28	S33	5	1288, 1049, 827, 612, 449
29	S35b	7	1103, 908, 791, 665, 638, 565, 433
30	S37	6	908, 791, 638, 565, 466, 433
31	S38b	5	1648, 1449, 1234, 1101, 953
32	S39	5	1222, 999, 757, 543, 403
33	S41	1	1283
34	S9, S18, S22, S34, S35a, S36, S38a		No RAPD bands produced

Table 4. Fragment length polymorphisms among the total

Range of fragment length (bp)	Total number of bands produced	Range of fragment length (bp)	Total number of bands produced
200-300	2	1700-1800	12
300-400	9	1800-1900	6
400-500	25	1900-2000	10
500-600	23	2000-2100	7
600-700	34	2100-2200	6
700-800	50	2200-2300	4
800-900	46	2300-2400	4
900-1000	21	2400-2500	1
1000-1100	17	2500-2600	4
1100-1200	13	2600-2700	1
1200-1300	32	2700-2800	1
1300-1400	14	2800-2900	1
1400-1500	17	3100-3200	1
1500-1600	20	3300-3400	1
1600-1700	8	3700-3800	1

B. thuringiensis isolates

Calculation of discriminatory index value (D)

Of 86 isolates typed, 72 different polymorphics were found. The discriminatory index value (D) was calculated using the formula:

The D value was found to be 0.9901.

Discussion

All the soil samples in this study were collected from high altitude mountain area - Khumbu Base Camp of Everest region, Nepal expecting mainly cold tolerating strains of B. thuringiensis. The genome of each B. thuringiensis is unique and is basic to all DNA analysis aimed at identification (Belkum et al. 1994). Based on this assumption RAPD-PCR was optimized to study genetic diversity of the B. thuringiensis isolates from Khumbu region of Nepal. As RAPD-PCR has higher discrimination power than any other conventional techniques (Lechner et al.1998, Daffonchio et al. 1999, Robert & Crawford 2000, Brousseau et al. 1993, Puenti- Redondo et al. 1999) and opens a new horizon with reproducible data, it is considered as a doorway for any genetic analysis to perform. RAPD assay resulted in a clear separation of the psychrotolerant B. cereus strains (Lechner et al. 1998).

A series of 100 decamer primers from codes 201 to 300 (obtained from UBC) were tested for RAPD-PCR. It has been shown that the optimal length of primers used in RAPD analysis is approximately eight nucleotides. Primers longer than 10 nucleotides have less discriminating power, which again is strongly dependant on the annealing temperature (Belkum 1994). Nine primers, with GC% of 50-80, were found to amplify the target sequences with reproducible polymorphism to differentiate the B. thuringiensis strains. The primers with high GC content (70-80%) were found to produce more polymorphisms compared to those of low GC content (50-60%). PCR products are visualized by ethidium bromide staining (0.2-0.5 μg/ml of gel) of electrophoretically separated DNA in agarose gel. Fingerprints are recorded as banding patterns and comparisons made by visual inspections using standard scales. Standard molecular markers (l DNA Hind III digest and f X 174 phage DNA type II digest) were used. Each primer amplified polymorphisms ranged from two to ten over a range of 300 bp to 3 kbps. The bands were found reproducible for different independent DNA preparations from respective B. thuringiensis strains.

Each primer yielded RAPD patterns that were unique to strains of the B. thuringiensis isolates to be differentiated subsequently. Primer 284 from the series was selected best to type the B. thuringiensis isolates for polymorphic study. Of the total amplified products, maximum number of the product size ranged from 300 bp to 2 kb; with highest number of 50 bands within 700 to 800 bp, followed by 46 bands within 800 to 900 bp, 34 bands within 600 to 700 bp and 32 bands within 1200 to 1300 bp. Similar band patterns suggest that the strains are closely related to each other within each group. However, the data must be interpretative with caution since PCR bands of similar size do not necessarily mean that the molecules are identical in sequence (Brousseau et al.1993). As the strains were isolated from high altitude mountain region of the country, the maximum amplified genomes may represent cold tolerant genes common to all or the crystal endotoxin producing genes. However, for the best knowledge, such study for cold tolerance was not found yet and the data were not compared to any reference but predicted to contain common or consensus bands (or may be sequences) for cold tolerance so as to adopt the organisms in a given ecological niche.

