Acknowledgement - 7th World Congress of Chemical Engineering Abstract Title (max 150 char): Abstract Text (max 2500 char): Topic

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2 7th World Congress of Chemical Engineering Acknowledgement - 7th World Congress of Chemical Engineering Family Name First Name Title (Prof, Dr, Mr, Mrs, Ms) Institution/Company Department Address (Including post/zip code) Country Telephone (e.g ) Fax (e.g ) Benyahia Farid Dr United Arab Emirates University Chemical and Petroleum Engineering PO Box Al Ain United Arab Emirates farid.benyahia@uaeu.ac.ae Abstract Title (max 150 char): Is immobilization a biomass retention technique only? Abstract Text (max 2500 char): Cell immobilization is a well known technique for biomass retention in several biochemical applications, in particular with slow growing cells like nitrifiers or when rates of reaction using suspended cells are slow. However, other immobilization benefits have been quoted in the literature without provision of hard evidence. These benefits include resistance to, load and thermal shocks as well as protection against toxic effects of heavy metals that might be present during treatment. In nitrification processes, there is hardly any data on these issues. In this investigation, fully reactivated Nitrosomonas cells immobilized in PVA using the freeze-thaw cross linking method, have been subjected to the presence of common heavy metals,, load and thermal shocks, and nitrification performance closely monitored. The results thus obtained on free and immobilized cells have clearly highlighted the merits of PVA immobilization beyond biomass retention and this work provides unique quantitative evidence of such merits. Topic 24 Engineering for Life - Biochemical Engineering - Abstract Authors file:///d /Farid%20Files/Research/papers/world% glasgow/nitrification_PVA/ACSL htm (1 of 2) [12/9/2004 8:13:25 PM]

3 7th World Congress of Chemical Engineering Farid Benyahia, United Arab Emirates University, Chemical and Petroleum Engineering, United Arab Emirates, (Presenting); A Embaby, United Arab Emirates University, Chemical and Petroleum Engineering, United Arab Emirates file:///d /Farid%20Files/Research/papers/world% glasgow/nitrification_PVA/ACSL htm (2 of 2) [12/9/2004 8:13:25 PM]

4 Is immobilization a biomass retention technique only? Farid Benyahia and Ahmed Embaby Department of Chemical and Petroleum Engineering United Arab Emirates University PO Box Al Ain UAE Abstract A series of load and shock experiments were carried out in a nitrification system involving Nitrosomonas bacteria immobilized in freeze-thaw crosslinked PVA matrices of various polymer content. This investigation has shown clearly and quantitatively the tolerance to load and shocks of PVA immobilized Nitrosomonas cells. Indeed, the cells nitrifying activity recovered in just 10 hours following shock treatment in all cases, and the 10% PVA support matrix gave the best overall results owing to its morphology. This investigation has clearly demonstrated in quantitative terms the benefits of PVA support matrices, beyond biomass retention. The results reported herein are of great industrial significance. Introduction Cell immobilization is a well known and now established technique for biomass retention in several biochemical applications. Indeed, this technique is reported to lead to higher volumetric productivity due to increased local cell density, to enable higher dilution rates to be used, to reduce disposal of used cells, to lead to improved reaction conditions inside the support matrix and to offer a protective environment to the cells. Whilst these virtues may be true and verified in some applications, in the vast majority of biochemical systems, they have yet to be proven qualitatively and quantitatively. In nitrification processes for example, where Nitrosomonas and Nitrobacter cells are very slow growing, this technique does offer an obvious advantage in terms of biomass retention. However, recent studies made Benyahia and Polomarkaki [1] have shown that immobilization of Nitrosomonas cells can be subject to quite severe internal mass transfer limitations. Although Benyahia and Polomarkaki's [1] investigation was carried out on alginate gel immobilized nitrifiers, other types of natural and synthetic gels or polymers have been reported as suitable support material [2,3]. The latter include gelatin, collagen, polyvinyl alcohol, polyethylene glycol, polyacrylamide and polycarbonates. These gels have variable mechanical strength and stability, and for potential large scale applications, mechanical properties become of paramount importance. Amongst stable gels, Polyvinyl alcohol (PVA) enjoys a good popularity in the research community [4,5,6]. However, this apparent stability was put to test by the present authors and was found to be relative to the method employed to cross link PVA. For example, the boric acid method [6] did not produce a stable gel despite claims in the literature. However, PVA crosslinked by the freeze-thaw method was found to be mechanically stable and in fact remained stable for quite a long time, making this hydrogel particularly suitable for extended applications.

