tag:blogger.com,1999:blog-29429659185703289482024-03-05T17:22:38.654+08:00MR BIOREACTORBioreactor Itu Menarikhttp://www.blogger.com/profile/02699912848197862358noreply@blogger.comBlogger13125tag:blogger.com,1999:blog-2942965918570328948.post-12181070886191872062012-12-24T11:41:00.004+08:002012-12-24T11:42:41.744+08:00Members of group 4 <a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjobI72wXexrnsd9BlIrYLo0vPTS47mv6WNEh-UONc1RKmDLxW2rifw6WYVeWQ2eXRnsCAMgrikyaTPXtJRxnvLJwyyL1z2OCCZvvS0VUuzv7OOUAc7xH3wUG3cxzZBZ8ArzDfY0CdAwSQ/s1600/DSC08164.JPG" imageanchor="1" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" height="240" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjobI72wXexrnsd9BlIrYLo0vPTS47mv6WNEh-UONc1RKmDLxW2rifw6WYVeWQ2eXRnsCAMgrikyaTPXtJRxnvLJwyyL1z2OCCZvvS0VUuzv7OOUAc7xH3wUG3cxzZBZ8ArzDfY0CdAwSQ/s320/DSC08164.JPG" width="320" /></a><span style="font-size: x-large;">Fauzan</span><br />
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<blockquote class="tr_bq">
<i><span style="font-size: large;">"It was one of the coolest experiments I have ever done!"</span><span style="font-size: x-large;"> </span></i></blockquote>
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhdaraw-xhKz5g3UUce-3wIj7yCO-T9VftirZYLG2RVV5mYzJzsWHXQ68S601CqgnJ37LM0qcvAWLT_OMqMG1WsdHpixBgtre_C6fAH0z-j-QVot9p4fPFOLvd3BBXWXPC34KBD_EEaZKU/s1600/DSC08157.JPG" imageanchor="1" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" height="240" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhdaraw-xhKz5g3UUce-3wIj7yCO-T9VftirZYLG2RVV5mYzJzsWHXQ68S601CqgnJ37LM0qcvAWLT_OMqMG1WsdHpixBgtre_C6fAH0z-j-QVot9p4fPFOLvd3BBXWXPC34KBD_EEaZKU/s320/DSC08157.JPG" width="320" /></a></div>
<span style="font-size: x-large;">Estevan</span><br />
<blockquote class="tr_bq">
<i><span style="font-size: large;">"Learnt a lot. First time handling such an expensive piece of equipment all by myself. But it won't be my last."</span></i></blockquote>
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<span style="font-size: x-large;"> Azie</span><br />
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<span style="font-size: large;"><i>"A marathon laboratory experiment isn't so bad after all. I really enjoy working as a team."</i></span></blockquote>
Bioreactor Itu Menarikhttp://www.blogger.com/profile/02699912848197862358noreply@blogger.com0tag:blogger.com,1999:blog-2942965918570328948.post-57992866100756160012012-12-24T10:16:00.001+08:002012-12-24T11:45:44.761+08:00Result Interpretation <div class="separator" style="clear: both; text-align: center;">
<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgrnn9o-STr9BFYeOYWj7iwvA_wl4GoSFS_2c8_Jlhq05aTwIaENAG0sR7Ls5qOR008wI0VXUNAvYJO2_y8pwBlXKyK0ivPHrLzDlx5TYJAc8C5iqDGyZZK6j6AkPRxRrwxe6dEKBQOdZ8/s1600/Untitled-1.jpg" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" height="456" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgrnn9o-STr9BFYeOYWj7iwvA_wl4GoSFS_2c8_Jlhq05aTwIaENAG0sR7Ls5qOR008wI0VXUNAvYJO2_y8pwBlXKyK0ivPHrLzDlx5TYJAc8C5iqDGyZZK6j6AkPRxRrwxe6dEKBQOdZ8/s640/Untitled-1.jpg" width="640" /></a></div>
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<span style="font-size: large;">This growth curve that we obtained from the experiment is not a common growth curve of the <i>Saccharomyces cerevisiae. </i></span><br />
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As we can see from the curve, the cell culture achieved 2 stages of log phase with a stationary phase in between.</span><br />
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<span style="font-size: large;">Interpretation for the growth curve is as below:</span><br />
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<u><span style="font-size: large;">Lag phase</span></u><br />
<span style="font-size: large;">Inoculation was done at 0th hour, and the cells went into lag phase for 2 hours. During this stage, the cells which were originated from a 500 ml shake flask were trying to adjust themselves to the new environment. Rate of growth of cells was low at this stage. Lag phase lasted for approximately 2 hours.</span><br />
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<u><span style="font-size: large;">Log phase 1</span></u><br />
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At around 2nd hour, the cell biomass started to exhibit rapid growth. At this stage, the glucose substrate in the fermenter was fully utilized by the cells for growth and cell division. We observed a gradual increment of optical density from 1.600 to 8.270 within 4 hours.</span><br />
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<u><span style="font-size: large;">Stationary phase</span></u><br />
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<span style="font-size: large;">During the 6th hour to 12th hour, we observed the cells were experiencing fluctuation of growth. The Optical density of the cell biomass was maintained at 7.360, 8.900 and 8.630 for 8th, 10th and 12th hour respectively. The initial assumption we made was that, the cell biomass had reached its maximum due to depletion of the glucose substrate.</span><br />
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<u><span style="font-size: large;">Log phase 2 </span></u><br />
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<span style="font-size: large;">At 12th hour, we increased the air flow rate of the fermenter from 1.5 vvm to 3.0 vvm to test for test on our initial assumption. We found out that, increasing the air flow rate had also promoted the cells to enter another log phase. It appeared that our initial assumption on the depletion of glucose that had limited the growth of the cells were inaccurate. The actual limiting factor that had limited the growth of Saccharomyces cerevisiae from 8th to 12th hour was the oxygen concentration in the fermenter. A rapid increment of cell biomass was observed from 12th to 14th hours, followed by a slower increment of cell biomass from 14th to 16th hour.</span><br />