Vogel et al. (1999) tested the usefulness of genomic typing methods viz. RAPD analysis and ribotyping with conventional serotyping for three collections of well defined clinical E. coli isolates and found that RAPD has the highest discriminatory capacity. Similarly, Sarkar et al. (2002) exploited the RAPD-PCR fingerprinting analysis and showed a high level of diversity of Bacillus spp and related genera. In order to determine the index of discrimination (Hunter 1990, Hunter & Gaston 1998), all the 86(N) crystal producing B. thuringiensis strains were classified into 71(s) types with two sets of five similar band patterns (n₁₄ and n₃₇), one set of two similar band pattern (n₄₅) and 68 sets of a single band pattern (n₁-n₁₃, n₁₅-n₃₆, n₃₈-n₄₄, and n₄₆-n₇₁). Altogether six B. thuringiensis isolates were not found to contain amplifying region in their genome using 284 primers and were classified in the 72nd type as n₇₂.

The RAPD-PCR, used to categorize the B. thuringiensis strains isolated from Khumbu region of Nepal, was found to discriminate the organisms with 99.01% confidence (D = 0.9901). The level of confidence satisfied the conclusion made by Vogel et al. (1999) to define RAPD analysis had the highest discriminatory capacity for typing E. coli isolates. Similarly, while working for RAPD-typing of Pseudomonas aeruginosa from cystic fibrosis patients, Mahenthiralingam et al. (1996) stated that, in general, despite alteration in the expression of mucoid exopolysaccharide, bacterial motility, and acquisition of a serum-sensitive phenotype, the RAPD fingerprints of sequential isolates remain stable, suggesting that these changes result from phenotypic adaptation of the primary colonizing isolates. With these findings, the polymorphisms set by RAPD to quantify the diversity of B. thuringiensis strain isolates from Khumbu region of Nepal can be confidently defined as rich one.

Acknowledgements

The authors express full gratitude to CNR (Italy’s National Research Council) for supporting this work; to Dr. Deepak Singh, Dr.Yogan Khatri and Dr. Rajindra Aryal for soil sample collection from the Everest region; and to Mr. Kiran Babu Tiwari for his sincere efforts and suggestions.

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Solid Waste Management

SOLID WASTE TYPES

Source	Typical waste generators	Types of solid wastes
Residential	Single and multifamily dwellings	Food wastes, paper, cardboard, plastics, textiles, leather, yard wastes, wood, glass, metals, ashes, special wastes (e.g., bulky items, consumer electronics, white goods, batteries, oil, tires), and household hazardous wastes.).
Industrial	Light and heavy manufacturing, fabrication, construction sites, power and chemical plants.	Housekeeping wastes, packaging, food wastes, construction and demolition materials, hazardous wastes, ashes, special wastes.
Commercial	Stores, hotels, restaurants, markets, office buildings, etc.	Paper, cardboard, plastics, wood, food wastes, glass, metals, special wastes, hazardous wastes.
Institutional	Schools, hospitals, prisons, government centers.	Same as commercial.
Construction and demolition	New construction sites, road repair, renovation sites, demolition of buildings	Wood, steel, concrete, dirt, etc.
Municipal services	Street cleaning, landscaping, parks, beaches, other recreational areas, water and wastewater treatment plants.	Street sweepings; landscape and tree trimmings; general wastes from parks, beaches, and other recreational areas; sludge.
Process (manufacturing, etc.)	Heavy and light manufacturing, refineries, chemical plants, power plants, mineral extraction and processing.	Industrial process wastes, scrap materials, off-specification products, slay, tailings.
Agriculture	Crops, orchards, vineyards, dairies, feedlots, farms.	Spoiled food wastes, agricultural wastes, hazardous wastes (e.g., pesticides).

SOLID WASTE MANAGEMENT

Management of solid waste is an important part which not only prevents the environmental pollution but also generates the valuable products for commercial uses e.g. ethanol, compost metabolites by utilizing special wastes and cooking gas.

