Biosorption of copper by activated sludge : a thesis presented in partial fulfilment of the requirements for the degree of Master of Philosophy in Environmental Engineering at Massey University, Palmerston North, New Zealand

Biosorption of copper by sludge from a lab-scale activated sludge was studied. S-typed isotherms were found in almost all cases. This revealed the importance of reversible sites on the cell surfaces. Hydroxyl groups on the neutral polymers of the cell surfaces were likely to be the biosorption sites. The equilibrium time of biosorption could be divided into two phases. The fast initial phase was observed within thirty minutes. The second phase went to an equilibrium after six hours. The biphasic equilibrium time was explained by the adsorption on the cell surfaces and active uptake, respectively. Freundlich isotherms were found to describe the biosorption fairly. From constants of Freundlich equation, it was found that unwashed sludge could biosorb about 16 mg copper per gram dry weight of sludge. Washing of sludge by various concentrations of EDTA and 0.85% NaCl did not show any difference from unwashed sludge. Anyway the optimum washing time in this study was three hours. The specific biosorptions were decreased after the long period of washing. The high concentration of EDTA (1% EDTA) gave the lowest biosorption capacity. Sludge characteristics play the most important role in copper biosorption. Type of organisms influenced the biosorption capacity. The population proportion was changed due to the operation conditions of the reactor and the biological interaction among species. Effects of hydraulic retention time (HRT) and solids retention time (SRT) were discussed. Although they could not control the biosorption directly, they influenced sludge characteristics and the performance of exocellular polymers. Behaviour of the lab-scale activated sludge was monitored during the operation period in order to compare the adsorption with the biological characteristics of sludge. At the high dilution rate (0.042 hr-1) the solids in the reactor fluctuated and did not reach a steady state after a prolonged period of six months. In contrast, the solids concentration of 0.021 hr-1 dilution rate went to a stable state after one month. The interrelationship of three groups of organisms in the reactor was proposed in order to explain the transient behaviour of the system. The combination of dilution and predation separated the fast and slow growing bacteria resulting in the instability of the system

Environmental Free-Living Amoebae Isolated from Soil in Khon Kaen, Thailand, Antagonize Burkholderia pseudomallei.

Presence of Burkholderia pseudomallei in soil and water is correlated with endemicity of melioidosis in Southeast Asia and northern Australia. Several biological and physico-chemical factors have been shown to influence persistence of B. pseudomallei in the environment of endemic areas. This study was the first to evaluate the interaction of B. pseudomallei with soil amoebae isolated from B. pseudomallei-positive soil site in Khon Kaen, Thailand. Four species of amoebae, Paravahlkampfia ustiana, Acanthamoeba sp., Naegleria pagei, and isolate A-ST39-E1, were isolated, cultured and identified based on morphology, movement and 18S rRNA gene sequence. Co-cultivation combined with a kanamycin-protection assay of B. pseudomallei with these amoebae at MOI 20 at 30°C were evaluated during 0-6 h using the plate count technique on Ashdown's agar. The fate of intracellular B. pseudomallei in these amoebae was also monitored by confocal laser scanning microscopy (CLSM) observation of the CellTracker™ Orange-B. pseudomallei stained cells. The results demonstrated the ability of P. ustiana, Acanthamoeba sp. and isolate A-ST39-E1 to graze B. pseudomallei. However, the number of internalized B. pseudomallei substantially decreased and the bacterial cells disappeared during the observation period, suggesting they had been digested. We found that B. pseudomallei promoted the growth of Acanthamoeba sp. and isolate A-ST39-E1 in co-cultures at MOI 100 at 30°C, 24 h. These findings indicated that P. ustiana, Acanthamoeba sp. and isolate A-ST39-E1 may prey upon B. pseudomallei rather than representing potential environmental reservoirs in which the bacteria can persist

Intracellular survival through time of <i>B</i>. <i>pseudomallei</i> in <i>P</i>. <i>ustiana</i> (A), <i>Acanthamoeba</i> sp. (B) and isolate A-ST39-E1 (C).

<p>Time zero represents 3 hours after <i>B</i>. <i>pseudomallei</i> feeding. Bars represent the standard errors of the means of duplicate, three times independent experiments, * <i>p</i> < 0. 0001 using ANOVA.</p

Numbers of <i>Acanthamoeba</i> sp. and isolate A-ST39-E1 over time (A-B and C-D respectively) after feeding with <i>B</i>. <i>pseudomallei</i> (▲) or <i>E</i>. <i>coli</i> (positive control) (■) or deprived of bacteria as a negative control (●).

<p>Graphs and figures show no significant differences between amoebae fed on <i>B</i>. <i>pseudomallei</i> and <i>E</i>. <i>coli</i>. However, numbers of amoebae in the negative control group were significantly lower than in the pother groups (<i>p</i> ≤ 0.0001). Data are mean ± SD from duplicates of the three independent experiments.</p

Specific primers for the 18s rRNA gene of amoebae.

<p>Specific primers for the 18s rRNA gene of amoebae.</p