Industrial and Environmental Biotechnology Chapter Abstracts
Chapter 1: Molecular Mechanisms Regulating the Catechol and Chlorocatechol Biodegradative Operons
Sudha A. Chugani and Ananda. M. Chakrabarty
Abstract
The conversion of catechol and 3-chlorocatechol to tricarboxylic acid (TCA) cycle intermediates in Pseudomonas putida is mediated by the catBCA and clcABD operons respectively. The two operons are regulated by the LysR homologues CatR and ClcR with cis,cis-muconate and 2-chloromuconate as the respective inducers. CatR and ClcR have 32.5% sequence identity and 43% similarity at the amino acid level and a comparison of the cat and clc systems suggests that the two regulators utilize similar transcriptional activation mechanisms. In vitro transcription analyses showed that this mechanism involves interactions between the regulators and the a-carboxy terminal domain (a-CTD) of RNA polymerase. In vitro transcription studies done utilizing mutant RNA polymerases suggest that in case of CatR the a-CTD may also interact with the DNA at the promoter region. In vitro transcription studies using different clcABD promoter region fragments demonstrated that the a-CTD interacts with the ClcR dimer bound to the promoter proximal site. Gel shift and footprinting analyses have shown that CatR has two binding sites in the catBCA promoter region. Another low affinity CatR binding site, the internal binding site (IBS) is located within the catB structural gene. Site directed mutagenesis studies demonstrated that this site repressed catBCA operon expression approximately 3-4 fold under inducing conditions in vivo. Footprinting and phasing experiments further indicated that the occupation of this site is mediated by cooperative interactions with CatR bound to the two upstream binding sites by looping of the intervening DNA. Growth of P. putida cells on succinate, citrate or fumarate results in repressed transcription from the clcABD promoter. It was further observed in in vitro transcription assays that fumarate, which is a TCA cycle intermediate, specifically abrogates clcA transcript formation. Both the catechol and chlorocatechol pathways feed into the TCA cycle; however, unlike the clcABD promoter the presence of succinate, citrate or fumarate did not affect expression from the catBCA promoter.
Chapter 2: Biotreatment of Phenolic Waste from a Typical Pharmaceutical Plant
S. Ahmed and A. Hameed
Abstract
Basic laboratory studies for the biotreatment of phenolic wastewater from a typical pharmaceutical plant were carried out. Some processes produce waste water containing phenol, about 12 g.l1 (9.33 g.l1, 4-nitrosophenol and 3.11 g.l1, phenol) with high concentrations of sulfates (11.8 g.l-1). Waste water was diluted ten-fold with sewage water, and incubated with Pseudomonas putida F1 (toluene-induced cells) in batch fermentation experiments. Virtually all phenolic concentrations were reduced after 70-80 hours. This biotreatment worked efficiently at 20-25°C, 37°C, and 42°C though at lower temperatures (10-15°C) only a 40% reduction in the phenolic concentration was achieved after 72 h of fermentation.
Chapter 3: Genes for Pentachlorophenol Degradation and the Bacteria That Contain Them
V. Saboo, M. Sullivan, V. Sobhon, and M.A. Gealt
Abstract
Pentachlorophenol (PCP) is a priority pollutant that has been used to preserve wood and as a precursor for synthesis of several biocides. Contamination is common near sites where wood was treated. The literature indicates that PCP is degraded aerobically as well as anaerobically. Several species initially reported to degrade PCP aerobically have now been reclassified Sphingomonas chlorophenolica. DNA sequences for genes involved in the biodegradation pathway are available. The PCP-4-monooxygenase, the product of the pcpB gene, has been identified as the initial (and probably most critical) gene in the pathway. This enzyme dechlorinates PCP to tetrachlorohydroquinone (TCHQ). Using PCR, different soil samples were examined for pcpB gene sequences. Sequences were found in soil from near a local rail yard, even when no PCP was demonstrated. Sequences were less common in soil samples collected farther from the rail yard site. These findings suggests acclimation of indigenous bacteria to degrade PCP happens in places that have likely been contaminated. This process acclimation requires bacterial acquisition of the degradative genes. Using hybridization techniques and PCR, new isolates able to grow on PCP at 50 µg/ml were obtained from contaminated soil. Several of these isolates contained the pcpB gene sequences but were not able to degrade PCP. Based on fatty acid analysis and general growth characteristic, these strains were demonstrated not to be S.chorophenolica. These results indicate that other species may have the potential to degrade PCP, but lack critical path way factors However it may be possible to get degradation without all currently identified gene sequences. At least one strain of S.chlorophenolica (the former Arthobacter RA2) apparently lacks the sequences of pcpC, the gene that dechlorinates CHQ. Thus, the presence of a partial pathway may enable some degradation to take place, but simply having sequences from the first gene is insufficient
Chapter 4: Toxic Metal Resistances: Molecular Biology and the Potential for Bioremediation
