Paulo Zoo Research Articles

New Iflavirus Species Characterized from Mosquitoes Captured in the Sao Paulo Zoological Facilities.

Metagenomic studies of mosquito viromes demonstrated a more diverse composition than just an exclusive composition of pathogenic arboviruses transmitted to humans. In our study, the virome of 866 female mosquitoes collected throughout 2020 at the São Paulo Zoo, located in the city of São Paulo/SP-Brazil, was obtained. Specifically, in this paper, we describe a new virus found by viral RNA extraction and next-generation MiSeq sequencing of a group of 23 specimens of Anopheles (Nys.) strodei. The complete genome with a length of 9709 nucleotides was characterized by a positive orientation and a single strand, with a single large ORF, which encodes a polyprotein of 2987 amino acids. The phylogenetic analysis showed an association with the viral family Iflaviridae and the Riboviria realm. We carried out comparisons with translated sequences of the capsid regions of other iflavirus, and the identities in relation to our sequence were below the minimum limit of 90%, indicating that possibly it is a new species of iflavirus. Our findings contribute to expanding knowledge of virome composition among mosquito species in Brazil and globally. Moreover, we provide a viral genome reference specific to this geographic region and Culicidae family of mosquitoes. This resource facilitates future in silico recognition and assembly of viral genomes within metagenomic datasets.

Establishing hematologic and biochemical reference intervals for the endangered Lear's macaw (Anodorhynchus leari).

The Lear's macaw, Anodorhynchus leari (Psittaciformes, Psittacidae), is an endemic species of the Caatinga region in Brazil. With only a few free-living individuals that are constantly under threat from agricultural clearance, hunting, and trapping, A. leari is classified as "endangered" by the Red Book of Endangered Brazilian Fauna and by the International Union for Conservation of Nature. At the same time, the number of individuals in captivity has been growing due to the efforts of biologists and veterinarians in zoological parks to reproduce this species. Thus, having species-specific reference intervals for hematologic and biochemical measurands is essential to assess the health status of these captive and wild birds and guarantee their reproductive success. This study aimed to determine reference intervals for hematologic and biochemical measurands for A. leari kept in captivity in São Paulo Zoo, Brazil. Blood samples from 32 individuals were selected based on inclusion and exclusion criteria. Only samples from adults and healthy individuals were included in this study. All statistical analyses were performed according to the American Society for Veterinary Clinical Pathology guidelines and available literature. Reference values and reference intervals were determined for the captive population of Anodorhynchus leari. This is the first study to establish reference intervals for A. leari, which can aid the veterinarian in the interpretation of laboratory test results, helping in the diagnosis, treatment, and monitoring of diseases of this endangered species of Brazilian fauna.

DNA Barcoding of Morphologically Characterized Mosquitoes Belonging to the Genus Mansonia from the Atlantic Forest and Brazilian Savanna

The identification of mosquito species is necessary for determining the entomological components of disease transmission. However, identification can be difficult in species that are morphologically similar. The cytochrome c oxidase subunit I (COI) DNA barcode region is considered a valuable and reliable diagnostic tool for mosquito species recognition, including those that belong to species complexes. Mansonia mosquitoes are found in forests near swampy areas. They are nocturnal and are highly attracted to light. Hematophagous adult females exhibit aggressive biting behavior and can become infected with and transmit pathogens during their feeding, including some epizootic viruses and avian malaria. In Brazil, twelve Mansonia species have been reported. In a recent study from the São Paulo Zoo in Brazil, three morphologically distinct species were collected and identified, namely: Mansonia (Mansonia) indubitans, Ma. (Man.) pseudotitillans and Ma. (Man.) titillans. However, confirmation of these species by molecular identification was unsuccessful due to a lack of COI sequences in the GenBank database. Thus, this research aimed to describe the COI DNA barcode sequences of some morphologically characterized Mansonia (Man.) species from Brazil and to determine their utility in delimiting species collected from the Atlantic Forest and Brazilian Savanna. Accordingly, we provide tools for the genetic identification of species that play a significant role in pathogen transmission in wildlife and potentially humans. We show that the delimitation of Mansonia species via five different approaches based on COI DNA sequences (BI, NJ, ASAP, bPTP and GMYC) yield basically the same groups identified by traditional taxonomy, and we provide the identification of specimens that were previously identified only up to the subgenus level. We also provide COI sequences from two Mansonia species that were not previously available in sequence databases, Ma. wilsoni and Ma. pseudotitillans, and thus contribute to the ongoing global effort to standardize DNA barcoding as a molecular means of species identification.

