Dr. Lanzhuang Chen
Division of Biotechnology and Breeding, Faculty of Environmental and Horticultural Science,
The Dean of Graduate School of Horticulture and Foods, Minami Kyushu University, Japan.


ISBN 978-93-90149-82-7 (Print)
ISBN 978-93-90149-83-4 (eBook)
DOI: 10.9734/bpi/imb/v1


This book covers key areas of microbiology and biotechnology. The contributions by the authors include total petroleum hydrocarbon, groundwater, bacteria, nutrients, natural attenuation, biodegradation,  poly-phosphate accumulating bacteria, poly-phosphate, phosphate removal, Pi index, ethanol stress, freeze-drying starter, yeasts, hydrophilins, traditional sorghum beer, biodiesel, diesel engine, engine performance, transesterification, protein expression, membrane proteins, cytochrome P450, galling index, incidence, meloidogyne species infection, population densities, bacteriocin, urinary catheter, antibiotics, etiological agents, antimicrobial therapy, urinary tract infection, antimicrobial susceptibility pattern, clinical samples, Mayaro virus, arbovirus, emerging disease, viral structure, immune response, pathogenesis, antibiotic sensitivity, hemodialysis, phb genes, EMP pathway, PP pathway, ED pathway, diatom, transgenic plant, petroleum based plastics. This book contains various materials suitable for students, researchers and academicians in the field of microbiology and biotechnology.


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This study described the role of aerobic microorganisms in the biodegradation of petroleum hydrocarbon contaminated groundwater. Groundwater sample treated with nutrients were compared with untreated water sample. The total hydrocarbon content removal in the water treated with nutrients and untreated water sample (natural attenuation) at day 56 showed percentage removal of 89 and 74, respectively. Total heterotrophic and total hydrocarbon utilizing bacterial count increased progressively in all experimental samples. Bacterial strains isolated include Bacillus sp., Arthrobacter sp., Micrococcus sp., Pseudomonas sp., Alcaligenes sp. and Flavobacterium sp. The total petroleum hydrocarbon reduced from 15.33955 mg/L to 3.90361 mg/L and 9.49634 mg/L, in water sample treated with nutrient and untreaed water sample, respectively at the end of day 56. The physicochemical analyses at day 56 were within the World Health Organization standard for drinking water. Total petroleum hydrocarbons in laboratory contaminated groundwater were utilized by the aerobic microorganisms present in the water samples, however, treatment with nutrient showed greater removal of the hydrocarbon pollutants.

Poly-phosphate accumulating bacteria (PAB) is an important bacterial group that can take up large amounts of phosphate and accumulate as intracellular poly-phosphate, contributing to biological phosphorus removal in waste-water treatment. The study was conducted to analyze a genetic diversity of PAB community isolated from samples of water and sludge of intensive catfish ponds and effluent water and sludge obtained from piggery waste-water treated by bio-digesters in the Mekong Delta, Vietnam. In a total of 439 isolates, there are 48 isolates that have the potential of accumulating intracellular poly-phosphate shown main characters included shaped like a rods or short rod-shaped; motile, twitching movements or non-motile; expression of poly-phosphate electron dense granules within the cells; accumulated poly-phosphate from poly-phosphate kinase gene 1; the content of intracellular poly-phosphate varied from 10-9 to 10-12 mg/cell. Based on phylogenetic tree for partial 16S rRNA genes, there are 22 strains isolated from intensive catfish ponds included in four classes: Bacilli, Actinobacteria, Beta-proteobacteria, Gamma-proteobacteria, and 26 strains isolated from piggery waste-water treated by bio-digesters included in four classes: Bacilli, Actinobacteria, Alpha-proteobacteria, Gamma-proteobacteria. The process of analysis and comparison of genetic diversity of 48 isolates showed 16S rRNA sequences have nucleotide regions of high variability interspersed with nucleotide regions of low variability. The variation of o and Pi index formed the different types of haplotype in population of PAB, there were 25 haplotypes (genotypes) from 48 sequences. The difference of the structure of haplotypes formed a high diversity between them (Hd=0.91). The levels of haplotype diversity created many genotypes for the genetic variation and the ability to adapt to the environment in the evolutionary process of bacterial strains capable ofhigh accumulating poly-P. Comparison of genetic diversity between populations of bacteria isolated from two sampling places, the results showed that the nucleotide and haplotype diversity of the strains isolated in piggery waste-water treated biodigesters (Pi=0.16, h=14) were lower and the genetic conservation was higher than isolated strains in intensive catfish ponds (Pi=0.18, h=16). This is scientific basis for the selection of the sample sources for the isolation and selection of poly-phosphate accumulating strains.