S. Silver, L.T. Phung, J.-F. Lo, and A. Gupta, A.
Abstract
Bacteria, plants and animals often possess the ability to tolerate elevated concentrations of toxic metal ions. In bacteria the resistance to metal ions is mostly conferred by products of genes present on plasmids, but chromosomal genes are also used. For example bacterial resistances to Ag+, AsO2-, AsO43-, Cd2+, Co2+, CrO42-, Cu2+, Hg2+, Ni2+, Pb2+, Sb3+, TeO32-, Tl+ and Zn2+ are plasmid encoded. These metal resistance systems are under continuing investigations and in-depth molecular understanding has developed for some. The understanding of bacterial metal resistance systems has been useful for both environmental sciences and molecular medicine. We describe here some of the better understood bacterial toxic metal resistance systems and their bioremediation potential. We also try to project how the molecular understanding of the resistance mechanisms is a prerequisite in order to utilize the resistances for bioremediation.
Chapter 5: Characterizations of Copper and Chromate Resistant Bacteria Isolated From Karachi Tanneries Effluents
Uzma Badar, Ramla Abbas and Nuzhat Ahmed
Abstract
Several copper and chromate resistant bacterial strains were isolated from the effluent of leather tanning industries in Korangi sector 7-A Karachi, Pakistan. They were isolated under the selective pressure of copper/chromium salt in order to characterize the essential micronutrient (copper) and extremely toxic heavy metal (chromium) resistant bacterial strains. These strains showed multiple metal and antibiotic resistances. On the basis of copper and chromate resistance two bacterial strains were selected for further studies i.e. CMG458 (Pseudomonas sp.) and CMG451 (Bacillus sp.) respectively. The strain CMG451 was found to carry plasmid DNA having a molecular weight of 14.68 kb. A sensitive derivative of CMG451 designated as CMG460 was obtained, the chromate resistance was not completely lost but it was reduced from 1.5mM to 0.1mM. The resistance to copper and chromate was inducible in both the strains. Both the strains showed accumulation of respective metal salts from the medium, CMG458 accumulated copper upto 26.95% while CMG451 accumulated chromium upto 22.68% from the solution whereas its cured derivative accumulated approx. two times more than the parental strain i.e. 41.89%. Consequently, the mechanism of resistance of CMG451 appeared to involve reduced accumulation/uptake of chromium and that of CMG458 accumulation of copper. CMG451 and its mutant CMG460 were found to reduce chromate to trivalent form, aerobically.
Chapter 6: Microbial Processes for Solubilization or Immobilization of Metals and Metalloids and Their Potential for Environmental Bioremediation
G.M. Gadd, T.A.M. Bridge, M.M. Gharieb, J.A. Sayer, and C. White
Abstract
Mechanisms of microbial solubilization and immobilization of metal(loid)s, radionuclides and related substances are of clear potential for bioremediation with some processes integral to the operation of several successful in situ and ex situ biotreatment methods. Although the biotechnological potential of most of these processes has only been explored at laboratory scale, some mechanisms, notably bioleaching, biosorption and precipitation, have been employed at a commercial scale. Autotrophic leaching is an established major process in mineral extraction and has also been applied to the treatment of contaminated land. As a process for immobilizing metals, precipitation of metals as sulphides has also achieved large-scale application. This chapter outlines some of the main mechanisms by which microorganisms effect changes in the speciation and mobility of metals including autotrophic and heterotrophic leaching, biosorption, metal precipitation by metal-reducing and sulphate-reducing bacteria, metal(loid) reduction and methylation, and, where possible, provides examples of field application. As well as the biotechnological significance, it should be emphasised that this work also provides understanding of the biogeochemistry of metal(loid) cycling in the environment and the central role of microorganisms in affecting metal mobility and transfer between different biotic and abiotic locations.