Nowadays management of hazardous waste is given keen interest in order to prevents the spread of infectious diseases by poor management

The management of waste comprises various stages which are given as:-

1. Collection

2. Transfer

3. Disposal

4. Potential methods of disposal

1. Collection Methods/Tranportation

Proper collection & transportation processes are essentials part of solid waste management system. Nearly 70-80% of total cost is required for collection & transportation of waste. The basic collection method in developing countries is from community collection point. The basic collection methods in Nepal are to collect in metal containers placed at several places in the city. However, metropolitan cities are also provided facilities to collect the waste to waste management unit. This facility is provided in some wards of Kathmandu, Pokhara & Biratnagar.

The simple & best methods to collect the waste comprise the daily collection of the waste especially during summer & rainy seasons. If wastes are not collected daily, it generates odor problem & chances of spreading of microbes into the environment (air & water).

In developed countries block collection & curbside collection are common methods.

A. Block Collection

Individuals bring waste in containers to a carryings vehicle which travels a regular route twice or thrice a week. The containers are emptied by vehicle crew & returned to the individuals.

B. Curbside Collection

The Waste is brought in containers & placed on the footway at special places, which is collected later by cranes of solid waste management department and leave the containers at sample place.

For transportation in a developing countries simple hard carte are effective in many places due to lack of manpower, wide streets & vehicles. However, special vehicles are in compound practice to collect the waste from containers & solid waste collection centre.

The waste in the tractors & trucks are covered with plastics in order to prevent spreading of waste into the air & transported to final disposal place.

In the developed countries, government provides containers to the individuals in order to collect biodegradable & non-biodegradable wastes separately. Peoples themselves separate the recyclable waste & non-recyclable & transfer it to the collection containers. This practice makes an easy economical disposal method / Recycle method.

3. Disposal of waste

The process of selection of right solid waste disposal method is a complex one due to heterogeneity of the urban waste, but an appropriate method can save money as well as reduces the chances of problems occurs in future.

The disposal methods should be selected in such a way that the present situations are fulfilled & future situations are anticipated. The methods should also provide opportunity for recycling of materials if possible and should not pollute the air, ground water, surface water & the land water.

Several disposal methods are being using the various parts of the world. Among the various methods, sanitary landfill is applied in developed countries while open dumping is practiced in developing countries.

A. Hog feeding

B. Open dumping

C. Incineration

D. Sanitary landfills

E. Composting

F. Pyrolysis

G. Pulverization

H. Controlled tipping

I. Pulverization combined with controlled tipping

J. Ocean Dumping and River Dumping

A. Hog feeding

It is an old method of solid waste (particularly kitchen waste) management practiced in many countries property controlled; waste can be converted into the wealth previously hogs were used as scavengers of raw garbage but the leads to the spread of several bacterial, viral & parasitic diseases to animal and human beings. This problem is overcome by steaming of garbage for ½-1 hours before feeding to the hogs. This method is applicable in the cities covered with rural areas where hog farming is possible. In many terai regions of Nepal hogs are stills (fed) with human excreta & raw garbage.

B. Open Dumping

Open dumping of solid waste is practiced extensively in developing countries because it is cheap & requires no planning. Generally low lying areas & out skirts of town and cities are used for this purpose open dumps course public health problems by encouraging the breeding of files, rats, mosquitoes and other pets. Files can transmit typhoid, fever, cholera, dysentery, tuberculosis, anthrax & other disease; rats which can transmit typhoid, leptospirosis, rickettsial pox & cockroaches & mosquitoes transmit malaria, yellow fever, dengue, encephalitis, filarial etc. They also become a source of objectionable odors & course air pollution when the wastes are buried in order to reduce their volume & conserve space.

C. Incineration

Incineration is also a commonly used method which involves the burning of waste a high temperature. After burning ashes, glass, metals & other unburned materials account for 25% of total waste. The remaining materials are to be needed disposal safety by after methods such as sanitary landfill or by dumping or recycling. Incineration leads to ambient air pollution if incinerators are not properly designed, equipped (operate) properly. This method is useful when availability of land is limited as well as safe disposal of hazardous waste such as waste from nursing homes, hospitals, laboratories. Incineration results into the formation of some air pollutants which are ash, SO₂, Hydrogen chloride & organic acids. New techniques for the handling of waste involves the separation of non-combustible part by magnetic or gravity separation methods & recycle them. This technique encourages the recycling of waste as well reduces the total cost needed for incineration. Incineration is widely applied techniques for the disposal of solid waste generated in the industries. The design of incinerator used is depend upon combustibility & nature of waste to be disposal. Another term commonly used for this technology is energy recovery or waste to energy because the heat derived from incineration refuse is a useful resource. The heat energy liberated could be used to generate electricity. Internationally well over 1500 waste to energy plants in Brazil, Japan & Western Europe generate electricity & reduce the investments needed for landfilling. Various types of incinerators widely used in the industries are multiple hearth rotary & flurdurd bed type reactors.