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<u><span style="font-size: large;">Decline phase</span></u><br />
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<span style="font-size: large;">Starting from 16th to 18th hour, optical density of the cell biomass started to show decreasing trend. At this stage, we had observed another uncommon phenomenon of cell growth where the microorganism had skipped the stationary phase and directly entered the declining phase. A possible rationale is that, the glucose substrate in the culture had really depleted this time. An increase in air flow rate afterwards didn't increase the optical density of the cell biomass. Another reasoning our group came up with was that, the build up of toxic in the culture has inhibited the growth of the cell biomass. However, future study and experimental testing would have to be done to prove that assumption.</span><br />
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<br />Bioreactor Itu Menarikhttp://www.blogger.com/profile/02699912848197862358noreply@blogger.com0tag:blogger.com,1999:blog-2942965918570328948.post-35322353239792612822012-12-24T09:28:00.001+08:002012-12-24T11:47:05.103+08:00Result<span style="font-size: large;">The following are the results we obtained from the analysis:</span><br />
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhzDeq1eCkuVlIpwW1kfy1x-qfJflfcLToi65bSJ7UiE_qbS3NaMT7j14CPV-9BeFSZo4ayTtv-uOA3rKt4TmAAacWBAcMHWZLmUoQu08JfCfrHlw-3uxLrJhyIKN5r4JKwadicPGIn7_E/s1600/Dilution+result.jpg" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" height="344" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhzDeq1eCkuVlIpwW1kfy1x-qfJflfcLToi65bSJ7UiE_qbS3NaMT7j14CPV-9BeFSZo4ayTtv-uOA3rKt4TmAAacWBAcMHWZLmUoQu08JfCfrHlw-3uxLrJhyIKN5r4JKwadicPGIn7_E/s640/Dilution+result.jpg" width="640" /></a></div>
Table 1: The result of the Optical Density of Cell biomass <i>Saccharomyces Cerevisiae</i> in 18 hours.<br />
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEj7fk9ZBAPML5h5CRPCw-6UdUbeK0PVzrNyagmWaO2a3I9qEZdIZG3sUaT2hvxHIBYFtGI-EtBo5szVdew_MSU2TgkHHVhCxqCjFbzKNf4jeyUzRBsT5fYVRi3pKWnDYjnRvLW4uvy-B1c/s1600/Growth.jpg" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" height="451" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEj7fk9ZBAPML5h5CRPCw-6UdUbeK0PVzrNyagmWaO2a3I9qEZdIZG3sUaT2hvxHIBYFtGI-EtBo5szVdew_MSU2TgkHHVhCxqCjFbzKNf4jeyUzRBsT5fYVRi3pKWnDYjnRvLW4uvy-B1c/s640/Growth.jpg" width="640" /></a>Graph 1 represent the grow curve of the <i>Saccharomyces cerevisiae</i></div>
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhM_fLV0AxnMC6SL0skcwVgy84uE1l83K2qriVxI3bqdkx_FErAuctWcgDQv9YMyDbA-oZymFCK0eaYNrY11eSY88lDgxBK-2DYxhbCo_04I9vp2d3Lf8katvJzQr_436Gr-Rr1M_kbu8o/s1600/gluc.jpg" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" height="421" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhM_fLV0AxnMC6SL0skcwVgy84uE1l83K2qriVxI3bqdkx_FErAuctWcgDQv9YMyDbA-oZymFCK0eaYNrY11eSY88lDgxBK-2DYxhbCo_04I9vp2d3Lf8katvJzQr_436Gr-Rr1M_kbu8o/s640/gluc.jpg" width="640" /></a></div>
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Graph 2: Glucose Concentration over Time. </div>
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<span style="font-size: large;">Note: The glucose concentration was only measurable until the 4th hours, later than that, the concentration of glucose was too low to be detected by the glucose level device. </span></div>
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjy4nsZZLKt9GENmH6AnT3H7aeNDTm_jUDNAZ7F0fMW8QgjBz_Ky2MFWOeEsJm87tT5vMPvRl1S-fmh_4KBQyiyzRcTTcifD4UCFxB28L3Vsy5P1bi2Pf69eQw49tYRwrpTGyRdSirJjRE/s1600/grawth.jpg" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" height="194" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjy4nsZZLKt9GENmH6AnT3H7aeNDTm_jUDNAZ7F0fMW8QgjBz_Ky2MFWOeEsJm87tT5vMPvRl1S-fmh_4KBQyiyzRcTTcifD4UCFxB28L3Vsy5P1bi2Pf69eQw49tYRwrpTGyRdSirJjRE/s640/grawth.jpg" width="640" /></a></div>
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Graph 2 is obtain from the Minifors Iris program that records and monitors the changes along the fermentation process</div>
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<span style="font-size: large;">For result interpretation, please refer to the post on "Result interpretation".</span></div>
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<i><br /></i>Bioreactor Itu Menarikhttp://www.blogger.com/profile/02699912848197862358noreply@blogger.com0tag:blogger.com,1999:blog-2942965918570328948.post-77642780661509405162012-12-24T00:29:00.002+08:002012-12-24T11:27:46.912+08:00Day 3 (Final day)<span style="font-size: large;">The fermentation was completed on the 3rd day. We got our last reading at 7.30am. All the analysis was completed. Mr. Sharman showed us how we could retrieve informations from the Iris software as well as to interpret the graph and data we obtained.</span><br />
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<span style="font-size: large;">Soon after that, we switched off the bioreactor and disassemble the parts to be cleaned and dried.</span><br />
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<span style="font-size: large;">We had also thanked and said goodbye to Mr. Sharman for being with us for the past few days. It was indeed a great experience working with Mr. Sharman. Hopefully, in the near future, we would still have the chance to meet up and share experiences again.</span><br />
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgG-9WxRstq_ndTmlIbdCc1sJkyGNba-1xe-Xe83fb9VlZtIVdshy2YzW_Db8yDyngYV5C1_7QyABNlwNAtN1Ia6vRkz3XLPZeOpYDV9xZbEA6m5hD33V6i6xFMoOLaR_Z5orCwsoqZYng/s1600/DSC08094.JPG" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" height="480" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgG-9WxRstq_ndTmlIbdCc1sJkyGNba-1xe-Xe83fb9VlZtIVdshy2YzW_Db8yDyngYV5C1_7QyABNlwNAtN1Ia6vRkz3XLPZeOpYDV9xZbEA6m5hD33V6i6xFMoOLaR_Z5orCwsoqZYng/s640/DSC08094.JPG" width="640" /></a></div>
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Sitting: Dr. Rosma, MR. Sharman, Pn. Wan Nadia (left to right)</div>
