ABSTRACT INVESTIGATION OF LIGHT ATTENUATION IN LAKE EYMIR Selen ATIKER M.Sc, Department of Environmental Engineering Supervisor: Assoc. Prof. Dr. Ayseg&uuml / l Aksoy Co-Supervisor: Assoc. Prof. Dr. Selim Sanin January 2012, 164 pages. Light penetration and attenuation has significant impact on the water quality of lakes. Algal activity, which is important for the levels of several water quality parameters, is dependent on light penetration besides availability of nutrients. In this study, change in light penetration and attenuation in Lake Eymir was studied. The relationships of extinction coefficient (ke), and water quality parameters were investigated. The effect of ke on Chl-a over nutrients were investigated. The water quality parameters measured were / total suspended solid (TSS), phosphate, ammonium, nitrate, photosynthetically active radiation (PAR), chlorophyll-a (Chl-a), Secchi disk depth and lake Depth. The measurements were conducted at five different stations in Lake Eymir. Secchi disk, PAR and lake depth measurements were done on site, while TSS, Chl-a and phosphate analyses were done in laboratory, using standard methods. Nitrate and ammonium analyses were conducted through laboratory kits. Linear and non-linear regression models of ke and Chl-a were developed to understand their relationships with the the measured parameters, using XLSTAT software. Analyses of the data at sampling stations revealed that Station 2 and 3 were the most representative stations in general. The model results indicated that ke is as important as nutrients for Chl-a abundance. Secchi disk and Chl-a are the most correlated parameters with ke. Moreover Secchi disk depth is nonlinearly correlated with ke, while linearly correlation is present between Chl-a and ke. &emsp

Cry3Aa is a 73 kDa protoxin toxic to insect larvae of Coleoptera order. It is processed to form a stable 65 kDa &amp / #61540 / -endotoxin by endogenous proteases. The first part of this study involved the determination of the patterns of biosynthesis of Coleoptera-specific &amp / #61540 / -endotoxin by a local isolate of Bacillus thuringiensis subsp. tenebrionis (Btt) in relation to its growth and sporulation. Among four different media compared (DSM, GYS, HCT and C2) Cry3Aa production was the highest in DSM, especially at 72nd h and 120th h of incubation. For improvement of Cry3Aa production, the effects of different carbon and nitrogen sources, inorganic phosphate and other mineral elements were tested. Increasing concentrations (5-10 g.L-1) of glucose or sucrose decreased the toxin yield probably by suppressing sporulation. Inorganic phosphate was found to have the most striking effect on toxin biosynthesis. 200 mM inorganic phosphate concentration resulted in 5 fold increase in Cry3Aa yield. Cry3Aa production was greatly reduced when various combinations of organic and inorganic nitrogen sources, especially ammonium sulphate and Casamino acids were replaced with Nutrient broth in DSM. The highest Cry3Aa production was obtained in the media containing 10-5-10-7 M MnCl2, 10-5 M FeSO4 and 5.10-4 M MgSO4, corresponding to their original concentrations in DSM. Decrease of iron concentration or its omission from the medium decreased the toxin yield. Toxin production capacity of our local isolate was compared with those of 30 different anti-Coleopteran Bt strains. Most of the strains producing this protein gave general protein banding patterns quite similar to that of our local isolate. Lastly, the cry3Aa gene of the Btt local isolate was PCR-amplified and cloned into the E. coli/Bacillus shuttle vector pNW33N. The recombinant plasmid was amplified in E. coli and the sequence of the cry3Aa was determined. Amino acid sequence deduced was found to be 97.4 %-99.2 % identical to the cry3Aa sequences (GenBank) of 10 different quaternary ranks. In this respect, the gene has to represent the 11th quaternary rank of the cry3Aa ones. The recombinant plasmid carrying cry3Aa gene was next used to transform Bs 168 as an intermediate host and low level of expression was seen.

