bugs in india

There are times when we walk in the wild and find something interesting. We look at it, and forget. Then there are times when we remember seeing it but not photographing it. That’s when we regret. I have been through such times, apparently not photographing something that is very common, but then thinking back and wondering if it’s so common, why didn’t I photograph it?

On a dull winter day I was browsing on the web, where I came across a picture of an interesting centipede. I had photographed a similar fellow during monsoon in 2008, when I had been on a trail at SGNP (read it HERE). I came home and forgot about this image. It never appeared on my blog either. So now, more than a year later I dig into heaps of albums and find this one image. Now I know what it was and I wish I had taken more photographs.

This made me look for some common but lesser known bugs. After researching online, I was amazed to see little to no information on some bugs found in our backyards. There are studies and specific research around these bugs, but little is known to the general public. The bugs I am going to talk about interest me, and I conclude them to be the least or little known bugs of Mumbai. These are the top six on my list. Starting with the commonest:

Spittlebug
Everyone who has been on nature trails during monsoon has seen this bug. That frothy, spit-like “shelter” is built by a bug nymph. It’s not hard to recognize it once you see it. Spittlebug or Spit bug is very common all around Mumbai. These “spit homes” are more so common during post-monsoon months starting August to October. The frothy structure is not just a home, but a safe zone. It is itchy if it comes in contact with the skin, it keeps the nymphs moist and protect against heat and cold.

Spittlebug Larva in it's foamy shelter

The Spittlebug is in fact a larval stage of insects in the family Hemiptera – the true bugs. The adults are referred to as Leafhoppers or Froghoppers. The adults are easily distinguishable from one another compared to their larvae. The family Cercopidae is most abundant. The adults are as commonly seen during post-monsoon months as the nymphs. Some species are considered pests since they damage cash crops.

Callitettix versicolor - seen at Nagla Block part of SGNP

The “Spittlebug” I find interesting is Callitettix versicolor. This mysterious bug is so common, it’s an irony that there is no information available on the internet. It belongs to Cercopidae, is a plant-sap sucker, and is most commonly seen during post-monsoon as are its other cousins. It is a beautiful conspicuous bug, fairly large in size compared to some Leafhoppers and prefers clinging onto grass blades. Although the nymph may be a pest, the adult is not known to be a pest on any plants, but there is a big lacuna in online literature on this fellow.

Cercopis sp.

There are several Cercopidae bugs unknown to us – with their real name buried deep in million different scientific papers. The above photograph was taken in 2006 with an SLR. It is probably a Cercopis sp. – another beautiful Spittlebug adult – seen at Karnala.

Vindhyan Bob
Skippers (Hesperiidae) are my favorite butterflies because of their cryptic appearance, and the fact that identifying them is tougher than identifying the Blues (Lycaenidae) butterflies. These butterflies are small, commonly seen during monsoon and post-monsoon, and love shady forests. The Hesperiidae of Mumbai is well documented, but there is little information available on some.

Arnetta vindhiana - Vindhyan Bob, identified by the two distinct spots in the
center of the forewing - seen in the picture

Vindhyan Bob is of particular interest to me because of its mystery. It is seen throughout the seasons, but is most common during Monsoon months. This little skipper has baffled scientists for its multiple seasonal forms that are confusing. The different seasonal forms were long thought to be different subspecies! But that’s not it. This endemic skipper of Western Ghats is one of the commonest skippers seen in Mumbai – particularly SGNP – including Yeoor and some part of IIT-Campus. The sad part is – its life history is unknown to us. There are no records on its larval host plant and larval stages. However, we can speculate how the caterpillar might look and which food plant it might prefer based on its cousins in the same genus, but the fact that this endemic butterfly of ours is still unknown is the reason why I included it in Little Known Bugs of Mumbai.

Mating pair of Vindhyan Bob

There are several successful butterfly rearing enthusiasts, who I hope will one day find a mysterious caterpillar that belongs to Vindhyan Bob. Until one of us hunts it down, we can only wait. I have seen a mating-pair during monsoon of 2008, but that’s just one-fourth of its life cycle.

