Saturday, 24 January 2015

Anaerobic Wastewater Treatment Troubleshooting : Sulphate reduction bacteria

1) Sulphate reducing bacteria that can only oxidize their substrate into acetate. i.e, desulfobacter strain, desulfovibrio strain, desulfobacterium strain, desulfosarcina strain, desulfotomaculum strain.
2) Sulphate reducing bacteria that can oxidize their substrate completely to carbon dioxide. i.e.,Desulfobulbus strain, desulfomonas strain, desulfovibrio strain.
3) Dissimilatory sulphate & Bisulphate reduction. i.e, Desulfovibrio vulgaris, desulfotomaculum strain, desulfovibrio gigas, desulfovibrio desulfuricans, desulfomicrobium, desulfomonile, desulfuramonas, desulfurella, sulfurospirillum, etc.
4) Chemotrophic aerobic  sulfur oxidation. i.e., thiobacillus thioparus and thiobacillus strain, pseudomonas strain. etc.
5) phototrophic anaerobic oxidation of sulfur compounds : chromatiaceae, rhodospirillaceae,etc.

Tuesday, 6 January 2015

Immobilization of Yeast Cells


Objective

To Immobilize active Yeast Cells in the Calcium Alginate Gel and to check the viability of cell by 
invertase activity


 
Introduction

The term ‘immobilization’ was first proposed at the first enzyme engineering conference in 1971.
 Immobilization often causes a dramatic change in the apparent measuring parameter of the enzyme
 catalyzed by Michalis – Menton constant, temp optima, pH optima, and effect of inhibitors may be 
changed when an enzyme in immobilized. The degree and nature of these changes not only depends
 on the immobilization but also on the enzyme reaction.
There are various methods available for immobilization of enzyme
  • Absorption
  • Covalent binding
  • Cross matching
  • Micro encapsulation
  • Polymerization
  • Gel entrapment
Advantages of enzyme- immobilization
  • Stability of the enzyme immobilization increases even in adverse condition.
  • Resistance of enzyme molecules against metal ions and other inhibitors can also be increased.
  • Enzyme can be used repeatedly and continuously for the conversion of substrate into products.
  • In immobilized condition enzyme can be stored for longer time
Requirement
  • Yeast potato Dextrose (YPD)/ Potato Dextrose Agar (PDA) medium (for cultivation of Yeast cells)
  • Sodium alginate solution (2.5% w/v in 0.1% NaCl)
  • Calcium Chloride (CaCl2 ) solution (0.05 N)
  • Gluteraldehyde (2.5% v/v)
  • Sucrose solution (1% w/v)
Procedure for immobilization of Yeast Cells
  • Grow the yeast cells in YPD/PDA medium
  • Keep it in shaker for 24hours at 100 rpm
  • filter out the yeast cells with the help of Whattman filter paper
  • Take 20 ml sodium alginate solution and add 3 ml yeast cell in it. Mix properly and incubate at room temp for 30 min. then add 3 ml of gluteraldehyde solution incubate at room temperature for 90 min. With the help of 10 ml pipette, drop wise add this mixture into the beaker containing 100 ml CaCl2 solution
  • Filter out the beads with the help of normal filter paper
  • Wash the beads 2-3 times with sterile D/W
  • Load these beads into thoroughly washed glass column/ beaker
  • Add 50ml of sterile 1% sucrose solution
  • After every 30 min interval collect the 1 ml of sample and estimate the amount of glucose by using Dinitro salilcylic acid method (DNSA) method
Procedure for Estimation of Glucose by Dinitro salilcylic acid method (DNSA) method
  • Prepare a standard solution of carbohydrate (here glucose) having concentration of 1.0 mg/ml
  • Take different volumes of glucose solution like 0.5, 1.0, 1.5 & 2.0 ml etc. into various tubes previously labeled as S1, S2, S3, S4 etc. respectively
  • One tube should be labeled as blank
  • Now, take three tubes and labels as U1, U2 & U3 & pipette out 3 different volumes of sucrose solution from reaction mixture from above experiment in this tube
  • Add distilled water in all tubes in such a way that the total volume will be 2.0 ml
  • Add 2.0 ml of DNSA reagent in all tubes. Mix it properly by reversing the tubes or by using magnetic stirrer
  • Keep all the tubes in boiling water bath for 10 minutes. Then allow it to cool down
  • Take absorbance at 520 nm (using green filter) and plot a standard curve
  • Calculate out the concentration of glucose produced in the reaction mixture of above experiment

Immobilization of Yeast Cells


Result
Increase in the concentration of  glucose from the sucrose with respect totime by Active yeast cell (Invertase) indicates that Yeast Cells immobilized and they are viable

Media Formulation

  • Thorough analysis is essential to establish a suitable medium for an individual fermentation process
  • All most all microbes need water, energy sources, sources of carbon and nitrogen, certain mineral elements and perhaps vitamins plus oxygen if microbes are aerobic
  • It is easy to devise a medium containing pure compounds on a small scale but this medium may be unsuitable for use in a large scale fermentation processes
  • Following are the criteria imperative to  consider while designing a medium for large scale production
~ The medium should support the maximum production of yield of product per gram of substrate used
~ It should promote maximum accumulation of  the product
~The maximum rate of product formation should be achieved 
~There should be minimum production of unwanted products
~Constituents of the medium should be available throughout the year at cheaper rate and nearby area
~There should not be any undesirable changes in the consistency of the medium during preparation of media and after sterilization
~There should not be any difficulty in the operations like aeration, agitation during the production process and downstream operations like detection, isolation, extraction, purification and waste treatment

Saturday, 3 January 2015

The Development of Inocula for Yeast Processes


Industrial fermentations utilizing yeasts are the brewing of beer, the production of Baker’s Yeast (biomass) and recent processes have also been established for the production of recombinant products.