Determining the Potentialities of Yeast Strains to be Used as Freeze- Drying Starters for the Production of Traditional Sorghum Beer in Côte d’Ivoire

Wahauwouélé Hermann Coulibaly, Fatoumata Camara, Koffi Maïzan Jean- Paul Bouatenin, Alfred K. Kouamé, Youan Charles Tra Bi

Innovations in Microbiology and Biotechnology Vol. 1, 30 August 2021, Page 30-46

The present study was aimed to select strains of Saccharomyces cerevisiae and Candida tropicalis based on their ability to tolerate ethanol toxicity and to evaluate the expression level of he hydrophillins encoding genes TIF 11 and YJL144W to express encoding for hydrophilins.

Freeze-drying is a popular dehydration method for preserving microorganisms. . In order to produce freeze-dried yeast starter culture for the brewing purpose of African traditional sorghum beer, we tested tolerance to ethanol stress and evaluated the relative expression level of genes TIF 11 and YJL144W encoding the hydrophilins. Among the strains tested, the best viability rate to ethanol stress (7.5% ethanol (v/v)) was found with Saccharomyces cerevisiae F12–7 and Candida tropicalis C0–7 respectively with 95% and 80%. For Saccharomyces cerevisiae strains, the strain F12-7, which had distinguished itself from other strains in previous tests, the TIF11 and YJL144W genes were the least expressed. For C. tropicalis strains, the statistical analyzes of the relative expression levels from the Tukey test revealed no difference between the strains for the 2 genes (P> 0.05). It is concluded that before freeze-drying starter production other investigations are needed in order to evaluate yeast performance. Also, the pathogenicity of Candida tropicalis strains will be studied for the using potentially non-pathogen strains.

Determination of Engine Performance and Emission with Neem Oil (NOME) Based Bio-Diesel

Tejaswita Kajale, Abhay Pawar, Channapatana .

Innovations in Microbiology and Biotechnology Vol. 1, 30 August 2021, Page 47-52

The present study aims to investigate the production of biodiesel from neem oil with a view to determine its performance in Internal Combustion engine (I.C. engine). The physicochemical properties of the biodiesel produced were also studied. The world is getting modernized and industrialized day by day. As a result vehicles and engines are increasing. But energy sources used in these engines are limited and decreasing gradually. This condition necessitates the use of biodiesel as an alternate fuel for diesel engines. Biodiesel is a non-toxic, biodegradable, and renewable alternative fuel that may be used in diesel engines with very minor changes. Biodiesel could be produced from low-cost Neem seed oils. Biodiesel performance and testing is done in C.I. engine. Neem oil was extracted from neem seed by solvent extraction. Refractive index, density, viscosity, ash content, Saphonification value, iodine number was studied. Biodiesel has been prepared from NEEM oil by esterification and transesterification. It was examined for physical and chemical properties and chemical properties. HC, CO, NOx, SOx, and particulate matter was studied. The conversion of the biodiesel fuel's energy to work was equal to that from diesel fuel. The results also clearly indicate that the engine running with biodiesel and blends have higher NOx emission by up to 20%. However, the emissions of the CI engine running on neat biodiesel (B100) were reduced by up to 15%, 40% and 30% for CO, CO2 and THC emissions respectively, as compared to diesel fuel at various operating conditions. Biodiesel is linked to lower HC, CO, and particle emissions. This is likely owing to the fact that biodiesel includes around ten percent oxygen by weight, which aids in the oxidation of these combustion products in the cylinder.