DerbidaeDerbidae is a family of bugs belonging to Hemiptera. The bug I am interested in is called – Derbid Planthoppers. It belongs to genus Proutista. I like to call it rabbit-eared fly. This bug is fairly common in all seasons if you have an eye for nature. It is mostly seen on palms, specifically Coconut palm, but also prefers shaded forests.

Derbid Planthopper

These bugs are plant sap-suckers in their adult stage, the nymphs are known to feed on fungi. There is another big lacuna on literature of these bugs. Seeing them in Mumbai is common, but seeking information is not. It is related to the planthoppers, as its name signifies, but they hold their wings straight up. They are cute, little interesting bugs that I know very less about.

Another unidentified Derbid Planthopper

Soldier Flies

Sargus sp. possibly metallinus

These flies are as common as Robber Flies. What’s not common is finding enough information on them. These are metallic, elongated flies with large eyes, covering most of the head. They are seen throughout the seasons. The soldier flies, specifically those found in Mumbai (or India) have no information available on the internet. But a quick search on Soldier Flies earns a good deal of information.


Melon flies

These flies are excellent mimics of wasps and bees. They belong to Stratiomyidae, a diverse family of flies. The most common I have seen is Sargus sp. This fly is most common of all, which is seen on most nature trails sitting patiently on leaf surfaces. The flight is very fast, to-and-fro, but they do not fly far off once disturbed.

Paper-wasp mimicking flies - Tephritidae flies

(Another probable Soldier Fly is a Paper Wasp mimic, picture shown above. It was seen at Yeoor in monsoon of 2009. It is one of the most interesting flies I have ever seen – which not only resembles a paper wasp, but congregates like one too.)

* This fly is now identified as a Melon-fly, Dacus sp., from the family Tephritidae. They are known to be serious pests on melons. Adults lay eggs under the "skin" of fruits, and larvae bore tunnels inside, thereby making the fruit rot sooner.

Kleptoparasitic FliesLet’s accept the fact that we don’t like observing flies as much as we like butterflies and spiders. There are several fly families that go unnoticed, but not all. The flies of the family Milichiidae are as common as spiders – because, where there are spiders – there are these flies, entitled Kleptoparasitic flies.

Uninvited Desmometopa sp. flies share Honey Bee prey of a Crab Spider

Kleptoparasitism is a form of parasitism, where one animal takes prey or food from another that has caught, collected or prepared the food – including stored food. The prey may not be stolen from its predator, but merely fed upon without predator’s consent. The Melichiidae flies are one best example, where they feed off the prey captured by other insects or spiders.

A Lynx feeding on a Honey Bee. Can you spot Milichiidae flies in the above picture?

This sort of behavior has been documented and photographed by many at SGNP. The flies hover around spiders and sit on the prey a spider is feeding on. This parasitic behavior ultimately gives the spider less nutrients, since the flies feed off the free food, sadly avoiding these flies is impossible.

Unidentified flies hike a ride on Lemon Pansy

There are several fly families that are kleptoparasitic, and Milichiidae is only one of them I have observed and photographed in SGNP. The flies sitting on this Lemon Pansy butterfly could probably be parasitic, but I have no idea – and never came across information on this kind of behavior – they are probably just hiking a ride ‘cause they are too lazy? Or are they parasitic?

Soil Centipede
One rare creature of the ground, soil centipedes are not that rare underground. It was this interesting centipede that made me wonder how many other creatures I overlooked in the past. I do not have a thing for centipedes, but a soil centipede is nothing like the ones I am afraid of.

Soil Centipede - being carried away by ants

Soil centipedes belong to Geophilomorpha – an Order under Chilopoda (Centipedes). They dwell underground or under rocks and are eyeless. These centipedes feed on bugs in the soil and are considered beneficial to the ecosystem. Seeing one above ground is rare, unless you turn the rocks or dig underground. Some species occur commonly in gardens, even plant pots, but that’s all we know about them. The soil centipedes also show an enormous lacuna in its information availability. The only reference I collected related to Indian Geophilomorpha was “Contributions to knowledge of the Chilopoda - Geophilomorpha of India” which is unfortunately not available online.