Brewing
Inocula for Yeast Process

  • Yeast can be used to inoculate a fresh batch of wort from previous fermentation or from propagator.
  • It is common practice in the British brewing industry to use the yeast from the previous fermentation.  
  • The brewing terms used to describe this process and 'crop', referring to the harvested yeast from the previous fermentation, and 'pitch', meaning to inoculate.
  • One of the major factors contributing to the continuation of this practice is the wort-based excise laws in the United Kingdom where duty is charged on the sugar consumed rather than the alcohol produced.
  • Thus, dedicated yeast propagation systems are expensive to operate because duty is charged on the sugar consumed by the yeast during growth.
  • The problems with this technique are chances of contamination and degeneration of strains, the most common problem with the degenerated cell is the change in the degree of flocculence and weakening of abilities of the yeast.
  • In breweries employing top fermentations in open fermenters these dangers are minimized by collecting yeast to be used for future pitching from 'middle skimmings’'.
  • As the head of yeast develops, the surface layer (the most flocculent and highly contaminated yeasts) is removed and discarded and the underlying cells (the 'middle skimmings') are harvested and used for subsequent pitching.
  • Therefore, the 'middle skimmings' contain cells which have the desired flocculence and which have been protected from contamination by the surface layer of the yeast head.
  • The pitching yeast may be treated to reduce the level of contaminating bacteria and remove protein and dead yeast cells by such treatments as reducing the pH of the slurry to 2.5 to 3, washing with water, washing with ammonium persulphate and treatment with antibiotics such as polymixin, penicillin and neomycin.
  • However, traditional open vessels are becoming rare and the bulk of beer is brewed using cylindro-conical fermenters.
  • In these systems the yeast flocculates and collects in the cone at the bottom of the fermenter where it is subject to the stresses of nutrient starvation, high ethanol concentration, low water activity, high carbon dioxide concentration and high pressure, which decreases the viability and physiological state of the yeast crop, would not be ideal for an inoculum.
  • The situation is further complicated by the fact that the harvested yeast is stored rapidly to about 1°, before it is used as inoculum suspending in beer and storing in the absence of oxygen.

  • Thus, we have the irregularity of oxygen being required for sterol synthesis; yet anaerobic conditions are required for ethanol production.
  • This irregularity is resolved traditionally by aerating the wort before inoculation.
  • The difficulties outlined above and the likelihood of strain degeneration and contamination mean that are rarely used for more than five to ten consecutive fermentations which necessitates the periodical production of a pure inoculum.
  • Pure inocula can be prepared by a yeast propagation scheme utilizing a 10% inoculum volume at each stage in the programme and employing conditions similar to those used during brewing.
  • Continuous aeration may be used during the propagation stage which seems to have little effect on the beer produced in the subsequent fermentation.
  • Yeast inoculum produced in this way would also be sterol rich, obviating the need for aerated wort.
  • The simplest type of propagator is a single stage system resembling an unstirred, aerated fermenter which is inoculated with a shake-flask culture developed from a single colony.
  • Two-stage systems propagator could be operated semi-continuously. It consisted of two linked vessels, 1.5 and 150 dm3 respectively.
  • The smaller vessel is filled with wort, sterilized, cooled, aerated and inoculated with a flask-grown culture. After growth for 3 to 4 days the culture was forced by air pressure into the second vessel which had been filled with sterilized, cooled wort and aerated.
  • An aliquot of 1.5 dm3 was forced back into the first vessel after mixing. In a further 3 to 4 days the larger vessel contained sufficient biomass to pitch a 1000 dm3 fermenter and the first vessel contained sufficient inoculum for another second stage.
  • However, although this procedure should produce a pure inoculum there is a danger of strain degeneration occurring in such a semi-continuous system.

Baker's Yeast

  • The commercial production of bakers' yeast involves the development of an inoculum through a large number of aerobic stages.
  • Although the production stages of the process may not be operated under strictly aseptic conditions a pure culture is used for the initial inoculum, thereby keeping contamination to a minimum in the early stages of growth.
  • The development of inoculum for the production of bakers' yeast involve eight stages, the first three being aseptic while the remaining stages were carried out in open vessels.
  • The yeast may be pumped from one stage to the next or the seed cultures may be centrifuged and washed before transfer, which reduces the level of contamination.
  • The yields obtained in the first five stages are relatively low because they are not fed-batch systems, whereas the last three stages are fed-batch.