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with 3rd year students of Bioprocess Technology Industry 2012.</div>
Bioreactor Itu Menarikhttp://www.blogger.com/profile/02699912848197862358noreply@blogger.com0tag:blogger.com,1999:blog-2942965918570328948.post-644301256589793912012-12-20T01:40:00.000+08:002012-12-24T00:41:11.040+08:00PO2 PROBE CALIBRATION<span style="font-size: large;">This process was shown by Mr.Shamam,</span><br />
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<span class="Apple-style-span" style="font-family: Georgia,"Times New Roman",serif; font-size: large;">He took the pO2 electrode and removed the green plastic end cap. </span></div>
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<span class="Apple-style-span" style="font-family: Georgia,"Times New Roman",serif; font-size: large;">Then, the bottom metal section was unscrewed to check whether there was liquid electrolyte in the membrane cartridge inside. </span></div>
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<span class="Apple-style-span" style="font-family: Georgia,"Times New Roman",serif; font-size: large;">There is a bottle provided from the company to top up the liquid electrolyte if the level of the liquid is low. </span></div>
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<span class="Apple-style-span" style="font-family: Georgia,"Times New Roman",serif; font-size: large;">After that, he replaced the end section, and fit it into the vessel vertically and secured it.</span></div>
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</span>Bioreactor Itu Menarikhttp://www.blogger.com/profile/02699912848197862358noreply@blogger.com0tag:blogger.com,1999:blog-2942965918570328948.post-31006502933268632702012-12-20T00:38:00.003+08:002012-12-20T01:06:31.323+08:00Inoculation<span style="font-family: Georgia;"></span><br />
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<span style="font-family: Georgia;"><u><span class="Apple-style-span" style="font-family: Times,"Times New Roman",serif;">MEDIA PREPARATION FOR INNOCULATION OF SACCHAROMYCES CEREVISIAE </span></u></span></div>
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<span class="Apple-style-span" style="background-color: blue;"><span class="Apple-style-span" style="color: yellow;"><span class="Apple-style-span" style="font-size: large;"><span class="Apple-style-span" style="font-family: Georgia,"Times New Roman",serif;"><div style="margin: 0px;">
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<span class="Apple-style-span" style="font-family: Georgia,"Times New Roman",serif;">After Mr. Shaman gave us some explanations about the components of the bioreactor and their functions, we had move to second part of our lab which was the preparation of media for the innoculation of <i>Saccharomyces Cerevisiae,</i> which is the yeast that we will use in this experiment. </span></div>
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<span class="Apple-style-span" style="font-family: Georgia,"Times New Roman",serif;">The media that we used in this experiment was YEPG medium which contain yeast extract (1%), Peptone (2%) and glucose (2%). The total medium that needed for the innoculation was 200ml, so the weight for yeast extract, peptone and glucose needed were :</span></div>
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<span class="Apple-style-span" style="font-family: Georgia,"Times New Roman",serif;">Yeast Extract (1%) = 2g</span></div>
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<span class="Apple-style-span" style="font-family: Georgia,"Times New Roman",serif;">Peptone (2%) = 4g</span></div>
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<span class="Apple-style-span" style="font-family: Georgia,"Times New Roman",serif;">Glucose (2%) = 4g</span></div>
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<span class="Apple-style-span" style="font-family: Georgia,"Times New Roman",serif;">and after that we dissolved them with distilled water.</span></div>
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjl9aHx3KFDVeN6hgz-erZPceD3CA8MhICQ0YTDUAFNOPurvQf4dIMjwQbfmkmXffbeSg78pfNqi3sW3H2XFi11oJjvJcbvrYd-0rdLoJ4H1bUgXL3fG6WFf3zFjSP_tYUzzL6V_1fDvHg/s1600/DSCN7253.JPG" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" height="320" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjl9aHx3KFDVeN6hgz-erZPceD3CA8MhICQ0YTDUAFNOPurvQf4dIMjwQbfmkmXffbeSg78pfNqi3sW3H2XFi11oJjvJcbvrYd-0rdLoJ4H1bUgXL3fG6WFf3zFjSP_tYUzzL6V_1fDvHg/s320/DSCN7253.JPG" width="240" /></a></div>
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Image 1: medium of innoculation</div>
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<span class="Apple-style-span" style="font-family: Georgia,"Times New Roman",serif;"><span class="Apple-style-span" style="font-family: Georgia,"Times New Roman",serif;">After the medium was done, the media was then autoclaved before the innoculation of the yeast step was taken.</span></span></div>
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<span class="Apple-style-span" style="font-family: Georgia,"Times New Roman",serif;"><span class="Apple-style-span" style="font-family: Georgia,"Times New Roman",serif;">After 3o minutes we prepared seed culture, we using sterile toothpicks to scrape up a single colony aseptically. The work will be completed by transferring the colony into the shake flask which contained medium. We left it overnight (18-24 hours) in the incubator shaker.</span></span></div>
</span>Bioreactor Itu Menarikhttp://www.blogger.com/profile/02699912848197862358noreply@blogger.com0tag:blogger.com,1999:blog-2942965918570328948.post-5491699895813610142012-12-19T18:11:00.000+08:002012-12-24T00:44:01.592+08:00How to prepare a bioreactor for autoclaving <span style="font-size: large;"> Before undergo the fermentation process bioreactor and all its compartment must be sterile first in autoclave to prevent contamination. Below shows all steps preparation a bioreactor for autoclaving.</span><br />