Chapter 7: Resistance and Accumulation of Heavy Metals by Indigenous Bacteria: Bioremediation
N. Ahmed, U. Badar, and S. Raihan
Abstract
Resistance to toxic heavy metals and their accumulation by bacteria is a wide spread phenomenon that can be exploited for the improvement of the environment. Metal resistant bacteria have developed very efficient and varying mechanisms for tolerating high levels of toxic metals and thus hold potential for controlling heavy metal pollution. In order to study the levels of metal resistance, the mechanisms involved, and locating the genetic determinants, several bacteria showing resistance to chromium, copper, lead, zinc, cadmium and nickel were isolated from effluents of local industries and studied. All isolates showed an effect of changing pH, temperature and growth media, on metal resistance. Studies on the isolates revealed that different mechanism of heavy metal resistance may be operative in the different isolates. In most isolates, metal resistance was found to be plasmid mediated and several isolates showed metal accumulation. This study provides some interesting information about metal resistance in Pakistani bacterial isolates, viz.
1. They are highly resistant to most metal salts and multiple metal resistance is common.
2. Smaller plasmids ranging from 2.6 kb to 10.35 kb were found to be associated with cadmium and nickel resistance.
3. The plasmid isolated from cadmium resistant CMG64 is of 10.35 kb, and encodes for the production of a 70.79 kDa protein, while plasmid from nickel resistant strain CMG61 is of 3.93 kb and produces a protein of 56.207 kDa.
Cadmium resistant strain CMG64 that showed highest accumulation was then used in a lab-scale bioreactor where biofilms of this strain were developed on locally available cheap support materials and successfully used for cadmium removal.
Chapter 8: Application of Microorganisms to the Decontamination of Heavy Metal-bearing Wastes
L. E. Macaskie, R. A. P. Thomas, and J. R. Lloyd
Abstract
Metal-bearing liquid industrial wastes can be remediated by biomineralization, whereby microbially-produced ligands precipitate with heavy metals as biomass-bound crystalline deposits. Ligands may be sulfide or phosphate, with metal (M) deposition to very high load as MS or MHPO4, respectively, e.g. by Citrobacter or Acinetobacter, which produce phosphate ligand via phosphatase-mediated cleavage of suitable phosphate substrates. Immobilized cells within flow-through columns gave steady-state metal removal. The Citrobacter enzyme occurs within the cellular periplasmic space and in association with the extracellular polymeric matrix, which provides foci for initiation and continuation of metal deposition and can buffer the cells against adverse conditions of the bulk flow, e.g. in application to acidic U mine drainage waters. Recalcitrant Ni2+ can be intercalated within biogenic HUO2PO4: a bioinorganic ion exchanger. Formation of mixed crystals also promotes removal of other 'difficult' metals such as Pu. High-valence metals such as Cr(VI) and Tc(VII) can be remediated via bioreduction to low valence, insoluble forms of metal oxides and hydroxides, e.g. 99Tc was removed using a reductase activity of Escherichia coli identified as the hydrogenase 3 component of the formate hydrogenlyase complex. Immobilized cells removed Tc continuously at the expense of H2. This activity extended to the sulfate-reducing bacteria, where the extensive hydrogenase activities of Desulfovibrio desulfuricans permitted very efficient removal of Tc (95%) with a short flow residence time (2h), and high radiotolerance of the enzyme(s). Heavy metals are often associated with organic ligands, reducing their availability for bioremediation. New bacterial cultures have been developed to degrade the ligands to liberate associated metals for bioremediation using the biomineralization techniques.
Chapter 9: Microbial Transformation of Morphine Alkaloids: Biological Routes for the Production of Semisynthetic Opiate Drugs
Deborah A. Rathbone, and Neil C. Bruce
Abstract
The morphine alkaloids are the major alkaloid components of the opium poppy Papaver somniferum and these compounds provide some of the most important painkilling drugs in clinical use. Semisynthetic opiates are derived from the naturally occurring alkaloids morphine, codeine and thebaine. However, like many bioactive compounds, these alkaloids possess a variety of different functional groups that generally require protection during chemical transformation, which often results in poor yields. Biotransformations can offer advantages over chemical routes and, to this end, we have now obtained a diverse range of microorganisms that are capable of transforming morphine and codeine to several important therapeutic compoun ds. The biochemical and genetic aspects of this metabolic study have now been directed towards engineering pathways in microorganisms for the efficient production of analgesic compounds.
Chapter 10: Influence of Ammonium Concentration on Citric Acid and Biomass Production from Sugarcane-molasses by 2-Deoxy-D-glucose-Resistant Mutant Strain of Aspergillus niger.