Yeast as a Versatile System for the Expression of Heterologous Proteins

Rajeev Soni

Innovations in Microbiology and Biotechnology Vol. 1, 30 August 2021, Page 53-58

The yeast, Saccharomyces cerevisiae has been extensively used for heterologous protein expression due to its ease of genetic manipulation, faster generation time, availability of integrative and episomal vectors and promoters that can be easily turned on and off using simple chemical moieties. In addition, being a simplest eukaryote, it offers several advantages in the expression of mammalian proteins regarding post-translational modifications and expression of proteins that are membrane bound. This article describes development of a strain for the expression of membrane bound human drug metabolism enzymes.

Root-knot Nematode Infections in Okra in the Middle Belt of Ghana

Yaw Danso

Innovations in Microbiology and Biotechnology Vol. 1, 30 August 2021, Page 59-66

A survey study was conducted in six major okra growing Districts of Ghana between August and December 2014. It was to assess the prevalence, incidence, and severity of root-knot disease in okra incited by root-knot nematodes. Fifty-four sole okra fields were purposively selected from 18 localities for the study. On an acre okra farm, 25 representative plants were sampled in an N-shaped pattern and assessed for incidence and degree of knotting following a rating scale of 0 to 10. Seventy-six percent of the okra fields assessed were found to be positive for root-knot nematodes infection. Seventy-eight, 67, 67, 89, 100, and 56% root-knot nematodes infection prevalence were recorded in the Kintampo North, Atebubu Amantin, Wenchi, Ejura Sekyedumase, Offinso North, and Atwima Nwabiagya Districts, respectively. The highest infection incidence (95%) was recorded at Sentiatia in the Offinso North District while the lowest (22%), at Amponsakrom in the Wenchi District. Galling indices ranged between 5 and 8 across the Districts. The root-knot nematodes infestation menace in okra was common across the study area. Sustainable management strategies must be designed by crop protectionists and disseminated among okra farmers against the pest to boost okra production in Ghana and beyond.

Investigating of the Relationships between Non-Culturability and Nisin Production of Lactococcus lactis

Yury D. Pakhomov, Larisa P. Blinkova, Olga V. Dmitrieva, Olga S. Berdyugina, Lidia G. Stoyanova

Innovations in Microbiology and Biotechnology Vol. 1, 30 August 2021, Page 67-81

We studied formation of non-culturable forms of three bacteriocin (nisin) producing strains of Lactococcus lactis subsp. lactis: MSU, 729 and F-116 under carbohydrate starvation stress. Two different types of inoculum were applied: A) unwashed cells with culture liquid, B) cells washed twice with normal 0,9% saline. Resulting total numbers of cells were 0.6 1.0×108 cells/ml for both types of inoculum. Population obtained using type A inoculum demonstrated active growth phase within first 1-5 days of incubation (up to 2.4×109 cells/ml) while those obtained using type B inoculum did not grow within that period. Type B population of strain MSU showed phenotypic dissociation that resulted in appearance of micro colonies. After that, we observed active growth phase (up to 5.2×109 cells/ml). Type B cultures of strains 729 and F-116 did not grow during the whole experiment. It was shown that type A population shifted into non-culturability faster than type B. This is due to differences in metabolic strategies and stress sensitivity of these types of population. After 1 year of incubation (383 days) culturability decreased by 3 orders of magnitude for type B (5 orders for type B population of strain MSU) and by 6 orders of magnitude for type A population. We also observed considerable reduction of cell size for type A population of strains 729 and F-116. Studies of bacteriocin activity showed that in type B population cells were up to 78 times more productive compared to those of type A cultures. This phenomenon can be explained by differences in survival strategies of population that use antibacterial potential of bacteriocins for their benefit.

Background: The etiological agents of catheter-associated urinary tract infection (CAUTI) are sundry and often multidrug-resistant. An early, appropriate antimicrobial therapy based on culture sensitivity report and implementation of infection control practices together play a key role in the management of CAUTI.

Objectives: 1. To find the incidence of CAUTI in intensive care unit (ICU) patients.
2. To identify etiological agents responsible for CAUTI.
3. To determine antimicrobial susceptibility pattern for bacterial agents obtained.

Methods: Urine samples from clinically suspected cases of CAUTI were obtained from patients with urinary catheters admitted in ICUs. Urine samples were subjected to wet mount, culture, and sensitivity testing. Responsible pathogens and their antimicrobial susceptibility pattern were obtained based on CSLI standards.