Many studies conducted abroad on Soil Centipedes cite the above reference, which is a clear fact that it is a priceless study on Geophilomorpha of India. It is our misfortune to not be able to find the document.

An unknown fly (probably Stomorhina sp.) or a deformed Chrysomya sp.?
Both belong to Calliphoridae (Blue Bottle Fly/Blow Flies)

The little known bugs are several that include spiders, moths, ants, beetles, bees and other insects. This is just a touch-up on the ones I find interesting, but know little about. Whether we will get know them or not is uncertain, since it might be too late to learn if all the habitats as we know fall under a serious threat. The current state of the habitats is not something I would argue upon. We all know what happened to Uran – a bird paradise, or we know what is happening to the mangroves elsewhere – or with the constant encroachment into the last forests of Mumbai, and we all regret on what’s happening and can do nothing about - simply because we-don’t-know-what-the-hell-we-can-do-without-getting-involved-with-police-and-politics.

By studying these animals, we will not only learn a bit more about the biodiversity of our surrounding, but it will be easier to assess the health of the ecosystem. These little flies and other creatures are at the base of a large food pyramid. The larva of Soldier Flies grow in damp, dead and decaying wood – excellent for breaking down the wood and passing the nutrients into the ground. The Milichiidae flies somehow control the extent of spider’s reign over the flowers (in place of bees). The Spittle bug larva and adults might be pests, but they also feed on weeds! Every organism is an indicator of the health of a habitat. Let’s look further into the thickets and find these creatures, and let them have their share of fame for the little but wonderful work they do to keep our planet going.

What does your “Little Known Bugs” list comprise of?

14 Pipetting Hacks to Become an Instant Expert

After a long day, you finally push back your chair, hang up your lab coat and take a well-deserved stretch. Time to go home! Your hand is aching, your thumb is quivering and your shoulder feels like it just ran a marathon. Another day in the lab, another day spent pipetting.

Some say it takes 10,000 hours of doing something to become an expert. We’d like to save you some of those hours, so here are 12 pipetting hacks to up your game.

Hack #1. No Lies, Not Even White Lies

Spinning the dial on your 1ml pipette to 753µl doesn’t mean you’re pipette is about to pick up 753µl of fluid. Pipettes have a measurement margin of error. This varies depending on the brand and you usually pay for increased accuracy. However, don’t take it for granted that your pipette has stayed true to its accuracy rating. Double-checking is a very good idea, especially if accuracy is paramount for your experiments. For a well-loved pipette, check its accuracy every 3 months. If you’re a less frequent pipette-r, annually is sufficient.

Hack #2. Keep Your Anger for the Gym

I’ve sat back helplessly as a colleague, perhaps frustrated at a conversation they just had or scared it would fall off, has rammed their pipette into tip after tip. SHOVE! JAB! STAB!

Ah! Poor pipette! They’re designed to fit the tip snugly but it should never be hard to get tips off. Tips should also never be hanging on for dear life. To put a tip on correctly, push the bottom of the pipette firmly into the tip. Try your best to use consistent force. Don’t tap or bang the pipette into the tip. Also, don’t pick up each tip individually and shove it on. Nobody has time for that! And you shouldn’t have to resort to it.

If your pipette has seen better days and faced far too much abuse from previous owners, the tips may fall off even though they seemed to be nicely fitted on. If this is the case, it’s time to go shopping (or at least hunting) for a replacement.

Hack #3. Don’t Harass Your Pipette

For some reason, folks seem convinced that the tiny, teeny, weeny bit of fluid remaining in the tip shouldn’t be there. Your pipette hasn’t forgotten to let it go. This is intentional. So never, ever, ever depress a pipette’s plunger a second time. Rookie move.