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<span style="font-family: Calibri; font-size: large;">1. First, the buffer pH 4.01 and buffer pH 7.01 was prepared.<o:p></o:p></span></div>
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<v:shape fillcolor="white [3201]" id="_x0000_s1026" strokecolor="#4f81bd [3204]" strokeweight="5pt" style="height: 21.75pt; left: 0px; margin-left: 225pt; margin-top: 6.55pt; position: absolute; text-align: left; width: 15pt; z-index: 251658240;" type="#_x0000_t67"><span style="font-family: Calibri; font-size: large;"><v:stroke linestyle="thickThin"><v:shadow color="#868686"><v:textbox style="layout-flow: vertical-ideographic;"></v:textbox></v:shadow></v:stroke></span></v:shape><span style="font-size: large;"><o:p><span style="font-family: Calibri;"> 2. </span></o:p><span style="font-family: Calibri;">Then the pH probe was took and the cap was removed <span style="mso-spacerun: yes;"> </span>on the bottom and platinum was scraped to make sure it is neutral.<o:p></o:p></span></span></div>
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjQaDpOwrXuwvy2RgoGmaVaip_vCvJzQviUG5Sf_NS22NjQ4ODx7cK885s-PeyBgRaAGuwVvl3AvWS5GI9vKF29o7wu6JtpqUrSxEQ2W6VFcRTdo3qc6b5IbpPiFyrwoXaYpvECn1wasX8/s1600/DSC08064.JPG" imageanchor="1" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" height="400" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjQaDpOwrXuwvy2RgoGmaVaip_vCvJzQviUG5Sf_NS22NjQ4ODx7cK885s-PeyBgRaAGuwVvl3AvWS5GI9vKF29o7wu6JtpqUrSxEQ2W6VFcRTdo3qc6b5IbpPiFyrwoXaYpvECn1wasX8/s400/DSC08064.JPG" width="300" /></a><span style="font-size: large;"><span style="font-family: Calibri;">3. The probe was put in the pH buffer to set the point of the calibration curve, C1 is pH 7.01 and C2 is pH 4.01.</span><span style="mso-fareast-language: EN-MY; mso-no-proof: yes;"><v:shape id="Picture_x0020_4" o:spid="_x0000_i1027" style="height: 31.5pt; mso-wrap-style: square; visibility: visible; width: 33pt;" type="#_x0000_t75"><span style="font-family: Calibri;"> <v:imagedata o:title="" src="file:///C:\Users\UNATWI~1\AppData\Local\Temp\msohtmlclip1\01\clip_image004.png"></v:imagedata></span></v:shape></span></span></div>
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<span style="font-family: Calibri; font-size: large;">4. When calibrated step, the pH probe <span style="mso-spacerun: yes;"> </span>need to insert together with the temperature probe to allow comparison.<o:p></o:p></span></div>
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<span style="font-family: Calibri; font-size: large;">5. The medium was poured<span style="mso-spacerun: yes;"> </span>into the vessel with the adding of 15ml distilled water to make sure that no evaporation lost during autoclaving the medium.<o:p></o:p></span></div>
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<span style="font-family: Calibri; font-size: large;">6. The pO2 electrode was took and the green plastic end cap ws removed. Then, the bottom metal section was unscrewed to check whether there was liquid electrolyte in the membrane cartridge inside. There is a bottle provided from the company to top up the liquid electrolyte if the level of the liquid is low. After that, the end section was replaced, and fit it into the vessel vertically and secured it.<o:p></o:p></span></div>
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<span style="font-family: Calibri; font-size: large;">7. The reagent which is antifoam liquid was prepared by adding around 10ml in the 250ml bottle. A silicone dip tube was connected to one metal pipe on the head plate was checked. The tubing between the bottle and the pump head is clamped off.<o:p></o:p></span></div>
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<span style="font-family: Calibri; font-size: large;">8. Then the other parts of the fermentor was set up, exit gas cooler with filter, inlet filter connected to sparger, free port for inoculation and sample device. Need to double check all these accessories already completely set up. Then need to use aluminium foil to close all the parts that will direct exposed to the air before autoclaved, so that the fermentor will remain sterile after taking out from the autoclave.<o:p></o:p></span></div>
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<span style="font-family: Calibri; font-size: large;">9.Bioreactor was ready to autoclave.<o:p></o:p></span></div>
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<br />Bioreactor Itu Menarikhttp://www.blogger.com/profile/02699912848197862358noreply@blogger.com5tag:blogger.com,1999:blog-2942965918570328948.post-49020995784807322542012-12-19T16:25:00.000+08:002012-12-20T00:14:57.895+08:00Parts of a bioreactor<span style="font-family: Times, "Times New Roman", serif;"> In the first day meet Mr.Shamam from HT Company at room 148, he was introduced all compartments of bioreactor. All this part is very important to maintain and standardize the fermentation process for small and large volume production. Below shows a list of compartments of the bioreactor and its functions that was introduced by Mr.Shamam.</span><br />
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<span style="font-family: Times, "Times New Roman", serif;"></span><br />
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<span style="font-family: Times, "Times New Roman", serif;"> Image 1: compartments inside the vessel of bioreactor</span></div>
<span style="font-family: Times, "Times New Roman", serif;"></span><br />
<span style="font-family: Times, "Times New Roman", serif;"> The function of the vessel is where the
fermentation take place and inside it has many of compartments to complete the fermentation process.</span><br />
<span style="font-family: Times, "Times New Roman", serif;"></span><br />
<strong><span style="font-family: Times, "Times New Roman", serif;">1. Head plate:</span></strong><br />
<span style="font-family: Times, "Times New Roman", serif;">to cover the top of vessel of bioreactor</span><br />
<span style="font-family: Times, "Times New Roman", serif;"></span><br />