Advantages	Disadvantages
1. Energy generation. 2. Volume reduction by 90% 3. Suitable from aesthetic hygienic and stand point 4. Requires small land area. 5. Inert materials such as ash is recovered,	1. Initial investment is high. 2. Required skilled manpower. 3. High Maintenance cost 4. Air pollution

D. Sanitary landfill

The landfill operation is essentially a biological method of waste treatment. The stabilization of waste may be divided into five distinct phases within overall process. During first phase of operation aerobic bacteria are dominant which consume oxygen. As a result of metabolism of bacteria temperature of the environmental increases. In the second phase anaerobic conditions established & hydrogen & CO₂ gas are evolved due to fermentation of organic substrates. In the third phase of growth methanogens active to produce methane & CO₂ gas. In the fourth phase methanogenic activity becomes stabilized. In the fifth phase the methanogenic activity decreases representation depletion of the organic matter & alternately the system returns to aerobic conditions within the landfills. The duration of each phase of prevailing environmental conditions & the nature of the waste. The end products of decomposition of wastes during phase three & four are mostly CO₂ & CH₄ gas along with a small amount of H₂S, NH₃ & water. During first year the amount of N₂ decreases as air cannot penetrate the soil to the seated cell. Methane production can usually be observed around 200 days after reduce disposal. During construction the landfills site must be designed in such a way that there leach ate should be drained in proper manner to avoid surface & ground water pollution. Escape rents for gases should be provided they do not build up to dangerous levels. Landfills gas is renewable source of energy & which can be used for cooking purpose.

Advantages

Disadvantages

1. Most economical method where land is available. 2. Minimum public health problem because files, rats & other pets are unable to breed in the covered waste. 3. Minimum fire hazards. 4. Can receive all types of waste, eliminating the necessity of separation of waste into degradable & non-biodegradable. 5. Finished landfills sites can be used for the development of parks, playground, golf courses etc.

1. Unavailability of suitable land especially rears the crowded area. 2. Proper operation & skilled manpower needed for daily operation. 3. Danger of surface & ground water pollution if not properly selected & designed. 4. Sealing materials used do not remain effective permanently 5. Leakage of gases may cause odor problems.

E. Composting

Composting of organic waste appears to offer an attractive alternative to landfill for decomposition of solid domestic and agricultural waste. In contrast to a sanitary landfill composting of refuse is an aerobic method of decomposing solid waste. It is microbial process that converts putrecible organic materials into stable humus like product that is reduced in bulk and can be used for soil improvement. The organisms included are bacteria which predominate at all stages, fungi which often appear after first week and actinomycetes which assist during final stages.

Initially the process starts with the mesophilic bacteria which oxidize the organic matter in the refuse to carbondioxide and heat the temperature rises to about 45°C and at that point the thermophilic bacteria take over and continue the decomposition. During this phase temperature further rises to about 60°C. During the operation the refuse is periodically turned over to allow the sufficient oxygen to penetrate to all parts of the material to support the aerobic life. After about 3 weeks the compost is stabilized and the end point of composting operation can be measured by a drop in temperature.

Composting process can be categorized into two types

1. Aerobic process
2. Anaerobic Fermentation process

E1. Aerobic process

In aerobic decomposition process microorganisms utilize oxygen to feed on the organic matter in the waste produce stable end product as well as their own biomass. The end product is humus like material which is extremely useful as plant nutrient. The aerobic fermentation process occurs very widely in nature and is the main way in which waste products from field and forest are converted into humus. Composting is accomplished in static piles aerated piles or continuous feed reactors.