Are American Black Vultures a Biological Threat for Captive Animals and Environmental Health? a Case Report in São Paulo Zoo

Are American Black Vultures a Biological Threat for Captive Animals and Environmental Health? a Case Report in São Paulo Zoo

Assessing Diversity, Plasmodium Infection and Blood Meal Sources in Mosquitoes (Diptera: Culicidae) from a Brazilian Zoological Park with Avian Malaria Transmission.

Simple SummaryZoological gardens in forest areas host a large diversity of vertebrate species (exotic and indigenous, free-living and captive, migrant and resident), resulting in an artificial proximity of animal species that would never share the same environment in natural conditions. The presence of mosquitoes enables the transmission of vector-borne pathogens, as is the case with avian malaria parasites. The mild infections in some bird species may become a serious threat to others that do not possess a natural resistance. Thus, the identification of the potential vectors of these parasites is important for future control of these agents, aiming at the bird species conservation. In this study, we collected over 2000 mosquitoes in São Paulo Zoo and analyzed them through molecular methods. Six different mosquito species yielded positive for the targeted parasite DNA. We demonstrated that these culicids had fed mainly on bird species and we reported three mosquito species that have never been previously incriminated as potential vectors of these parasites, enabling the use of more specific measures for vigilance and mosquito control.Avian malaria parasites are widespread parasites transmitted by Culicidae insects belonging to different genera. Even though several studies have been conducted recently, there is still a lack of information about potential vectors of Plasmodium parasites, especially in Neotropical regions. Former studies with free-living and captive animals in São Paulo Zoo showed the presence of several Plasmodium and Haemoproteus species. In 2015, a pilot study was conducted at the zoo to collect mosquitoes in order to find out (i) which species of Culicidae are present in the study area, (ii) what are their blood meal sources, and (iii) to which Plasmodium species might they be potential vectors. Mosquitoes were morphologically and molecularly identified. Blood meal source and haemosporidian DNA were identified using molecular protocols. A total of 25 Culicidae species were identified, and 6 of them were positive for Plasmodium/Haemoproteus DNA. Ten mosquito species had their source of blood meal identified, which were mainly birds, including some species that were positive for haemosporidian parasites in the former study mentioned. This study allowed us to expand the list of potential vectors of avian malaria parasites and to improve our knowledge of the evolutionary and ecological relationships between the highly diverse communities of birds, parasites, and vectors present at São Paulo Zoo.

Nutritional and Protein Characterization of Leachate from the São Paulo Zoo composing Unit

In recent years the, Foundation Zoological Park of Sao Paulo (FZPSP) implemented and put into operation the Organic.

Feral Cats: Parasitic Reservoirs in Our Zoos?

Up until the recent past, zoos served limited function, primarily existing for entertainment value. Today’s zoos, however, are serving many roles, chief among them: species conservation of captive animals. The biggest zoo in Brazil, S?o Paulo Zoological Park Foundation, has among its 2000 animals and many species of wild cats. The presence of domestic cats living freely in zoos is common and can be a source of spreading disease. The aim of this study was to verify the variety and prevalence of parasites found in the feces of felids (feral and wild) living in the S?o Paulo Zoo. The results of this parasitological analysis have been obtained from the laboratory of clinical analysis and correspond to the 4-year period beginning January/2009 and ending December/2012. Eight species of parasites were identified in the feces of captive wild cats and three in the feces of feral cats. For those captives, Toxocara cati (7.95%) had the highest prevalence, followed by Toxascaris leonina (7.58%), Isospora sp. (2.03%), Hymenolepis nana (0.92%), Eimeria sp., Giardia sp. and Blastocystis sp. (0.37% each) and Ascaris sp. (0.18%). Among the feral cats, we found Toxocara cati (59.26%), Giardia sp. (22.22%) and Isospora sp. (11.11%). For the captive group, we also distinguished natives from exotic species, finding native species to be more frequently parasitized than the exotic ones. Key to our findings, though, was the fact that a few parasite species were found among all groups of felids, specifically (Toxocara cati, Giardia sp. and Isospora sp). Further research is needed, however, to confirm that transmission of these parasites is occurring between and among these groups.