<strong><span style="font-family: Times, "Times New Roman", serif;">2. Antifoam probe:</span></strong><br />
<span style="font-family: Times, "Times New Roman", serif;">to prevent contamination in fermentation process</span><br />
<span style="font-family: Times, "Times New Roman", serif;"></span><br />
<strong><span style="font-family: Times, "Times New Roman", serif;">3. Stirrer shaft seal:</span></strong><br />
<span style="font-family: Times, "Times New Roman", serif;">To hold the stirrer and head plate</span><br />
<span lang="EN" style="font-family: Times, "Times New Roman", serif; line-height: 115%; mso-ansi-language: EN; mso-bidi-language: AR-SA; mso-fareast-font-family: "Times New Roman"; mso-fareast-language: EN-MY;"></span><br />
<strong><span style="font-family: Times, "Times New Roman", serif;">4. Acid/base probe:</span></strong><br />
<span style="font-family: Times, "Times New Roman", serif;">A flow to add acid and bases during fermentation process</span><br />
<span style="font-family: Times, "Times New Roman", serif;"></span><br />
<strong><span style="font-family: Times, "Times New Roman", serif;">5. Shaft:</span></strong><br />
<span lang="EN" style="font-family: Times, "Times New Roman", serif; line-height: 115%; mso-ansi-language: EN; mso-bidi-language: AR-SA; mso-fareast-font-family: "Times New Roman"; mso-fareast-language: EN-MY;">Not allow the medium to escape or microorganism enter</span><br />
<span lang="EN" style="font-family: Times, "Times New Roman", serif; line-height: 115%; mso-ansi-language: EN; mso-bidi-language: AR-SA; mso-fareast-font-family: "Times New Roman"; mso-fareast-language: EN-MY;"></span><br />
<strong><span style="font-family: Times, "Times New Roman", serif;">6. 2.5 glass jar bioreactor:</span></strong><br />
<span style="font-family: Times, "Times New Roman", serif;">Material of the vessel of bioreactor</span><br />
<strong><span style="font-family: Times, "Times New Roman", serif;"></span></strong><br />
<strong><span style="font-family: Times, "Times New Roman", serif;">7. Baffles:</span></strong><br />
<span style="background-color: white;"><span style="color: black;"><span style="color: white; language: en-US; mso-ascii-font-family: "Book Antiqua"; mso-ascii-theme-font: minor-latin; mso-bidi-font-family: +mn-cs; mso-bidi-theme-font: minor-bidi; mso-color-index: 1; mso-fareast-font-family: +mn-ea; mso-fareast-theme-font: minor-fareast; mso-font-kerning: 12.0pt; mso-style-textfill-fill-alpha: 100.0%; mso-style-textfill-fill-color: white; mso-style-textfill-fill-themecolor: text1; mso-style-textfill-type: solid;"><span style="background-color: white; color: black; font-family: Times, "Times New Roman", serif;">To prevent a whirl pool effect that could impede proper mixing</span></span></span></span><br />
<span style="background-color: white;"><span style="color: black;"><span style="color: white; font-family: Times, "Times New Roman", serif; language: en-US; mso-ascii-font-family: "Book Antiqua"; mso-ascii-theme-font: minor-latin; mso-bidi-font-family: +mn-cs; mso-bidi-theme-font: minor-bidi; mso-color-index: 1; mso-fareast-font-family: +mn-ea; mso-fareast-theme-font: minor-fareast; mso-font-kerning: 12.0pt; mso-style-textfill-fill-alpha: 100.0%; mso-style-textfill-fill-color: white; mso-style-textfill-fill-themecolor: text1; mso-style-textfill-type: solid;">per mixing</span></span></span><br />
<strong><span style="font-family: Times, "Times New Roman", serif;">8. Sampling port:</span></strong><br />
<span style="font-family: Times, "Times New Roman", serif;">A valve to get the sample of the fermentation</span><br />
<span style="font-family: Times, "Times New Roman", serif;"></span><br />
<span style="font-family: Times, "Times New Roman", serif;"><strong>9. 6- bladed disk impeller:
</strong><span lang="EN" style="line-height: 150%; mso-ansi-language: EN; mso-hansi-font-family: Calibri;"><span style="mso-list: Ignore;"><span style="font-family: "Times New Roman"; font-size-adjust: none; font-stretch: normal; font-style: normal; font-variant: normal; font-weight: normal; line-height: normal;"> </span></span></span></span><br />
<span lang="EN" style="font-family: Times, "Times New Roman", serif; line-height: 150%; mso-ansi-language: EN;">a. To diminish the size of air bubbles to give a bigger
interfacial area for O<span style="font-size: small;"><sub>2</sub> transfer and to decrease diffusion path.</span></span><br />
<div style="text-align: left;">
<span lang="EN" style="line-height: 150%; mso-ansi-language: EN;"></span><span lang="EN" style="line-height: 150%; mso-ansi-language: EN; mso-hansi-font-family: Calibri;"><o:p><span lang="EN" style="font-family: Times, "Times New Roman", serif; line-height: 115%; mso-ansi-language: EN; mso-bidi-language: AR-SA; mso-fareast-font-family: "Times New Roman"; mso-fareast-language: EN-MY;">b. To
maintain uniform environment throughout the vessel content.</span></o:p></span></div>
<br />
<span style="font-family: Times, "Times New Roman", serif;">
<strong>10. Air sparger:</strong></span><br />
<span style="font-family: Times, "Times New Roman", serif;"><span lang="EN" style="line-height: 115%; mso-ansi-language: EN; mso-bidi-language: AR-SA; mso-fareast-font-family: "Times New Roman"; mso-fareast-language: EN-MY;">To
ensure better dispersal of air</span><span lang="EN" style="line-height: 115%; mso-ansi-language: EN; mso-bidi-language: AR-SA; mso-fareast-font-family: "Times New Roman"; mso-fareast-language: EN-MY; mso-hansi-font-family: Calibri;">.</span></span><br />
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<span style="font-family: Times, "Times New Roman", serif;"> Image 2: All fittings on head plate of the bioreactor</span></div>