S. Parvez, R. Shahid, M. N. Ahmed, and M. I. Rajoka
Abstract
The effect of nitrogen concentration on citric acid production by a mutant derivative of Aspergillus niger designated RP-7 from sugarcane molasses containing 15% total sugars was studied by batch cultivation process. Addition of 0. 6% 0f NH4NO3 improved the citric acid yield by 3-times. The nitrogen source had very little effect on biomass concentration. Further increase in citric acid and biomass was not observed by increasing the concentration of ammonium nitrate. Use of ammonium nitrate shortened the time of fermentation from 7 days to 5 days. The process productivity was compared with wild strain of A. niger 280, and the best organisms reported in literature. Under these conditions NH4NO3 antagonized the inhibition of phosphofructokinase by citrate itself and regulated the carbon flow to citric acid synthesis.
Chapter 11: Tissue and Cell Culture for Sugarcane Improvement
A. Khatri, I.A. Khan, S. H. Siddiqui, M. Ahmad, M. H. Khanzada, N. A. Dahar,andR. Khan
Abstract
For inducing somaclonal variation, two sugarcane (Saccharum sp. hybrid) clones viz. BL4 and AEC81-8415 were used for callusing and regeneration of plantlets. Immature leaf segments were used for callus induction on MS medium supplemented with 4mg/l of 2,4-D. Differentiation of shoots was induced on MS medium supplemented with 2mg/l each of IAA, IBA and kinetin whereas rooting was induced on 1/2 strength MS medium supplemented with 1m/l IBA. Plantlets with well-developed roots were successfully established in the soil. These plants showed variability for qualitative and quantitative traits.
Chapter 12: Seed Potato Production Strategy Through Tissue Culture
Azra Quraishi and Mussarat Bhatti
Abstract
Inadequate or non-availability of quality seed potato has always remained a major constraint in meeting the requisite seed requirement of Pakistan. Consequently, the country relied on imported seed. There was a need to increase the quantity of pre-basic seed of indigenous potato cultivars by exploiting the proven potential of tissue culture and rapid multiplication. This paper reports on the appropriate strategy using novel biotechnological approaches to meet the seed requirement of the country.
Chapter 13: Applications of Molecular Methods for Typing Bacteria
Salma A. Ghori and Warren B. Grubb
Abstract
In order to monitor the spread of bacteria, particularly pathogens and antibiotic-resistant bacteria, between the environment, animals and humans, it is imperative that methods are available to type bacteria and trace their spread. Discussed are the major methods which are available. These will be dealt with under two main headings, phenotypic and genotypic. The phenotypic methods include antimicrobial susceptibility profiles, bacteriophage typing, multilocus-enzyme electrophoresis, protein profiles and immunoblotting. The genotypic methods include plasmid and chromosomal DNA analysis using restriction enzyme digestion and Southern blotting, polymerase chain reaction, pulsed-field gel electrophoresis (PFGE) and DNA sequencing. Of these, PFGE has proven to be particularly useful. Genotypic methods are proving more advantageous as they are more stable, reproducible and discriminatory than phenotypic methods. A combination of methods is often the most effective.
Chapter 14: Plasma Protein Studies of Patients with Mental Disorders and Normal Individuals
Talat Haider and Umar Jawaid
Abstract
Nowadays scientists in psychiatry have focused on the study of genes and their role in development of mental illness. Family, twin and adoption studies suggested that mental disorder like Schizophrenia, Manic Depressive Psychosis (MDP), Addiction, Neuroses, and many others are genetically inherited and tend to run in the families. The role of neurohormones in disordered mental activity, changes of enzyme activity and cell metabolism help to explain changes in blood and urine associated with some mental illness. In this study blood sample were collected from 11 normal and 66 patients of mental illness. The mean ages of on set for Schizophrenia, MDP, and Addiction group were 27.24, 30.11 and 23 years respectively. Plasma protein concentrations were measured both qualitatively and quantitatively. The average concentrations of plasma protein were 1533.6, 1465.8, 1421.5 and 1421.0 (mg/ml) for normal, Schizophrenics, MDP patients and addicts respectively. The results were analyzed by 4 x 2 factorial in CRD. Significant differences were noted for the plasma protein concentration of different clinical conditions. However non-significant differences were observed between different psychological disorders. Age effect and clinical condition x age interaction, both were non significant. Qualitative separation of plasma proteins were also performed using PAGE. Further work is required to relate the patient's plasma protein levels of enzymes involved in the regulation of the different neurotransmitters such as dopamine, adrenaline, noradrenaline and serotinin, that have very active role in determining the pathophysiology of individuals.
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