Giardia spp., ten years of parasitological data in the biggest zoo of Latin America

Giardia spp. (Diplomonadida: Hexamitidae) is an important and widely studied protozoan parasite with worldwide distribution. Nowadays have six described species, and the most important probably is Giardia duodenalis due to the zoonotical potential that some assemblages have. Many studies analysing samples from wild animals have detected Giardia in captive environment, including the zoonotic type. The aim of this study was to determine the prevalence of Giardia sp. in wild captive animals at São Paulo Zoo, using conventional parasitological techniques (direct smear, passive flotation with saturated sodium chloride solution and simple gravity sedimentation), from 2006 to 2016. In total, 7066 coprological exams were performed during this period with samples from mammals, birds, reptiles and amphibians. The prevalence of Giardia infections was of 1.5% (103/7066). Mammals had the higher prevalence of infections with 2% (77/3872), followed by birds with 1.1% (25/2186) and reptiles with only one positive sample (1/894). All samples from amphibians were negative. Representatives of thirteen families presented positive results for this parasite: Dromaidae, Phasianidae, Ramphastidae, Cervidae, Giraffidae, Canidae, Felidae, Herpestidae, Myrmecophagidae, Callithrichidae, Cebidae, Hylobatidae and Dipsadidae. This study presents the first report of Giardia sp. in Pavo muticus and Brachyteles arachnoides. Infections were prevalent in Cebidae and Ramphastidae species. The findings of this study highlight the importance of identifying which Giardia assemblage are involved in the infections and if they may have a zoonotic potential.

A Tropical Composting Operation Unit at São Paulo Zoo as a Source of Bacterial Proteolytic Enzymes

Composting operation systems are valuable sources of microorganisms and enzymes. This work reports the assessment of proteolytic enzymes from cultivable bacteria isolated from a composting facility of the São Paulo Zoo Park (SPZPF), São Paulo, Brazil. Three hundred bacterial isolates were obtained and identified based on 16S rRNA gene as belonging to 13 different genera. The most common genus among the isolates was Bacillus (67%); some of which show high proteolytic activity in their culture media. Biochemical assays of hydrolytic activities using FRET peptides as substrates allowed the characterization of a repertoire of serine proteases and metalloproteases with different molecular weights secreted by Bacillus strains isolated from composting. Furthermore, thermostable serine and metalloproteases were detected in the composting leachate, which might be of interest for industrial applications.

Ticks infesting captive and free-roaming wild animal species at the São Paulo Zoo, São Paulo, Brazil

Ticks are ectoparasites of worldwide distribution that affect vertebrates and can transmit pathogens to animals and humans. The Zoological Park Foundation of São Paulo (FPZSP) is located in a Conservation Unit in one of the most important remaining fragments of the Atlantic Rainforest biome in the suburbs of São Paulo, Brazil. The FPZSP houses more than 3,000 wild animals on exhibit, in breeding programs and in environmental education programs, and also attracts migratory birds and free-roaming wildlife. This study focused on identifying the diversity of tick species that infest captive and free-roaming animals at the FPZSP. The collection of ticks kept at the FPZSP contains 523 specimens that were collected from different host species between 1990 and 2017. Ten tick species were found. In addition, Amblyomma aureolatum (Pallas) was found on stray cats living in the Atlantic forest fragment in the FPZSP. This study reveals a low occurrence of parasitism in captive animals and a high diversity of tick species collected from hosts in this Atlantic forest fragment, contributing information about host-parasite relationships and potential vectors of zoonotic diseases, since the vectors of Brazilian spotted fever, A. aureolatum and Amblyomma sculptum Berlese, were found in some hosts.

Genome-Centric Analysis of a Thermophilic and Cellulolytic Bacterial Consortium Derived from Composting.