Results: A total of 640 patients were having an indwelling urinary catheter with an aggregate of 5199 catheter-days in 6 months duration. 19 were culture positive out of 45 clinically suspected cases of CAUTI. The CAUTI rate was 3.65 cases per 1000 catheter days. The most predominant etiological agents responsible for CAUTI were Citrobacter species (26.3%) followed by P. aeruginosa (21.1%) and E. coli (21.1%). 2(18.2%) ESBL producing Gram-negative bacteria were obtained among Enterobacteriaciae. The most effective antibiotics were ceftazidime/clavulinic acid, colistin, and meropenam for Enterobacteriaciae; piperacillin/tazobactam for P. aeruginosa; linezolid and vancomycin for Enterococcus species.

An Analysis of Data of Enterococci Species and Its Antibiogram at a Tertiary Care Hospital in Karnataka

Trupti B. Naik, Vijaykumar Mane, Amruthkishan K. Upadhya

Innovations in Microbiology and Biotechnology Vol. 1, 30 August 2021, Page 89-97

Background: Enterococci were previously thought to be less virulent in healthy people, but they have recently emerged as important opportunistic pathogens, particularly in hospitalized patients, due to their ability to colonize the gastrointestinal tract for long periods, which is a key factor in the development of drug resistance and has become a major treatment roadblock.

Objectives: To determine the species of Enterococci isolated from different clinical samples. 2. To determine the antimicrobial susceptibility pattern of Enterococci species.

Methods and Materials: A cross-sectional study was carried out at a private tertiary care hospital in the Shivamogga district of Karnataka, using secondary data of Enterococcal species isolated from various clinical samples such as urine, blood, pus, sputum, and sterile body fluids that were maintained in the Microbiology laboratory registers for a period of one year from January 2014 to December 2014.

Results: Out of 66 enterococcal isolates from various clinical samples majority i.e. 32 (48.48%) were found in urine, followed by pus 22 (33.33%). The majority of Enterococcus was seen in females 34 (51.51%). Enterococcus faecalis was the most common isolate, accounting for 56 (84.84%) of the total. Sensitivity to Linezolid and Vancomycin was found in all of the isolates. Maximum resistance was seen against Penicillin i.e. 26 (76.47%).

Conclusion: Given the general trend of rising drug resistance and the occurrence of a wide range of Enterococcus species, ongoing surveillance of antimicrobial resistance in enterococci is required to propose suitable therapy.

Study on Mayaro Virus Disease: Symptoms and Treatments

Salvatore G. De-Simone, Paloma Napoleão-Pego, Larissa Rodrigues Gomes, Guilherme C. Lechuga, David W. Provance-Jr

Innovations in Microbiology and Biotechnology Vol. 1, 30 August 2021, Page 98-113

This chapter aimed to review the various aspects related to symptoms, pathogenesis, treatment, and diagnosis of fever caused by the Mayaro virus. Mayaro virus produces a nonspecific, sublethal disease in man with symptoms often confused with chikungunya, dengue, and another virus. The symptoms of arthralgia, often associated with these viral infections, can cause an incapacitating disability. To date, outbreaks have been localized and sporadic within the Pan-Amazonia forest since its first isolation in 1954 [Trinidad and Tobago]. Thus, the available literature is diverse, scarce, and dispersed. Mayaro virus is an alphavirus, which is phylogenetically related to the Semliki forest antigenic complex. In the New World, Mayaro and the related UNA viruses are the only members of this complex isolated. The genome of Mayaro consists of single-stranded RNA with a positive charge and a length of 12 kb that can be subdivided into genomic and subgenomic regions that encode nonstructural and structural proteins, respectively. Mayaro has shown remarkable plasticity in hosts for vertebrate infections but high specificity invertebrates towards the family Culicidae [mosquitoes]. Risk factors for disease are forested areas in northern South America and the rainy season. Two genotypes of MAYV have been identified, L [restricted to Belterra, Brazil] and D [widely distributed in the Pan-Amazonia]. The enzootic cycle is similar to the jungle cycle of yellow fever, which involves Haemagogus mosquitoes and monkeys as reservoirs. Of concern is the potential spread of the virus by the involvement of other secondary vectors and hosts such as Aedes aegypti, Aedes albopictus, and Aedes scapularis that have been shown experimentally to transmit the virus efficiently. With the observed high viremia levels of infected individuals, a significant risk exists for an emerging disease in urban, rural, and peridomestic locations close to enzootic foci of the Mayaro virus.