Hack #4. Finding the Right Angle

Repeat after me: aspirate at 90 degrees, dispense at 45 degrees, aspirate at 90 degrees, dispense at 45 degrees, aspirate at 90 degrees, dispense at 45 degrees. This pipette dance should become so second nature to you that anything else seems utterly bizarre and strange. When you pick up the liquid, hold the pipette at 90 degrees straight above the liquid. When you push it out, do so at a 45 degree angle and allow it to run down the side of the receptacle. Beautiful.

Hack #5. Don’t Rush Your Pipetting

Yes, it’s 8pm and you haven’t had lunch yet. Yes, your PI wanted the results yesterday. Yes, you need to vacate the tissue culture hood because you’re overtime and someone else has booked it. But no, you cannot rush your pipetting!

Aspirating at 100 miles per hour can cause fluid to fly into your pipette leading to contamination of your equipment, incorrect volumes pipetted, rusting of your pipette innards, and many other things that you don’t want. So take a breath or take a break. Your pipette will thank you for it!

Hack #6. No Sharesies

There is always someone in the lab who thinks it’s totally fine to go off with other people’s things. Your pipettes are only as clean and reliable as you can account for, so don’t share your pipettes. Or if you do, make the process so miserable no one will ask (photos, contracts, rules, time limits, whatever it takes). Worst case, have decoy pipettes that you care less about. I’m serious. Ain’t nobody got time for thieving labmates!

Hack #7. Don’t Get Airy Fairy

Be careful to aspirate liquid and not air. Simple, but key. Aspirating air can cause fluid to jump into the neck of your pipette, *coughs*.

Hack #8. No Deepsea Diving

There is no need to bury your tip way down into the fluid. Take the liquid from the surface being careful to follow hack #7. This minimizes the chances of your pipette touching the sides of your container. Back in class, we were told this help avoid contamination but exactly how is a very good question!

Hack #9. Sticky Situations

Sometimes when your volumes are super small, a drop can cling to the pipette tip. To discharge this tight-clinger, keep the plunger fully down, touch the drop onto the side of the container and if it still sticks, gently tap the side of the container until it drops. Don’t be tempted to go against hack #3.

Hack #10. Annual Holiday

We all need a break. Time to relax. Get ourselves back in order. Your pipette isn’t an exception. Send it off to be serviced at least once a year. Extra bonus points if you do so every 6 months.

If you drop it, send it off early or at least follow hack #1. Also, while you get yourself a coffee every day to stay on top form, your pipette prefers at least daily rubdowns with 70% ethanol. To each his own.

Hack #11. Take a Stand on Drawers

You like sleeping lying down. Well, your pipette likes sleeping standing up. Don’t store them in a drawer on their sides. Keep them in a pipette stand or make your own using binder clips. If anything has gotten in to the pipette, gravity will let it come out. Also pipette trees look cool but don’t spin them too fast, pipettes get dizzy.

Hack #12 The Right Tip for the Job

Make sure the tips you buy are compatible with the pipettes you have. Not all brands fit all. Also, make sure you’re using the correct sized tip for your pipette. There is no one size fits all tip either!

Hack #13. Pre-Wet Your Tip

Now here is one fact to know if you really want to show off at the office party!

Pre-wetting increases the humidity within the tip. This minimizes evaporation of the solution (unless you’re working in high ambient humidity).

Hack #14. Just Like Disapparition

Harry Potter taught us that to disappear and reappear somewhere else, you need only remember the three D’s of disapparition: Destination, Determination and Deliberation. Pipetting is the same.

Destination: know where your liquid is going to end up and have that destination ready to go with lids off etc.

Determination: don’t get fidgety halfway and hold the pipette in midair while you adjust things around.

Deliberation: don’t hang around in any container too long. Commit to what you started and, without rushing, move promptly. The longer containers are open, the more chance there is of them getting contaminated, spilt or anything else.

If you follow these three D’s, you’re basically a pipetting witch/wizard.

Follow these rules and you’ll be pipetting with the best of them and don’t forget to ice your hand.

FERMENTATION

A fermentation process requires a fermenter for successful production because it provides the following facilities for the process such as contamination free environment, specific temperature maintenance, maintenance of agitation and aeration, pH control, monitoring Dissolved Oxygen (DO), ports for nutrient and reagent feeding, ports for inoculation and
sampling, fittings and geometry for scale up, minimize liquid loss and growth facility for wide range of organisms.