Microbial consortia selected from complex lignocellulolytic microbial communities are promising alternatives to deconstruct plant waste, since synergistic action of different enzymes is required for full degradation of plant biomass in biorefining applications. Culture enrichment also facilitates the study of interactions among consortium members, and can be a good source of novel microbial species. Here, we used a sample from a plant waste composting operation in the São Paulo Zoo (Brazil) as inoculum to obtain a thermophilic aerobic consortium enriched through multiple passages at 60°C in carboxymethylcellulose as sole carbon source. The microbial community composition of this consortium was investigated by shotgun metagenomics and genome-centric analysis. Six near-complete (over 90%) genomes were reconstructed. Similarity and phylogenetic analyses show that four of these six genomes are novel, with the following hypothesized identifications: a new Thermobacillus species; the first Bacillus thermozeamaize genome (for which currently only 16S sequences are available) or else the first representative of a new family in the Bacillales order; the first representative of a new genus in the Paenibacillaceae family; and the first representative of a new deep-branching family in the Clostridia class. The reconstructed genomes from known species were identified as Geobacillus thermoglucosidasius and Caldibacillus debilis. The metabolic potential of these recovered genomes based on COG and CAZy analyses show that these genomes encode several glycoside hydrolases (GHs) as well as other genes related to lignocellulose breakdown. The new Thermobacillus species stands out for being the richest in diversity and abundance of GHs, possessing the greatest potential for biomass degradation among the six recovered genomes. We also investigated the presence and activity of the organisms corresponding to these genomes in the composting operation from which the consortium was built, using compost metagenome and metatranscriptome datasets generated in a previous study. We obtained strong evidence that five of the six recovered genomes are indeed present and active in that composting process. We have thus discovered three (perhaps four) new thermophillic bacterial species that add to the increasing repertoire of known lignocellulose degraders, whose biotechnological potential can now be investigated in further studies.

Diversity and distribution of avian malaria and related haemosporidian parasites in captive birds from a Brazilian\xa0megalopolis

BackgroundThe role of zoos in conservation programmes has increased significantly in last decades, and the health of captive animals is essential to guarantee success of such programmes. However, zoo birds suffer from parasitic infections, which often are caused by malaria parasites and related haemosporidians. Studies determining the occurrence and diversity of these parasites, aiming better understanding infection influence on fitness of captive birds, are limited.MethodsIn 2011–2015, the prevalence and diversity of Plasmodium spp. and Haemoproteus spp. was examined in blood samples of 677 captive birds from the São Paulo Zoo, the largest zoo in Latin America. Molecular and microscopic diagnostic methods were used in parallel to detect and identify these infections.ResultsThe overall prevalence of haemosporidians was 12.6%. Parasites were mostly detected by the molecular diagnosis, indicating that many birds harbour subclinical or abortive infections. In this project, birds of 17 orders (almost half of all the orders currently accepted in taxonomy of birds), 29 families, and 122 species, were tested, detecting positive individuals in 27% of bird species. Birds from the Anatidae were the most prevalently infected (64.7% of all infected animals). In all, infections with parasites of the genus Plasmodium (overall prevalence 97.6%) predominated when compared to those of the genus Haemoproteus (2.4%). In total, 14 cytochrome b (cytb) lineages of Plasmodium spp. and 2 cytb lineages of Haemoproteus spp. were recorded. Eight lineages were new. One of the reported lineages was broad generalist while others were reported in single or a few species of birds. Molecular characterization of Haemoproteus ortalidum was developed.ConclusionThis study shows that many species of birds are at risk in captivity. It is difficult to stop haemosporidian parasite transmission in zoos, but is possible to reduce the infection rate by treating the infected animals or/and while keeping them in facilities free from mosquitoes. Protocols of quarantine should be implemented whenever an animal is transferred between bird maintaining institutions. This is the first survey of haemosporidians in captive birds from different orders maintained in zoos. It is worth emphasizing the necessity of applying practices to control these parasites in management and husbandry of animals in captivity.

Occurrence of endoparasites in Ramphastidae (Aves: Piciformes) in São Paulo Zoo

Summary Ramphastidae (Aves: Piciformes) are animals recognized for their exuberant colours and long bill, with distribution range from south Mexico to north Argentina. They are important seed dispersers eating little vertebrates eventually. When in captivity, animals usually live in limited spaces, with high density and near by species that do not share a evolution history, facilitating the occurrence of infectious diseases for what they may not have a competent immune system against, including the parasitic ones. This study analyzed the endoparasites that occur in captive Ramphastidae at São Paulo Zoo in the period January 2009 to September 2011. Seven species of toucans and toucanets had parasitological results positive for Trichuridae nematodes, Eimeria sp., Giardia sp., non sporulated coccidian oocists and unidentified nematode eggs, and microfilarie was the only hemoparasite found.