Study on Bacteremia in Hemodialytic Patients and Antibiotics Susceptibility Pattern

Shrestha Prasansah, Pokharel Nabaraj, Pant Anil Dev

Innovations in Microbiology and Biotechnology Vol. 1, 30 August 2021, Page 114-120

Background: Bacteremia is the presence of bacteria in the blood stream. Because hemodialysis patients are immunocompromised, bacterial blood stream infection is the most common cause of death. In hemodialytic patients, blood stream infections are mainly due to Gram-positive cocci. Patients undergoing hemodialysis are especially vulnerable if the infection is caused by Staphylococcus aureus that accounts more than 8% mortality rate. 

Methods: A cross-sectional study was carried out at National Kidney Center of Nepal for duration of six months. Blood samples were collected from the individuals undergoing hemodialysis treatment. The collected blood samples were inoculated in brain heart infusion (BHI) biphasic medium in a blood culture bottle and incubated aerobically at 37°c for 18-24 hours (up to 1 week if necessary). It was then sub-cultured on blood agar plate where colony morphology and hemolytic reactions were observed. In order to identify the isolate organisms, a series of biochemical assays were performed. The modified Kirby Bauer technique was used to conduct the antimicrobial susceptibility test. The SPSS software was used to record and evaluate all the data collected.

Results: The study comprised of a total of hundred patients. Gram-positive bacteria were found to have highest frequency (61.5%). Gram-positive bacteria such as Staphylococcus aureus (26.9%) and CoNS (88.4%) were isolated in high frequency, as were Gram-negative bacteria such as Escherichia coli (3.8%), Klebsiella pneumoniae (3.8%), and Pseudomonas aeruginosa (3.8%) were also isolated. Staphylococcus aureus was shown to be the most prevalent Gram-positive isolate among hemodialysis patients. Most of these S. aureus strains were reported to be amoxicillin resistant but cefoxitin sensitive Methicillin resistance was detected in 37.5% of the 16 S. aureus isolates. For most Gram-positive bacteria, amoxicillin was shown to be the least effective drug, whereas ciprofloxacin was found to the most effective.

Conclusion: The purpose of the study is to understand the prevalence of bacteria in hemodialytic patients as well as the antibiogram of the isolates as it is the first to study about bloodstream infections in hemodialytic patients in Nepal. These results might provide a foundation to explore more about bacteriological infection study in hemodialytic patients and antibiogram.

Petroleum based plastics and their derivatives that are non-biodegradable cannot be ignored at all because they have been using in every aspect of our daily life such as applications in packaging, textile, agriculture, electronics, medical, building construction, injection and moulding. Consumption of these materials are growing day-by-day that are ultimately increasing environmental pollution, global warming, and waste management cost as well as threatening the biodiversity and life on earth. Since our earth is the only planet that contains life among the countless planets in outer space, this world needs to be protected by reducing the pollution and implementing other regulatory measures. Therefore, biodegradable plastics should be used as alternative to non-biodegradable plastics. Poly-3-hydroxybutyrate (PHB) has been extensively studied and is the best-characterized biodegradable plastic within the poly-hydroxyalkanoates family. It is used to make a wide range of household and packaging products, as well as medical products. Although biodegradable PHB is environmentally friendly and does not require fossil resources, it has traditionally been exceedingly expensive to produce utilising bacterial fermentation processes involving recombinant E. coli. For efficient PHB synthesis, recombinant diatoms and transgenic plants have also been investigated. But, increasing PHB yield at the theoretical maximum level has been proved extremely difficult that prohibits its industrial scale production. To address these problems, the objective of this chapter is to focus the importance on the metabolic pathway manipulations in recombinant E. coli. The main advantage of using genetically engineered E. coli is that PHB granules are not degraded once synthesized since they lack PHB degradation pathways unlike native producers. Other benefits of employing recombinant E. coli include their capacity to (i) use a wide range of inexpensive carbon sources, (ii) accumulate huge amounts of polymers with better productivity, (iii) maintain high-cell density fermentation, and (iv) recover the PHB very easily.Since no single strategy has been proved to be sufficient enough to produce PHB industrially until today, this chapter has also shed light on developing the advanced and integrated approaches for efficient PHB production in order to compete with non-biodegradable petrochemical plastics.