Aseptic environment or contamination is defined as protection against entry of unwanted organisms. Containment is defined as prevention of escape of viable cells from the process. Both these environment is provided by a fermenter where ever required. Contamination is applicable in all process whereas containment is necessary when pathogenic organism is used for the fermentation process. The containment level varies based on the pathogenicity of the organism
used.

Some organism are termed GRAS ie. Generally Recognized As Safe. Criteria for assessment of hazardous organism are known pathogenicity of organism, virulence level, number of organisms required to initiate infection, routes of infection, known incidence of infection, local existence of vectors and reserves of micro organisms, volume of organisms used in process, techniques used for cultivation and harvesting and prophylaxis and treatment facility. Based on all the criteria if an organism is termed pathogenic the containment of the fermentation process is
maintained. Good industrial large scale practice (GILSP) involves safe and highly productive
organism for the process.

Depending on the type of product, the concentration levels it is produced and the purity desired, the fermentation stage might constitute anywhere between 5-50% of the total fixed and operating costs of the process. Therefore, optimal design and operation of bioreactor frequently dominates the overall technological and economic performance of the process.

In any biological process, the following are unique features.
(a) The concentrations of starting materials (substrates) and products in the reaction mixture are frequently low; both the substrates and the products may inhibit the process. Cell growth, the structure of intracellular enzymes, and product formation depend on the nutritional needs of the cell (salts, oxygen) and on the maintenance of optimum biological conditions (temperature, concentration of reactants, and pH) with in narrow limits.

(b) Certain substances inhibitors effectors, precursors, metabolic products influence the rate and the mechanism of the reactions and intracellular regulation.

(c) Microorganisms can metabolize unconventional or even contaminated raw materials (cellulose, molasses, mineral oil, starch, ores, wastewater, exhaust air, biogenic waste), a process which is frequently carried out in highly viscous, non-Newtonian media.

(d) In contrast to isolated enzymes or chemical catalysts, microorganisms adapt the structure and activity of their enzymes to the process conditions, whereby selectivity and productivity can change. Mutations of the microorganisms can occur under sub optimal biological conditions.

(e) Microorganisms are frequently sensitive to strong shear stress and to thermal and chemical influences.

(f) Reactions generally occur in gas-liquid -solid systems, the liquid phase usually being aqueous.

(g) The microbial mass can increase as biochemical conversion progresses. Effects such as growth on the walls, flocculation, or autolysis of microorganisms can occur during the reaction.

(h) Continuous bioreactors often exhibit complicated dynamic behavior. Due to above mentioned demands made by biological systems on their environment, there is no universal bioreactor.

However, the general requirements of the bioreactor are as follows:
         (1) The design and construction of biochemical reactors must preclude foreign contamination (sterility). Furthermore, monoseptic conditions should be maintained during the fermentation and ensure containment.

         (2) Optimal mixing with low, uniform shear achieved by proper designing of agitator and aerator

         (3) Adequate mass transfer (oxygen) achieved by monitoring the speed of agitator and agitator

         (4) Clearly defined flow conditions that can be maintained by proper opening valves and monitoring devices

         (5) Feeding of substrate with prevention of under or overdosing by proper feed ports and monitoring

         (6) Suspension of solids

         (7) Gentle heat transfer

         (8) Compliance with design requirements such as: ability to be sterilized; simple construction; simple measuring, control, regulating techniques; scaleup; flexibility; long term stability; compatibility with up- downstream processes; antifoaming measures.

AN IDEAL FERMENTOR

BODY CONSTRUCTION
Construction materials differ with small scale, pilot and large scale. In small scale for
vessel construction glass or stainless steel may be used. For pilot and large scale process, stainless steel (>4% chromium), mild steel (coated with glass or epoxy material), wood, plastic or concrete may be used as vessel construction material. Any vessel used should not have any corners and smooth surface is essential. The construction material must be non toxic and corrosion proof.