5 If biomass can be retained in a stable hydrogel matrix like crosslinked PVA, are there other desirable attributes that can make this technology genuinely attractive for large scale applications in waste water treatment processes? For example, support matrices have been reported to offer some form of protective environment to entrapped cells. However, this was never proven in quantitative terms. It is therefore the objective of this paper to shed some light on the merit of cell immobilization in terms of resistance of Nitrosomonas cells immobilized in crosslinked PVA, to ammonia load shock and shock during a nitrification reaction. The outcome of these tests is extremely important in determining the merits of support material for cell immobilization and in shedding some light on cell shock recovery. Experimental Aqueous solutions of PVA with 3 different polymer contents were prepared: 10%, 15% and 20% (all w/w). The preparation method is tedious since PVA is difficult to dissolve in water which has to be warmed first. When the PVA solution cooled down to about 28 ºC, 2% w/w bacterial slurry (commercial Nitrosomonas cells) was added to the PVA solutions and mixed thoroughly. The PVA-biomass thick slurry was poured into rectangular molds and put in a freezer at -20 ºC for 24 hours. They were subsequently removed from the freezer and put in a refrigerator at 4-6 ºC to slowly thaw, then replaced into the freezer for other cycles of freezing thawing. There were 4 such cycles. Upon completion of the freeze-thawing cycles, the gel with immobilized Nitrosomonas cells were cut into small cubes, ready to be used in nitrification tests. The presence of viable Nitrosomonas cells was determined by activity tests, and a series of Scanning Electron Microscopy (SEM) images were taken to visualize bacterial cells and the porous structure of PVA support matrix. Figure 1 for example depicts colonies of Nitrosomonas cells in a section of the 10% PVA gel. The load and shock tolerance tests were conducted in a battery of 5 aerated (to O 2 saturation) bubble column reactors (1 Liter capacity), each with an individual experiment. All reactors had an initial ammonia concentration of 100 ppm, and 42 cubes of PVA (of various % polymer content) with immobilized Nitrosomonas cells (equivalent to 2 grams of cell slurry or g dry biomass). The 3 load and 3 shock tests were executed by pulse inputs into the bubble reactors of either ammonium carbonate solution to raise the ammonia concentration from 100 ppm to 500 ppm, or, of a dilute hydrochloric solution, to drop the reactor content to 5. The shock inputs were applied after 110 to 115 hours of continuous nitrification in the batch bubble column reactors described above. In all experiments, there were no growth nutrients supplement. The sixth experiment was conducted after completing 5 concurrent runs with the battery of 5 bioreactors described above. The nitrification reaction was monitored by measurement of nitrite liberation rates employing a standard spectrophotometric method for nitrogen nitrite determination. The was measured online throughout the nitrification reaction by means of a meter. The main experimental variable was the PVA % content of the gel matrix, and the experimental parameters were load shock and shock. These will constitute the basis for discussion in the following sections, and run tags describing the experimental variables and parameters will be presented in the results section.

6 Results and discussion Samples of the nitrification medium were taken from each reactor for the measurement of nitrite liberation, whilst was continuously measured and recorded. Figure 1 shows clearly that Nitrosomonas cells survived the harsh conditions they were exposed to during the freeze-thaw immobilization procedure. Figures 2 to 7 depict the nitrite liberation and of reaction solutions before and after applying the pulse inputs that constituted the load and shocks described in the previous section. In all cases, it can be seen that the bacteria did recover from the shocks after about 10 hours. This "10-hour" transition stage can be considered as an adaptation period to the new reaction conditions (both higher load and lower ). The fact that Nitrosomonas cells resumed their nitrification function after the shock inputs, clearly indicates their ability to tolerate extreme conditions in their immobilized state. We have experimental evidence in this work. A closer look at Figures 2 to 4, not only show evidence of load shock tolerance, but also show a large increase in the rate of nitrite production, after shock application. The percentage rate changes are indicated in Table 1. The highest rate increase is that of run PV-SL1 where 10% PVA was employed. The SEM image of a section of this gel is depicted in Figure 8 and show a morphology consisting of a mix of small and large pores, thus enabling easy transport of nutrients/products into or out of the support matrix where Nitrosomonas cells are immobilized. The bacteria were able to adjust to the higher concentrations of ammonia by producing proportionately larger nitrite quantities, as seen in Table 1, and the morphology of the 10% PVA support facilitated this process. The rate changes in runs PV-SL2 and PV-SL3 were significantly less than that of PV-SL1, but suggested that after 10 hours, immobilized Nitrosomonas resumed their nitrification function, albeit in a reduced capacity. This is probably due to the more tightly packed or closed structure (ie: narrow pores or fewer pores) of the 15% and 20% PVA matrices, as depicted in Figures 9 and 10. The SEM imagery and experimental trends are compatible. Figures 5 to 7 show a similar tolerance trend in the shock tests. The carbonate environment buffering capacity was able to readjust the level close to the optimum nitrifying range and the bacteria were back in action after a similar adaptation period observed in the load shock tests (about 10 hours). For the shock experiments, a reduction in nitrite liberation rate was observed in run PV-PH1 (10% PVA). In this particular case, the open morphology of 10% PVA allowed sufficient acid to quickly penetrate pores where Nitrosomonas cell clusters were attached to cause a significant local drop in, and therefore a reduction in bacterial activity due to the known low inhibition effect. As for the other runs (PV-PH2 and PV-PH3), increases in nitrite formation rates can be seen from Table 1. It seems that the more closed structure (narrow pores or fewer pores) of the 15% and 20% PVA prevented the acid from reaching the bacteria in the PVA matrix in sufficient amount so as to have a drastic effect. Figures 9 and 10 show the morphology of a section of 15% and 20% PVA respectively. In these tests, the trends are also compatible with the SEM imagery.