Bacterial Treatment of Drill Cuttings: Experimental Investigation

S. E. Ibekwe, G. C. Okpokwasili

Innovations in Microbiology and Biotechnology Vol. 1, 30 August 2021, Page 128-139

During exploration and development drilling operations, the presence of polycyclic aromatic hydrocarbons (PAH) from crude oil and gas condensate, ferrochrome lignosulphate, and lead compounds in drill cuttings and drilling mud additives is a source of concern for the environment. This investigation was prompted by the environmental toxicants discovered during drilling operations. This study was focused on using scientific and technological methods that are both efficient and cost-effective in lowering chemical toxicants in the environment to levels below the permissible limit, in order to fulfill the millennium objective of a sustainable environment. This goal was achieved by inducing PAH and TPH breakdown in the hydrocarbon using microorganisms found in drill cuttings. The physiochemical factors that promote the proliferation of hydrocarbon-using bacteria have been identified. Throughout the investigation, microbial populations, total petroleum hydrocarbon (TPH), and PAH levels were measured at regular intervals. Drill cutting (Dc) from an oil well in the Usan field was sampled for bioremediation treatment. Using plastic bowls, the five treatment choices Dc, Dc+S, Dc+F, Dc+S+F+D, and Dc+D were established in triplicates in distinct cells. A total of 15 plastic bowls with a diameter of 35cm and a depth of 11cm were used. Throughout the study period, the setup was sampled at regular intervals for analysis. Four treatment alternatives were biostimulated with soil (S), NPK fertiliser (F), or Gold Crew dispersant (D), with controls (DC) and heat-treated (hDC).For each treatment choice, 40g of treatment material was added to 2 kg (wet weight) of drill cutting. A 56-day study period was used to investigate the bioremediation process. On day 0, the unaffected treatment (Dc) had the greatest heterotrophic bacterial count (4.5 x 105 cfu/g), whereas Dc+D had the lowest (3.0 x 103 cfu/g) on day 56.

Dc+D had the highest count (6.5x 103 cfu/g) on day 28 and the lowest level (2.6 x 102 cfu/g) on day 56, according to the hydrocarbon utilizing bacterial count. On day 0, total petroleum hydrocarbons (TPH) ranged from 33.22 to 46.00 mg/kg for all treatment choices, whereas polycyclic aromatic hydrocarbons (PAH) ranged from 3.51 to 6.4 mg/kg.  In all the treatment option by day 56, the TPH was <8.0mg/kg and PAH <2.5mg/kg. By day 56, the percentages of biodegradation of TPH as measured with GC-FID were Dc(71.82%), Dc+S(77.09%), Dc+F(83.58%), Dc+S+F+D(79.95%), Dc+D(81.58%) and heat-treated (30.56%). PAH percentage degradation rates were as follows: Dc (49.92%), Dc+S (52.35%), Dc+F (86.09%), Dc+S+F+D (64.74%), Dc+D (74.20%) and heat-treated (2.23%). Dc+F gave the highest percentage degradation for both TPH and PAH. Fifty-two hydrocarbons were produced using bacterial isolates. Bacillus spp. (26.92%), Proteus Sp. (1.92%), Pseudomonas spp. (7.36%), Alcaligenes spp. (5.77%), Micrococcus spp. (7.55%), Acinetobacter sp. (1.92%), Aeromonas spp. (21.15%), and unidentified bacteria were among the bacteria isolated (28.85 percent ).Many of the bacterial isolates displayed hydrocarbon degradative capability, according to a screen test for degradative potential. As indicated in the heat-treated control, the drill cuttings analysed could be remediated utilising microbiological agents, and environmental conditions (abiotic factors) had a role in hydrocarbon alternation.