The static pile process is simple but relatively slow, requiring many months for stabilization. Insects breeding and odor problem during static pile method can be controlled by covering the piles with a layer of soil, finished compost or wood chips. Under favorable conditions, self heating causes to rise in temperature to 55-60° C or above in 2-3 days. After few days temperature is gradually decreased. Oxygen concentration in the compost pile is five times lower than ambient air. Periodic turning of the compost piles help to saturate the waste with oxygen and uniform mixing. After thermophilic process, a curing phase at mesophilic temperature starts for several month. Hence, large land area is required due to slowness of the process.

The aerated pile process is substantially faster process through improved aeration. The aerated pile process involves the suction of air through perforated pipes buried inside the compost pile. This design, Beltsville process, achieves at least partial oxygenation of pile, but temperature control is inadequate. The temperature rises unto 70-80°C. This model is improved by Rutgers model which reverses the airflow from suction to injection. Thermostats placed inside the pile controls blower operation, starting when temperature exceeds 60°C. The injection at air not only oxygenates the pile but cools it sufficiently to avoid temperature rises. The heat generated by the biodegradation process is effectively used in evaporating water which results into dried and stable compost. The aerated pile process completes in three weeks.

In continuous feed reactor process composting is carried out in a bioreactor. It requires about 20,000 cubic feet of air per ton of organic matter/day for efficient composting. This process forms a uniform & stable product but also requires a high initial investment. Composting in a reactor is accomplished in 2-4 days. A part or the entire reactor is maintained at thermophilic temperature using the heat produced in the composting process. After processing in the reactor, the product requires “curing” for about a month prior to packaging.

Regardless of the process design, conducting the composting process in the thermophilic temperature range is desirable because it speeds the process and destroys pathogens that may be present in feacal matter and in sewage sludge. The aerobic oxidation process catalyzed by microorganisms produce heat which increases temperature to 76-78°C. This temperature is inhibitory to biodegradation process i.e. for microbes because the maxiamal thermophilic activity of microbes occurs between 52-63°C. Aeration or turning or periodic cool water spraying can reduce temperature to an optimum level.

The composting process is initiated by mesophilic heterotrophs. As temperature rises, they are replaced by thermophilic forms. Thermophilic bacteria predominating compost piles are B. steriothermophilus, Thermomonospera, Thermoactinomyces, Clostridrum, and Thermocellum. Important fungi in composting process are Geotrichum condidum, Aspergillus fumigatus, Mucor pusillus, Chaetomium thermophile, Thermoascus auranticus and Torula thermophila etc.

Factors affecting composting:-

1. Moisture: - Optimum 50-60% moisture is essential. Above 70% or more interferes with aeration and lowers self heating capacity.

2. C:N ratio(Carbon: nitrogen ration):- Should not be greater than 40:1. Lower nitrogen content should not permit formation of sufficient microbial biomass. Excessive nitrogen content (25:1) tends to volatilization of ammonia causing odor problems and lowers the fertilizer value of compost. So optimum is about 27 to 30:1.

3. Temperature: - Optimum is around 60°C at which thermophilic activity is favoured. Temperature above 50-60°C is an essential to operate thermophilic activity and to kill pathogens (cells and cysts).

4. Aeration: - Most important to maintain temperature and hence most stable compost.

Steps in composting: -

E1. Separation of compostible and non-compostible:-

Organic- compostible, inorganic – non-compostible

Magnetic device separates tin, iron and other metals.

Manual or gravity separation method is used to separate glasses, bottles, rubber, plastic etc.

Separation at source is more economical than separation at collection centre or composting centre.

E2. Shredding: -

Once most of non-compostible materials have been removed, the rest is shred. This is necessary to give the materials a sufficiently large area for the microbial attack to proceed readily. Hammer mills are generally employed for shredding which reduce the size.

E3. Blending for composting:-

Blending causes to adjust C:N ratio. Some materials with poor nitrogen content (waste plant products, saw dust etc.) are blended with materials having very high C.N ration (human or animal excreta, slaughterhouse waste, etc). Soil is also added to adjust the moisture.

E4. Composting:-

Aerated pites or bioreactors (mentioned above).

Nusoil process: - The pulverized matter then goes to a vertical digester where decomposition takes place. The digester is a circular unit has seven sections. The waste moves down through each section of the digester. It is kept for about one day in each section & air flow rate & water addition are regulated so that decomposition takes place under optimum condition. The digestion process is completed in seven days & the resultant compost is satisfactory for direct field application without addition of supplementary nutrients.