Parasitological surveillance in a rat (Rattus norvegicus) colony in São Paulo Zoo animal house

Rattus norvegicus (Mammalia: Rodentia) is a widespread and synanthropic rodent, broadly used in medical experiments. It can also be used for feeding captive animals in zoos. Parasitological surveys are important to guarantee the health of both the animals and the staff responsible for their management. The aim of this study was to identify intestinal parasites of Rattus norvegicus offered as food to captive animals from São Paulo Zoo, and demonstrate the importance of sanitary hurdling, disease control and biosecurity. The identified protozoan parasites were Eimeria sp., Entamoeba sp., Spironucleus sp., Giardia sp., Tritrichomonas sp., Chilomastix sp., unidentified cysts and non-sporulated coccidians oocysts (Isospora/Eimeria). The following helminths were found: Syphacia muris, Rodentolepis nana and Aspiculuris tetraptera.

Microbial community structure and dynamics in thermophilic composting viewed through metagenomics and metatranscriptomics.

Composting is a promising source of new organisms and thermostable enzymes that may be helpful in environmental management and industrial processes. Here we present results of metagenomic- and metatranscriptomic-based analyses of a large composting operation in the São Paulo Zoo Park. This composting exhibits a sustained thermophilic profile (50 °C to 75 °C), which seems to preclude fungal activity. The main novelty of our study is the combination of time-series sampling with shotgun DNA, 16S rRNA gene amplicon, and metatranscriptome high-throughput sequencing, enabling an unprecedented detailed view of microbial community structure, dynamics, and function in this ecosystem. The time-series data showed that the turning procedure has a strong impact on the compost microbiota, restoring to a certain extent the population profile seen at the beginning of the process; and that lignocellulosic biomass deconstruction occurs synergistically and sequentially, with hemicellulose being degraded preferentially to cellulose and lignin. Moreover, our sequencing data allowed near-complete genome reconstruction of five bacterial species previously found in biomass-degrading environments and of a novel biodegrading bacterial species, likely a new genus in the order Bacillales. The data and analyses provided are a rich source for additional investigations of thermophilic composting microbiology.

Capillary electrophoresis coupled to contactless conductivity detection for analysis of amino acids of agricultural interest in composting

Electrophoresis 2016, 37, 2449–2457. DOI: 10.1002/elps.201600302 If an average household produces weekly several pounds of organic garbage, imagine the volume of a large zoo like São Paulo Zoo. The amount of organic waste, from feeding left overs, to plants, manure and even dead animals, is enough to produce up to 120 tons monthly. If all this organic matter was sent to dumping grounds, this would create a serious environmental problem. Composting is a sustainable approach by the São Paulo Zoo to manage animal and vegetal waste generated in the Zoo's premises. The resulting compost is often used as organic fertiliser for the production of vegetables, which is further used to feed the animals. The compost produced provides many forms of minerals and also amino acids (AA) that are used by plants as nutrients. Since most amino acids absorb only slightly or not at all in the UV wavelengths, we developed a method for the determination of AA of agricultural interest in the compost samples. Due to the complexity of these samples, we used ion exchange chromatography for purification of AA prior analysis. The proposed CZE-C4Dmethod allowed a separation of the AA in a short analysis time (less than 3.0 min) without the need for derivatization, with sensitivity and reproducibility suitable for such samples. Photograph: Paulo Gil

Characterization of mycobacteria and mycobacteriophages isolated from compost at the São Paulo Zoo Park Foundation in Brazil and creation of the new mycobacteriophage Cluster U.

BackgroundA large collection of sequenced mycobacteriophages capable of infecting a single host strain of Mycobacterium smegmatis shows considerable genomic diversity with dozens of distinctive types (clusters) and extensive variation within those sharing evident nucleotide sequence similarity. Here we profiled the mycobacterial components of a large composting system at the São Paulo zoo.ResultsWe isolated and sequenced eight mycobacteriophages using Mycobacterium smegmatis mc2155 as a host. None of these eight phages infected any of mycobacterial strains isolated from the same materials. The phage isolates span considerable genomic diversity, including two phages (Barriga, Nhonho) related to Subcluster A1 phages, two Cluster B phages (Pops, Subcluster B1; Godines, Subcluster B2), three Subcluster F1 phages (Florinda, Girafales, and Quico), and Madruga, a relative of phage Patience with which it constitutes the new Cluster U. Interestingly, the two Subcluster A1 phages and the three Subcluster F1 phages have genomic relationships indicating relatively recent evolution within a geographically isolated niche in the composting system.ConclusionsWe predict that composting systems such as those used to obtain these mycobacteriophages will be a rich source for the isolation of additional phages that will expand our view of bacteriophage diversity and evolution.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0734-3) contains supplementary material, which is available to authorized users.