Glass vessel (borosilicate glass)
Type I – glass vessel round or flat bottom with top plate. It can be sterilized by autoclaving and the largest diameter is 60cm.
Type II – glass vessel flat bottom with top and bottom stainless steel plate. This type is used in in situ sterilization process and the largest diameter 30cm.

Stainless steel
Stainless steel is used as vessel construction material with the following modifications,
1. >4% chromium (atleast 10-13%) may be added
2. film of thin hydrous oxide - non-porous, continuous, self healing, corrosion resistance
3. inclusion of nickel - improves engineering
4. presence of molybdenum - resistance to halogen salts, brine, sea water
5. tungsten, silicone - improve resistance
Thickness of vessel should be increased with scale. Side plates have lower thickness than top and bottom plates. Top and bottom plate are hemispherical to withstand pressures.

SEALING
Sealing between top plate and vessel is an important criteria to maintain airtight condition, aseptic and containment. Sealing have to be done between three types of surfaces viz. between glass-glass, glass- metal and metal-metal. There are three types of sealing. They are gasket, lipseal and ‘O’ ring. This sealing ensures tight joint in spite of expansion of vessel material during fermentation. The materials used for sealing may be fabric-nitryl or butyl rubbers. The seals should be changed after finite time. There are two way of sealing in O ring type simple sealing and double sealing with steam between two seals.

BAFFLES
Baffles are metal strips that prevent vortex formation around the walls of the vessel. These metal strips attached radially to the wall for every 1/10th of vessel diameter. Usually 4 baffles are present but when the vessel diameter is over 3dm3 around 6-8 baffles are used. There should be enough gap between wall and baffle so that scouring action around vessel is facilitated. This movement minimizes microbial growth on baffles and fermentation walls. If needed cooling
coils may be attached to baffles.

AERATION SYSTEM (SPARGER)
Sparger is a device for introducing air into fermenter. Aeration provides sufficient oxygen for organism in the fermenter. Fine bubble aerators must be used. Large bubbles will have less surface area than smaller bubbles which will facilitate oxygen transfer to a greater extent. Agitation is not required when aeration provides enough agitation which is the case Air lift fermenter. But this is possible with only for medium with low viscosity and low total solids. For aeration to provide agitation the vessel height/diameter ratio (aspect ration) should be 5:1. Air supply to sparger should be supplied through filter.

There are three types of sparger viz. porous sparger, orifice sparger and nozzle sparger.
1. Porous sparger: made of sintered glass, ceramics or metal. It is used only in lab scale-non agitated vessel. The size of the bubble formed is 10-100 times larger than pore size. There is a pressure drop across the sparger and the holes tend to be blocked by growth which is the limitation of porous sparger.
2. Orifice sparger: used in small stirred fermenter. It is a perforated pipe kept below the  impeller in the form of crosses or rings. The size should be ~ ¾ of impeller diameter. Air holes drilled on the under surfaces of the tubes and the holes should be atleast 6mm diameter. This type of sparger is used mostly with agitation. It is also used with out agitation in some cases like yeast manufacture, effluent treatment and production of SCP.
3. Nozzle sparger: Mostly used in large scale. It is single open/partially closed pipe positioned centrally below the impeller. When air is passed through this pipe there is lower pressure loss and does not get blocked.
4. Combined sparger agitator: This is air supply via hallow agitator shaft. The air is emitted through holes in the disc or blades of agitator.

EXIT GAS COOLER
Similar to liebig condenser, condenses the moisture from the exhaust gas in the fermenter. This removes as much moisture as possible from the gas leaving the fermenter and prevent excess fluid loss.