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEggLPyeMjLXbRoF35jHRmjHL_gK5b2nwvcNVQu2LDbCvmbjVhK54lM2CNcfkBAfMMl1PFHjOV-OJy01F-oY8g-rvkJ7hPvZYmd50idVt6hXqjIJitSLorqCjqz_hFmrEMfLM16JssjmP28/s1600/DSCN7195.JPG" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><span style="font-family: Times, "Times New Roman", serif;"><img border="0" height="320" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEggLPyeMjLXbRoF35jHRmjHL_gK5b2nwvcNVQu2LDbCvmbjVhK54lM2CNcfkBAfMMl1PFHjOV-OJy01F-oY8g-rvkJ7hPvZYmd50idVt6hXqjIJitSLorqCjqz_hFmrEMfLM16JssjmP28/s320/DSCN7195.JPG" width="240" /></span></a></div>
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<span style="font-family: Times, "Times New Roman", serif;"> Image 3: rotameter</span></div>
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<span style="font-family: Times, "Times New Roman", serif;"><span style="mso-fareast-language: EN-MY; mso-no-proof: yes;"><v:shapetype coordsize="21600,21600" filled="f" id="_x0000_t75" o:preferrelative="t" o:spt="75" path="m@4@5l@4@11@9@11@9@5xe" stroked="f">
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</v:f></v:f></v:f></v:formulas></v:stroke></v:shapetype></span>The rotameter is the means of manually
controlling the amount of air entering the vessel and for controlling the gas
flow rate.</span></div>
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<o:p><span style="font-family: Times, "Times New Roman", serif;">Image 4: holder of reagent bottles</span></o:p></div>
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<span style="font-family: Times, "Times New Roman", serif;"><span style="mso-bidi-font-family: Calibri; mso-bidi-theme-font: minor-latin; mso-fareast-font-family: Calibri; mso-fareast-theme-font: minor-latin;"><span style="mso-list: Ignore;"><span style="font-family: "Times New Roman"; font-size-adjust: none; font-stretch: normal; font-style: normal; font-variant: normal; font-weight: normal; line-height: normal;">
</span></span></span>The function of this holder is where the reagent
bottles are usually placed during </span></div>
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<span style="font-family: Times, "Times New Roman", serif;"> the fermentation.</span></div>
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhZV-Tk2FdDqm_eNYtzYjxxy54fNFQV83qM3xGFGqymB7FCkpOXFOLEofzK-D7xDxqfkPl84C-avhh_y5hskPssiaRObQ4yK1vCZNbZMF9mGScbWQ4CGof0kVFUAI1FtrwGdpLbVXUwDlQ/s1600/PA051860.JPG" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><span style="font-family: Times, "Times New Roman", serif;"><img border="0" height="296" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhZV-Tk2FdDqm_eNYtzYjxxy54fNFQV83qM3xGFGqymB7FCkpOXFOLEofzK-D7xDxqfkPl84C-avhh_y5hskPssiaRObQ4yK1vCZNbZMF9mGScbWQ4CGof0kVFUAI1FtrwGdpLbVXUwDlQ/s320/PA051860.JPG" width="320" /></span></a></div>
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<span style="font-family: Times, "Times New Roman", serif;"> Image 5: reagent bottles</span></div>
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<span style="font-family: Times, "Times New Roman", serif;"><span style="mso-bidi-font-family: Calibri; mso-bidi-theme-font: minor-latin; mso-fareast-font-family: Calibri; mso-fareast-theme-font: minor-latin;"><span style="mso-list: Ignore;"><span style="font-family: "Times New Roman"; font-size-adjust: none; font-stretch: normal; font-style: normal; font-variant: normal; font-weight: normal; line-height: normal;">
</span></span></span>The reagent bottles that usually needed during
the fermentation take place are, reagent bottle for base, antifoam and acid,
but in this experiment we do not need acid because we dealing with
microorganism, which is yeast (<i style="mso-bidi-font-style: normal;">Saccharomyces
Cerevisiae</i>) which usually will produce acid trough out the process.<o:p></o:p></span></div>
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<span style="font-family: Times, "Times New Roman", serif;"> Image 6: pO2 electrode</span></div>
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</span></span></span>The pO2 electrode should already be connected to
the bioreactor base unit which has been powered for at least 2 hours to ensure
the electrode is polarized. <o:p></o:p></span></div>
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<span style="mso-bidi-font-family: Calibri; mso-bidi-theme-font: minor-latin; mso-fareast-font-family: Calibri; mso-fareast-theme-font: minor-latin;"><span style="mso-list: Ignore;"><span style="font-family: "Times New Roman"; font-size-adjust: none; font-stretch: normal; font-style: normal; font-variant: normal; font-weight: normal; line-height: normal;"><span style="font-family: Times, "Times New Roman", serif;"> Image 7: Air compressor</span></span></span></span></div>
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The function of a</span></span></span>ir compressor<span style="mso-spacerun: yes;">
</span>is used to provide air during fermentation process.<o:p></o:p></span></div>
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<span style="font-family: Times, "Times New Roman", serif;"> Thats all for the introducing session with bioreactor compartments. Then, we are ready to go the experiment operation.</span></div>
Bioreactor Itu Menarikhttp://www.blogger.com/profile/02699912848197862358noreply@blogger.com3tag:blogger.com,1999:blog-2942965918570328948.post-21629063373196610742012-12-19T15:22:00.002+08:002012-12-27T15:19:56.901+08:00The sweet treat at KFC<span style="font-size: large;">Besides working, we were also invited to have a little enjoyment together at a nearby restaurant. </span><br />
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<span style="font-size: large;">It was really enjoyable that all the third year students, Pn Wan Nadia, Dr. Rosma and the postgraduate students who helped in the laboratory were able to gather for a lunch treat by Mr. Sharman. </span><br />
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEh3fpkdeJABf4oMyjEgGBgstU7Kmq34Rw9JfrwBKSEL2V2XU1K17OrxyvgutxgJ7durFkp-zOfxLaT3XH-ggwNU40Xuv-LA25TcBqff4JLreFG9VgzY6axotF2RcKQksYeCbPvnZw1Z7rM/s1600/DSC08086.JPG" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" height="480" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEh3fpkdeJABf4oMyjEgGBgstU7Kmq34Rw9JfrwBKSEL2V2XU1K17OrxyvgutxgJ7durFkp-zOfxLaT3XH-ggwNU40Xuv-LA25TcBqff4JLreFG9VgzY6axotF2RcKQksYeCbPvnZw1Z7rM/s640/DSC08086.JPG" width="640" /></a></div>