E5. Curring & packaging:-

F. Pyrolysis: -

Pyrolysis is a method of solid waste disposal which results into chemical conversion of refuse to get new chemical compounds. Pyrolysis is operated using intense heat to cause chemical changes but not combustion. In Pyrolysis, the refuse is heated in an oxygen free environment during which most organic substrates can be split through a combination of thermal cracking and condensation reactions into gaseous, liquid and solid fractions. Pyrolysis is carried out in different reactors and three major component fractions resulting from pyrolysis are;

1. A gas stream generated by pyrolysis may contain primarily hydrogen, methane, carbon monoxide etc. depending upon organic characteristics of the material being pyrolysed.
2. A fraction that consists of tar and/or oil stream that is liquid at room temperature. It consists of chemicals like acetic acid, acetone and methanol.
3. Solid fraction containing pure carbon plus inert materials.

G. Pulverization: -

Pulverization is really a step involved in disposal of refuse by sanitary landfill, composting or other techniques. It is the process of making small pieces of solid waste by pressing or cutting the separation conducted by the use of rotating drum machines. The pulverized waste can be easily disposed by applying other techniques.

G1. Rotating dram machines: - These pulverize the waste by attrition. Water is added to the crude refuse and mixes, is then passed into the revolving drums which have a circular, an octagonal or a hexagonal crops section. The drums operate either in a batch or continuous basis with number of rotations depending upon types of refuse. The drums usually contain a separation device for materials which cannot be broken down: -mainly plastics, rubbers and metals etc. The final materials that come out of drums normally contain a moisture 50-60% and density of 0.5-0.6kg/dm­­­³_, much higher than the original density of refuse. Drum-type pulverizers usually have a capacity of about 10-12 tons/hr and require about 35kw of power to operate. It is provided with magnetic separation to remove iron and tin.

G2. Hamper mills: - Hammer milling is operated either a dry or wet process. Hammer mills are of two types i.e. fixed hammer and swing-hammer type. The hammer mills can process maximum about 30 tons/day. It consists basically a horizontal rotor which carries a number of swinging or fixed hammers. It consists of impact plates and grates through which the processed refuse is finally ejected. Size of pulverized particles is about 5-8 cm in diameter. Metals are separated by shredding and iron is recovered by magnetic separators. Power consumption is about 150-200kw.

H. Controlled tipping: -

This method allows permanent deposition of waste on land, sealed in cells isolated from the surface with layers of earth. Controlled tipping is generally practiced in conjugation with land reclamation schemes, to reclaim marshes, exhausted quarries and gravel pits for agricultural, industrial and recreational uses. If not properly designed it will create water pollution problem and invoke the breeding of pests. If the reclaimed area is to be used for agriculture purpose, non-biodegradable compounds should be removed.

I. Pulverization and controlled tipping:-

Although cost is high it has few advantages: -

a) Less earth cover with required than unpulverized waste.

b) Rat, mice, insect larvae destroyed.

c) Land can be used for agriculture purpose.

d) Very less time will required for complete degradation of waste.

J. Ocean dumping/River dumping: -

It is one of the oldest methods and nowadays it is completely restricted.

4. Potential methods of disposal

1. Utilization.

2. Recovery and recycling: - paper, metal, plastic, glass.

Education and awareness

Education and awareness in the area of waste and waste management is increasingly important from a global perspective of resource management. The Talloires Declaration is a declaration for sustainability concerned about the unprecedented scale and speed of environmental pollution and degradation, and the depletion of natural resources. Local, regional, and global air pollution; accumulation and distribution of toxic wastes; destruction and depletion of forests, soil, and water; depletion of the ozone layer and emission of "green house" gases threaten the survival of humans and thousands of other living species, the integrity of the earth and its biodiversity, the security of nations, and the heritage of future generations. Several universities have implemented the Talloires Declaration by establishing environmental management and waste management programs, e.g. the waste management university project. University and vocational education are promoted by various organizations, e.g. WAMITAB and Chartered Institution of Wastes Management. Many supermarkets encourage customers to use their reverse vending machines to deposit used purchased containers and receive a refund from the recycling fees. Brands that manufacture such machines include Tomra and Envipco.