AGITATION
Agitation provides uniform suspension of cells in homogenous nutrient medium. This agitation provides bulk fluid and gas phase mixing, air dispersion, facilitates oxygen transfer and heat transfer and uniform environment through out the vessel. There are four classes, namely Disc turbine, Vaned disc, Open turbine of variable pitch and Marine impeller.
Disc turbine prevents flooding by air bubbles. Flooding occurs when the air bubble is not properly dispersed the air pocket is formed one area. Flooded only at 120min/hour of air discharge when disc turbine is used. When open turbine and propeller are used the medium is flooded at 21min per hour of air discharge. Rushton disc turbine with 1/3 of fermentor diameter has been optimum for some fermentation process. Now recent designs of agitator have been introduced. Scaba is a new design of agitator that can handle high flow rate before flooding and has Radial flow. But this is not ideal for top to bottom mixing. Prochem maxflow agitator has low power conception with high hydrodynamic thrust. This design has increased downward pumping capacity of blades. In this design agitator/ vessel diameter ratio is 0.4. Appoximately 66% less power requirement even when viscous and oxygen transfer efficiency improved. Intermig agitator has two units. Unlike the earlier design agitator/ vessel diameter ratio is 0.6-0.7. For this agitator larger air sparger is used and top to bottom mixing not efficient. New turbine designs with dual impeller have been introduced. One for gas disperser and other for aiding circulation with multirod mixing.

STIRRER GLANDS AND BEARINGS
The entry point of stirrer into fermenter may be from top to bottom or sides. Mostly used from bottom so that that leaves more space for entry ports on top. There are four types of stirrer glands and bearings.
1) Stuffing box
a. sealed by several layers of packing rings of asbestos or cotton yarn-pressed against the
shaft by a gland follower
b. At high speeds- packing wears – pressure should be applied to ensure tightness
c. Difficult to sterilize- satisfactory heat penetration
d. Sufficient for GILSP containment

2) Mechanical seal
a. 2 parts; i) stationary in the bearing housing, ii) other rotates on the shaft.
b. Two parts pressed together by springs or expanding bellows
c. Steam condensate use to lubricate and cool seals
d. safe for containment
e. double mechanical seal for level 2
f. at level 2 and 3, the condensate is piped to a kill tank
g. Disinfectants flushed through the seal
h. steam condensate outlet monitoring indicates any seal failure

3) Magnetic drives (some animal cell cultures)
a. shaft does not pierce the vessel
b. two magnets- one driving, held in bearing in housing on outside of head plate and one driven, placed on one end of impeller shaft held in bearing in suitable housing
c. ceramic magnets –magnetic power cross 16mm gap
d. 300 – 2000 rpm rotation possible

4) Simple bush seals
Disadvantage of double seals are more difficult to assemble, difficult to detect failure of seal from normal and dead spaces and seals leading to contamination. Hence simple bush seal is preferred in some cases.

VALVES AND STEAM TRAPS
Addition valves
There are four types of addition valves viz.
(a) Simple ON and OFF,
(b) For coarse control,
(c) Accurate adjustment and
(d) Safety valve-flow in one direction.

There are different models of valves.
1. Opening and closing, raising or lowering of blocking unit
a. Gate valve - a sliding disc move in / out of flow path by a turn of the stem
b. Globe valve - horizontal disc / plug – raised / lowered
c. Piston valve - similar to globe valve except a piston controls flow
d. Needle valve - similar to globe valve except disc replaced with tapered plug / needle

2. Drilled sphere / plug
a. Plug valve - parallel / tapered plug with orifice – on 90 turn closes / open the flow path
b. Ball valve - similar to plug valve – except a ball (ss) with orifice replaces the plug

3. Disc rotating between bearings
Butterfly valve - a disc rotates about a shaft – closes against seal to stop flow

4. Rubber diaphragm / tube pinching
a. Diaphragm valve - similar to pinch valve – except not pinching, but pushing from one side against a diaphragm
b. Pinch valve - flexible sleeve closed by a pair of pinch bars (rubber, neoprene etc.)
Based on the four type of applications, the valves are chosen.
ON /OFF application – Globe, Butterfly
Crude flow control – Gate valve
Accurate control – Needle valve
Very sterile operation – Pinch / Diaphragm

Check valves
Valves used to prevent accidental reversal flow of liquid or gas due to break down. There are three types – swing check, lift check, combined stop and check.