Some yeasts secrete polypeptide toxins, which are lethal to other sensitive yeast cells, gram-positive pathogenic bacteria and pathogenic fungi. Therefore these are designated as killer toxins. Killer toxins are suggested as potent antimicrobial agents especially for the protection of fermentation process against contaminating yeasts, biological control of undesirable yeasts in the preservation of foods. Moreover they are promising antimicrobial agents in the medical field / due to immune system suppressing diseases like AIDS, there is an increase in the incidence of fungal diseases and current antimycotics have low selectivity and severe side effects. In this study our aim was to explain the cytocidal effect and enzymatic properties of K5 type yeast killer protein, which is secreted by Pichia anomala NCYC 434 cells, and known to have a broad range of killing spectrum. Competitive inhibition of the toxin with cell wall polysaccharides showed that primary binding site of toxin is &amp / #946 / -1,3-glucans of sensitive cells. Toxin showed exo-&amp / #946 / -1,3-glucanase activity which causes loss of cell wall rigidity leading cell death. Km and Vmax were found to be 0,3 mg/ml and 372,3 &micro / mol/min/mg for laminarin hydrolysis. The toxin exerted its cytocidal effect after 2 h contact with the target cells. Toxin production was found to be dependent on &amp / #946 / -1,3-glucan content of the media. Toxin activity was completely inhibited by Hg+2 ,while several metal ions and DTT increased the activity to different extends. Our findings revealed the characteristics of K5 type killer toxin which will help for its possible uses in near future.

In this study, the soluble protein fraction of P. chrysosporium grown under standard conditions was analyzed by using 2D-PAGE approach and a 2-D reference map was constructed. 910 spots could be separated and detected on Coomassie-stained 2-D gels by the help of Delta2D image analysis software. 720 spots could be cut from the master gel and were subjected to MALDI-TOF MS analysis followed by MASCOT search. A total of 517 spots out of 720 were assigned to specific accession numbers from the P. chrysosporium genome database. Further analysis of the data revealed 314 different gene products (distinct ORFs). The theoretical pI and MW values were plotted against the experimental migration distances. Results indicated the existence of 124 protein spots whose horizontal migration differed significantly from the expected migration according to the calculated pI values and 52 spots with an apparent molecular weight that is significantly different from their theoretical molecular weight. While protein modification could be predicted by these analyses, the main support was the presence of multiple spots of the same gene product. As much as 118 ORFs yielded multiple spots on the master gel, corresponding to 37.5% of the all distinct ORFs identified in this work. The relative abundance of each of the 517 identified polypeptides was calculated in terms of spot intensity. The majority of the most abundant proteins were found to be housekeeping ones. When the relative distribution of the proteins into four main functional categories was taken into consideration, &ldquo / Metabolism&rdquo / appeared the most important category with a share of 50.6% among identified proteins. However, among the functional classes, &ldquo / Posttranslational modifications, protein turnover, chaperones&rdquo / which is listed under the main category &ldquo / Cellular Processing and Signalling&rdquo / was represented by the highest number (104) of the identified proteins. Only 6 of the proteins listed in this study were assigned to hypothetical proteins. Out of the 314 identified gene products shown in P. chrysosporium, 29 were predicted to have a signal peptide sequence according to the SignalP algorithm. By making a WoLF PSORT search, subcellular localization of the proteins was predicted. Accordingly, 147 of the proteins were predicted to be located in cytoplasm. The phosphorylated proteins of P. chrysosporium were detected by ProQ phosphoprotein staining of the 2-D gel. 380 out of 910 distinct protein spots (40%) were found to be phosphorylated in exponentially growing cells of P. chrysosporium. Of these spots, 96 could be matched to the identified proteins.