Hemosporidian parasites of free-living birds in the São Paulo Zoo, Brazil

Numerous studies addressed the diversity of bird Plasmodium and Haemoproteus parasites. However, a few have been carried out in continental avian hotspot regions such as Brazil, a country with markedly different biomes, including Amazon, Brazilian Savanna, Atlantic Forest, Caatinga, Pantanal, and Pampas. We present the first study on hemosporidian (Haemosporida) parasites in free-living birds from an Atlantic Forest fragment where more than 80 avian species have been reported. Within this area, the São Paulo Zoo locates, and it is the fourth largest zoo in the world and the largest in Latin America. A total of 133 free-living bird samples representing 12 species were collected in the zoo, with the overall hemosporidian prevalence of 18 % by PCR-based diagnostics. Twenty-four positive PCR signals were reported from four different bird species, including migratory ones. Columba livia, an urban species, considered nowadays a pest in big cities, showed 100 % prevalence of Haemoproteus spp., mainly Haemoproteus columbae. We discuss the epidemiological importance of new parasites introduced by migratory birds in the São Paulo Zoo area and the risk it poses to the captive species, which are natives or exotics. We also warn about the influence these parasites can have on the biodiversity and the structure of host populations by altering the competitive interaction between the free-living and the captive birds.

Halotolerant bacteria in the São Paulo Zoo composting process and their hydrolases and bioproducts.

Halophilic microorganisms are able to grow in the presence of salt and are also excellent source of enzymes and biotechnological products, such as exopolysaccharides (EPSs) and polyhydroxyalkanoates (PHAs). Salt-tolerant bacteria were screened in the Organic Composting Production Unit (OCPU) of São Paulo Zoological Park Foundation, which processes 4 ton/day of organic residues including plant matter from the Atlantic Rain Forest, animal manure and carcasses and mud from water treatment. Among the screened microorganisms, eight halotolerant bacteria grew at NaCl concentrations up to 4 M. These cultures were classified based on phylogenetic characteristics and comparative partial 16S rRNA gene sequence analysis as belonging to the genera Staphylococcus, Bacillus and Brevibacterium. The results of this study describe the ability of these halotolerant bacteria to produce some classes of hydrolases, namely, lipases, proteases, amylases and cellulases, and biopolymers. The strain characterized as of Brevibacterium avium presented cellulase and amylase activities up to 4 M NaCl and also produced EPSs and PHAs. These results indicate the biotechnological potential of certain microorganisms recovered from the composting process, including halotolerant species, which have the ability to produce enzymes and biopolymers, offering new perspectives for environmental and industrial applications.

English

As the world population increases, the need for energy resources increases and for decades, petroleum has sustained this demand. Oil spill during petroleum extraction and processing has a negative impact on the environment. Methods that can decrease the impact of xenobiotic compounds on the environment have been developed. Among many bioremediation methods described, microbial remediation is of great importance. Our previous work demonstrated that Organic Waste Composting Unit (OWCU) located at the São Paulo Zoo Foundation (FPZSP) had a great diversity of microorganism. In the present work, we viewed this environment searching for xenobiotic degrading microorganisms by sampling the composting at various stages of the process with the aim of isolating the bacteria that would break down n-hexadecane, a model compound for hydrocarbon degradation. Two bacterial collections were assembled and tested in a 96-well plate model using n-hexadecane as a sole carbon source. Among the 418 isolates screened, eight were selected based on their ability to assimilate n-hexadecane. Molecular identification revealed their genus and species which are associated with xenobiotic degradation activities in different microbial consortia. However, these microorganisms have not being isolated from the same transforming process. Future studies with these isolates may shed light on the bacteria hydrocarbon degradation mechanism.   Key words: Composting, crude oil degrading microorganism, bioremediation, n-hexadecane degrading bacteria.