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<span style="font-size: large;">The chicken tasted especially delicious that day. We wonder whether it was because the chickens were provided free for us or it was due to the fact that we were having it together that makes it so. Maybe it was both. =)</span><br />
<br />Bioreactor Itu Menarikhttp://www.blogger.com/profile/02699912848197862358noreply@blogger.com0tag:blogger.com,1999:blog-2942965918570328948.post-6153302655342908992012-12-18T12:52:00.001+08:002012-12-20T00:13:42.973+08:00Day 2 (Part II)<div>
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<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: left; margin-right: 1em; text-align: left;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEi3tYMThUeKsa2_51f4eN28q3njLvygbip4Hc0nS27wSP3KjjCoC9LwxDg00oI3znhBgYiQbJyr5iz_F8P98hre1nnNNjUep30xqagX-kYi_51fP6KyvAqYreI6w1T_7J1FfHRTunLW6DI/s1600/DSC08171.JPG" imageanchor="1" style="clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" height="320" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEi3tYMThUeKsa2_51f4eN28q3njLvygbip4Hc0nS27wSP3KjjCoC9LwxDg00oI3znhBgYiQbJyr5iz_F8P98hre1nnNNjUep30xqagX-kYi_51fP6KyvAqYreI6w1T_7J1FfHRTunLW6DI/s320/DSC08171.JPG" width="240" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><span style="font-size: small;">Diagram 1: Fauzan measuring the Optical density of the sample</span></td></tr>
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<span style="font-size: large;">After the inoculation, the rest of the day was about sampling. The sampling was done at 2 hours interval. About 10 ml of culture was collected each time into a test tube. The cell biomass of the culture was monitor by measuring the optical density of the culture using a UV-Vis spectrophotometer. To monitor the glucose consumption by the cells, glucose tests were done using digital glucose testing device. Readings were recorded for both the optical density measurements as well as the glucose concentration in the samples.</span><br />
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<span style="font-size: large;">The sampling was carried out in a total of 18 hours, from 10.30 pm on 20/11/2012 (Tuesday) to 6.30 pm on 21/11/2012 (Wednesday). A total of 10 samples were collected and analysed.</span><br />
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<span style="font-size: large;">For the first time, we had our overnight experience in the lab. Through this experiment, we could see that teamwork is really important. We took turn, sacrificed our sleep time to make sure that the fermentation process was going smoothly throughout the night and sampling and analysis were done at the specific time. It was great experience!</span><br />
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<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEg3-o1fy4Cku4-ObQXYiaeH2wmdGRVQTwDHp7TmSAG5Ss7HgtpJ1zZlPYkPYczscq9s7KMR-I1ObxU36w_IcmqCpZ2ppfbqoJHtaLQaIfYJwI-oN3ysZDV-UmNTs72ynmbJHfzq2W74FgA/s1600/tube%252Bdilution%252Btest.jpg" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" height="240" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEg3-o1fy4Cku4-ObQXYiaeH2wmdGRVQTwDHp7TmSAG5Ss7HgtpJ1zZlPYkPYczscq9s7KMR-I1ObxU36w_IcmqCpZ2ppfbqoJHtaLQaIfYJwI-oN3ysZDV-UmNTs72ynmbJHfzq2W74FgA/s320/tube%252Bdilution%252Btest.jpg" width="320" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><span style="font-size: small;">Diagram 2: Samples collected to be analysed </span></td></tr>
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<br />Bioreactor Itu Menarikhttp://www.blogger.com/profile/02699912848197862358noreply@blogger.com0tag:blogger.com,1999:blog-2942965918570328948.post-37749173050311273092012-12-18T09:57:00.002+08:002012-12-18T10:07:14.669+08:00Day 2 (Part I)<div class="separator" style="clear: both; text-align: center;">
<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjTX0TJXzrIRckbu1ss9_XAdjRYH2zPfRIVDzAxjbxw9f_QGR06VO1jsOeZ2_3AdIy034Ms5gGxZlwDAPcfdmt8sLyrRmmtWLfnMHSfZG6W_4kn2tYZ2BPNO1W-VEzIZkW058WEwMrNyIE/s1600/DSC08069.jpg" imageanchor="1" style="clear: right; float: right; margin-bottom: 1em; margin-left: 1em;"><img border="0" height="240" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjTX0TJXzrIRckbu1ss9_XAdjRYH2zPfRIVDzAxjbxw9f_QGR06VO1jsOeZ2_3AdIy034Ms5gGxZlwDAPcfdmt8sLyrRmmtWLfnMHSfZG6W_4kn2tYZ2BPNO1W-VEzIZkW058WEwMrNyIE/s320/DSC08069.jpg" width="320" /></a></div>
<span style="font-size: large;">The day started with briefing on the inoculation process. It is crucial to carry out the aseptic techniques during the inoculation process to keep the contamination risk to the minimal.</span><br />
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<span style="font-size: large;">With Mr. Sharman's supervision, we inoculated the yeast culture into the bioreactor around 10.30am.</span><br />
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<span style="font-size: large;">Oxygen told by Mr. Sharman is one of the main factors that control the growth rate of the culture cells in the fermenter. The higher the oxygen transfer rate, the higher the cell growth rate.</span><br />
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<span style="font-size: large;">The Oxygen transfer rate which is as the KLa is controlled by a few parameters; the rotatory speed of the impeller, the air flow rate, concentration of the oxygen supplied and the pressure in the vessel.</span><br />
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<span style="font-size: large;">While using Minifors for the fermentation, we can easily manipulate 3 of the above parameters, which are the rotatory speed (rpm), the air flow rate (vvm) and the concentration of oxygen supplied (either we obtain oxygen from the air or from the oxygen tank).</span><br />