&alpha / -Galactosidase is an exo-glycosidase that hydrolyses non-reducing, &alpha / -1,6-linked &alpha / -galactose units from oligosaccharides, galactomannans, and galactolipids. &alpha / -Galactosidase activity has biotechnological, industrial, and medical importance. &alpha / -Galactosidase from A. fumigatus IMI 385708, in particular, can catalyse unique hydrolysis and transgalactosylation reactions on polymeric substrates. In this study, &alpha / -galactosidase of the human pathogen A. fumigatus IMI 385708 was first produced in a GRAS organism, Aspergillus sojae. For this aim, &alpha / -galactosidase gene (aglB) of A. fumigatus IMI 385708 was ligated onto pAN52-4 vector (Acc. No: Z32699) and transformed into Aspergillus sojae ATCC11906, under the control of the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter (gpdA) of A. nidulans and the signal sequence of glucoamylase gene (glaA) of A. niger. This allowed high level of &alpha / -galactosidase production on glucose instead of locust bean gum (2.45 U/mL), corresponding to a 3-fold increase in volumetric production. Next, using response surface methodology, carbon and nitrogen sources and agitation speed were optimized (10.5% molasses (w/v) / 1.3% NH4NO3 (w/v) / 276 rpm). Compared to non-optimized cultivation, a further 4-fold increase in &alpha / -galactosidase production (10.4 U/mL) was achieved. Recombinant &alpha / -galactosidase was purified 18.7-fold using Anion Exchange and Hydrophobic Interaction Chromatography with an overall yield of 56% and 64.7 U/mg protein. The Vmax and Km values for the hydrolysis of p-nitrophenyl &alpha / -D-galactopyranoside were 78 U/mg protein and 0.45 mM, respectively. Optimum pH and temperature for &alpha / -galactosidase activity were between pH 4&ndash / 6 and 50&ndash / 60 &deg / C, respectively. Among the tested chemical agents, Ag+, Hg2+, and Fe2+ drastically decreased the activity, while biotin, I+1, Mn+2, Pb+2, Li+1, and Mg+2 enhanced between 12&ndash / 29%. To analyse the influence of osmotic stress as a means of further inducing &alpha / -galactosidase production, salt was added into the complete growth medium. In addition to enzyme production, fungal growth and morphology were analysed for both &lsquo / salt-adapted&rsquo / and &lsquo / salt non-adapted&rsquo / A. sojae Ta1 cells in the presence of KCl, MgCl2, MgSO4, NaCl, and Na2SO4 at 1 M and 2 M. Accordingly, 3-fold increase in &alpha / -galactosidase production was achieved by non-adapted cells in the presence of 1 M NaCl. Exposure of A. sojae Ta1 cells to salt resulted in predominantly mycelial form, rather than the pellet form observed under normal conditions. Finally, the transgalactosylation ability of &alpha / -galactosidase was studied. &alpha / -Galactosidase efficiently catalysed galactose transfer to different monosaccharides and disaccharides in the presence of pNP&alpha / Gal as monitored by TLC, ESI-MS, and HPLC.

Yogurt is a characteristic fermented dairy product of Turkey and Bulgaria and its popularity has been increasing all over the world. Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus (Lactobacillus bulgaricus) are used together as starter culture in production of yogurt. The objective of this study was to isolate and characterize yogurt cultures from traditionally produced yogurts (i.e. produced without using commercial starter cultures) and to search the genotypic diversity within traditional S. thermophilus isolates. Yogurt cultures were isolated from traditionally produced yogurts collected from different regions of Turkey and identified biochemically. Acidification ability of the isolates was examined and the cultures giving best acidifying rates were further subjected to a selection in terms of their acetaldehyde production ability. Then, phage resistance and proteolytic activity of chosen isolates were tested. Finally, twenty-five L. bulgaricus and twenty-two S. thermophilus isolates were selected as cultures having best technological properties. Furthermore, subtyping studies were carried out to indicate strain diversity among isolates. S. thermophilus was selected as target organism for subtyping in this study. Clustered regularly interspaced short palindromic repeats (CRISPR) loci are highly polymorphic genetic regions, which are composed of partially palindromic direct repeats interspaced by sequences called spacers. In order to characterize S. thermophilus isolates genotypically, CRISPR1 locus of the isolates were analyzed. Additionally, nineteen isolates selected after CRISPR1 analysis were characterized using multilocus sequence typing (MLST). This provided to compare CRISPR1 analysis with MLST as a typing method. According to CRISPR1 analysis S. thermophilus isolates were grouped into 6 main clusters with a total of 15 sub-clusters. MLST results demonstrated an evolutionary relationship among these strains compatible with that derived from the CRISPR1 analysis.