7 Table 1: liberation rate changes before and after shock inputs Run tag Rate before shock application Rate after shock application % Rate change (ppm NO 2 - /hr -1 /mg -1 dry (ppm NO 2 - /hr -1 /mg -1 dry biomass) biomass) PV-SL % PV -SL % PV -SL % PV-PH % PV-PH % PV-PH % Conclusion The series of load and shock tests conducted in a nitrification reaction system involving Nitrosomonas cells immobilized in cross-linked PVA gels demonstrated clearly the protective environment that the support matrix offers. The nitrifying biomass was able to recover from load and shocks in about 10 hours, owing to the protection the PVA encapsulation offered. The % PVA in the support matrix did have an effect on the extent of cell activity recovery. The 10% PVA matrix was found to offer the best recovery performance because of the morphology of PVA gel in that percentage polymer gel. This investigation produced remarkable results on post shock recovery of Nitrosomonas cells, previously unreported. This is seen as a very significant contribution towards exploiting the potential of cell immobilization, beyond biomass retention. References 1 Benyahia, F. and Polomarkaki, R., "Mass transfer and kinetic studies under no cell growth conditions in nitrification using alginate gel immobilized Nitrosomonas" Process Biochemistry vol 40, pp (2005). 2 Norton, S. and D'Amore, T., "Physiological effects of yeast cell immobilization: application for brewing", Enzyme Microb. Technol. Vol 16, pp (1994). 3 Park, J.K., and Chang, H.N., "Microencapsulation of microbial cells", Biotechnol. Adv. Vol 18, pp (2000). 4 Chang, C.C. and Tseng, K.S., "Immobilization of Alcaligens Eutrophus using PVA crosslinked with sodium nitrate", Biotech. Tech. vol 12(12) pp (1998). 5 Ariga, O., Takagi, H., Nishizawa, H. and Sano, Y., "Immobilization of microorganisms with PVA hardened by iterative freezing and thawing", J. of Fermentation, vol 65 pp (1987).

8 6 Hashimoto, S. and Furukawa, K., "Immobilization of activated sludge by PVA- Boric acid method", Biotechnology and Bioengineering, vol 30 pp52-59 (1987). Acknowledgement The authors would like to acknowledge the financial support of the UAE University to this work, under grant /02 and a Master Scholarship to Mr Ahmed Embaby, the co-author of this paper Figure 1: Colonies of Nitrosomonas in the PVA gel (10% PVA)

9 Run tag: PV-SL1 liberation Load shock added here Time (hr) Figure 2: load shock test with the 10% PVA support Run tag: PV-SL Load shock added here Time (hr) Figure3 : Load shock test with 10 15% PVA support

10 Run tag: PV-SL liberation Load shock added here Time (hr) Figure 4: Load shock test with the 20% PVA support Run tag: PV-PH shock load added here liberated (ppm) Time (hr) Figure 5: shock test with the 10% PVA support

11 Run Tag: PV-PH2 liberated (ppm) shock load added here Time (hr) Figure 6: shock test with the 15% PVA support Run tag: PV-PH3 liberated (ppm) shock added here Time (hr) Figure 7: shock load with the 20% PVA support

12 Figure 8: Cross section of the 10% PVA gel, showing size of pores Figure 9: Cross section of the 15% PVA gel, showing size of pores

13 Figure 10: Cross section of the 20% PVA gel, showing few pores