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<span style="font-size: large;">Minifors is built in such a way that makes controlling the fermentation process easier and more efficient. The cascade mode is making use of an intelligent technology that constantly monitor the oxygen uptake rate by the cells in the fermenter. When the oxygen update rate by the cells increases, the concentration of the oxygen in the fermenter decreases, this change is detected by the oxygen regulatory system of the fermenter. With the cascade mode technology, rotatory speed will be increased spontaneously. Thus, the oxygen diffusing rate to the media will increase as well. </span><br />
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<span style="font-size: large;">Further reading on:</span><br />
<span style="font-size: large;">1. Inoculation process, please refer to the post on "Inoculation"</span><br />
<span style="font-size: large;">2. Calibration of the PO</span>2 <span style="font-size: large;">probe, please refer to the post on "PO</span>2<span style="font-size: large;"> probe Calibration"</span><br />
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Bioreactor Itu Menarikhttp://www.blogger.com/profile/02699912848197862358noreply@blogger.com0tag:blogger.com,1999:blog-2942965918570328948.post-60404440336096568392012-12-18T02:51:00.001+08:002012-12-18T02:53:40.082+08:00Day 1<br />
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<b><span lang="EN-MY">Introduction of bioreactor by Mr. Sharman, the Asian South Pacific Regional Manager from INFORS HT Company.</span></b></h2>
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<span style="font-size: large;">The first day of the workshop, we were greeted with Mr. Sharman, a very humorous Indian man who was the speaker and mentor for our bioreactor workshop. </span></div>
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<span style="font-size: large;">The day started with a little introduction of Mr. Sharman himself follow by general background of the INFORS HT company.</span></div>
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<span style="font-size: large;">Next, we were given a tour on the anatomy of Minifors bioreactor. Functions of every single part of the bioreactor were explained in great details. </span></div>
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<span style="font-size: large;">We were given a brief lessons on the basics of scaling up process of a shake flask to a lab scale/ seed fermentor. </span></div>
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<span style="font-size: large;">"The parameters of fermentation such as, the temperature, pH, aeration, agitation and antifoam are the important things that we must take note throughout the fermentation process." - Mr. Sharman</span></div>
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<span style="font-size: large;">After the presentation, we were shown the demo of how to disassemble as well as to prep the bioreactor for steam sterilization in the autoclave. </span></div>
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<span style="font-size: large;">For the details of:</span></div>
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<span style="font-size: large;">1. Functions of each part of the bioreactor please refer to the post on "Bioreactor parts".</span></div>
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<span style="font-size: large;">2. Sterilization please refer to the post on "How to prep a bioreactor for autoclaving"</span></div>
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Bioreactor Itu Menarikhttp://www.blogger.com/profile/02699912848197862358noreply@blogger.com0tag:blogger.com,1999:blog-2942965918570328948.post-9078692026454172232012-12-16T23:43:00.001+08:002012-12-24T11:47:44.831+08:00Minifors Bioreactor <div style="text-align: center;">
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<span style="background-color: white; font-family: Georgia, Times New Roman, serif; font-size: large;">Minifors Bioreactor</span></h2>
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<span style="-webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; background-color: white; display: inline !important; float: none; font-family: Georgia, Times New Roman, serif; font-size: large; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: 17px; orphans: 2; text-align: left; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px;">Hi ! Welcome and thank you for visiting our blog ! </span></div>
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<span style="line-height: 17px;"><span style="font-family: Georgia, Times New Roman, serif; font-size: large;">In this blog, you will read about our 3 days Bioreactor Workshop in the Bioprcessing Technology lab, operating on the very stylish, high-tech., table-top Continuous Stir Tank Bioreactor. </span></span></div>
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<span style="line-height: 17px;"><span style="font-family: Georgia, Times New Roman, serif; font-size: large;">We must say that the experience is both wonderful and beneficial. We all enjoyed working with the "little smart glass jar". </span></span></div>
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<span style="line-height: 17px;"><span style="font-family: Georgia, Times New Roman, serif; font-size: large;">We would also like to take this chance to express our gratitude to Pn. Wan Nadiah, the lecturer of IBG 302 Bioreactor Operation for her work on arranging such an excellent workshop. To, Mr. Sharman, we couldn't thank you enough for travelling all the way from Kuala Lumpur, spending your time and sharing your precious knowledge with us. </span></span></div>
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<span style="line-height: 17px;"><span style="font-family: Georgia, Times New Roman, serif; font-size: large;">Do enjoy spending your time to look around. </span></span><br />
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Bioreactor Itu Menarikhttp://www.blogger.com/profile/02699912848197862358noreply@blogger.com0