Density of fecal coliforms (FC) such as Escherichia coli is the most commonly used indicator of fecal pathogen content of biosolids. When biosolids are disposed off or used for soil amendment, they pose public health risks. So far anaerobic digesters have been considered to be an effective treatment option for pathogen and FC reduction in biosolids. However, recent studies revealed that there is a significant re-growth and reactivation of indicator organisms in biosolids upon dewatering by centrifugation. Although the exact mechanism of FC reactivation is yet to be understood, a few extensive recent studies strongly suggest that FC go into a viable but non-culturable (VBNC) state during anaerobic digestion. Therefore, quantitative detection of live cells among the total in biosolids samples, without using culturing-based approaches, is highly critical from a public health risk assessment perspective. Since recent investigations proved the significant re-growth and reactivation of indicator organisms. Persistence of DNA in the environment after cell death in the range of days to weeks limits the application of DNA-based approaches for the detection of live bacteria. Using selective nucleic acid intercalating dyes such as ethidium monoazide (EMA) and propidium monoazide (PMA) is one of the alternative approaches to detect and quantify the viable cells by quantitative PCR. These compounds have the ability to penetrate only into dead cells with compromised membrane integrity. They intercalate in the DNA via photo-inducible azide groups and in turn inhibit DNA amplification during PCR reactions. PMA has been successfully used in different studies and microorganisms but it has not been evaluated sufficiently for the complex environmental samples such as biosolids. In this study Escherichia coli ATCC 25922 and uidA gene were used as model organism and as target sequence respectively in absolute quantification method with real-time PCR. Experiments with the known quantities of live and dead cell mixtures showed that PMA treatment inhibits PCR amplification from dead cells with over 99% efficiency. The results of this study conclusively demonstrated that PMA-modified PCR could be successfully applied to the biosolids when total suspended solid (TSS) concentration is 2000 mg/L or below.

The development of insecticides resistance among many insect species and the ecological damage occasionally caused by the lack of specificity in the toxic effects of insecticides have provided the impetus to seek alternative methods of insect control. This observation led to the development of bioinsecticides based on the insecticidal action Bacillus sphaericus (Bs), Bacillus turingiensis (Bt). The discovery of biolarvicidal actions of Bacillus thuringiensis and Bacillus sphaericus opened a new perspective for insect control. In the first part of the study was initiated to determine a suitable fermentation medium formulation and optimal fermentation conditions for large scale, low cost production of Bs. Bs 2362 was tested in whey and soy flour based media. These media was reformulized form of NYSM (Nutrient Broth Yeast Extract Sporulation Medium). Soy flour based medium, SYSM, gave the promising results in terms of cell yield, sporulation frequency and toxin production. In the second part of the study, fermentation productivity anlaysis of a local isolate Bacillus thuringiensis subsp. kurstaki 81 was evaluated. In order to compare different C:N ratios (1:1, 2:1, 4:1, 8:1, 10:1 20:1 and 30:1) of YSM medium. Btk 81 were run for 72 h and cell growth, sporulation and toxin protein profile of Btk 81 were determined for each. When all the quantitative toxin data for both glucose and sucrose varying C:N ratios were compared, it was determined that the crystal protein concentrations had the highest value in sucrose based medium when C:N ratio was 10:1. Regulation by C:N ratio of crystal protein biosynthesis was investigated for improving the production of this protein by our third candidate strain Bacillus thuringiensis subsp. israelensis ONR60. The experiments were performed by using TBL medium, at three different C:N ratios, 2:1, 4:1 and 8:1 respectively. In view of the cell growth characteristics and bioassy results, TBL medium designed with 2:1 C:N ratio was chosen as the best for further steps. In addition, running time of the culture determined as 60 hours as was also determined in the previous experiment. As the last step of this study, the pre-determined optimal conditions were applied to a 30L batch type fermentor for toxin production by using Bacillus thuringiensis subsp. israelensis ONR60. Unfortunately, the toxicity was not satisfactory, being much below the level of that expected as based on the results of the laboratory scale studies.