Pressure control valves
These types of valves are used for two purposes.
a) Pressure reduction
b) Pressure retaining

Safety valve
There are types of safety valve by which the increase in pressure is released. They are,
a) A spindle lifted from its seating against the pressure – releases pressure
b) Bursting / rupturing of discs to release pressure
In case of releasing the gas, the escaping gas must be treated before release.

STEAM TRAPS
This steam trap is important to remove any steam condensate. There are two components viz. valve and seat assembly and open / close device. The operation of the component is based on, 10
i) density of fluid : A float (ball / bucket) float in water, sink in steam. When it floats it closes and when it sinks it opens the valve
ii) temperature of fluid : It has water / alcohol mixture which senses the change in temperature. This mixture expands in hot steam and closes the valve. When it contracts in cool water opens the valve.
iii) kinetic effect of fluid in motion : if a low density steam is flowing it will be high velocity. Like wise high density will flow with low velocity.The conversion of pressure energy into kinetic energy control the opening and closing

ibec indian biological engineering competition

Last date for submission: February 25, 2017

Purpose
This competition is to encourage and support student teams from India to participate in the world-wide synthetic biology competition, the iGEM [international Genetically Engineered Machine]

Competition details

• Research Concepts invited to build genetically engineered systems using standard interchangeable parts (promoters, terminators, plasmid backbone, reporter elements etc.) and operate them in living system.
• The cells so produced should exhibit novel and salient properties for potential applications.

Who can apply?
Undergraduate and postgraduate students teams (maximum 10) along with a mentor with credible experience in synthetic biology.

Selection criteria

• Non-obviousness and potential applications emerging from the proposed concept of the engineered systems
• Rational design of biological components that include construction of standard parts (DNA, RNA, Protein), devices (modules e.g. operon, transcription factor complex) and circuits (pathways/networks).
• Standards molecular biology studies that do not map to the rational design approach will not be selected

Upto five selected teams will be supported to register their research idea for submission to the iGEM competition

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Forms for submission is available at: PDF/ Doc
Last date for submission: February 25, 2017

For further enquiries contact:
Dr Kalaivani Ganesan,
Scientist ‘D’,
Department of Biotechnology
email: k.ganesan@nic.in

Scaleup criteria for Bioreactor 

(1) Microbial Cell Process:

Theoretically the following criteria were assumed suitable basis for scale up of bioreactors.

A. Constant power input per unit volume (P/V = constant).

B. Constant KLa

C. Constant mixing quality

D. Constant momentum factor (MF = ND. NWL (D – W)) = constant)

E. Similar drop size distribution (ds = constant)

F. Constant impeller tip speed (π NDi = constant)

G. Constant mixing rate number = (N/K)(Di/Dt)α constant

Short accounts of scaling up of bioreactors of fermenters on the basis of these criteria are discussed below;

1. Constant power input per unit volume:

For scaling up based on these criteria it is necessary to consider whether it is gassed or non-gassed system. From the work of Rushton and his associates, for geometrically similar, fully baffled vessels with turbulent conditions, it may be noted that if scale up should be based on maintaining a constant power input per unit volume considering no gassing in the system one may have the following.

:

(i) Non-gassed System:

When there is no gassing in the system then based on constant P/V has the power number, Np as

It was also apparent that in an oil drop dispersion system ds could serve as important criteria for scale up. The value of “a” in equation 8.34 depends on the type of agitator used in the system. For turbine impeller a= -0.6 whereas for draft tube fermented its value ranges between -0.15 to -0.35.

6. Constant impeller tip speed:

It was rather surprising to recognize that most of the data on impeller tip speeds collected were in the range 5-7 m sec-1 indicating the predominant importance of this parameter in scale up. Specially in antibiotic production plants constancy in tip speed (π NDi) was encountered in several cases. For scale up

In equation (8.37) N represents impeller tip speed and Di1 impeller diameter

7. Constant mixing rate number:

In scale up of bioreactors mixing time is often used as criteria. Mixing time (tmix), defined as the period of time required for the homogeneous distribution of a small volume of pulsating material in the bulk of the liquid, was used for scale up to adjust proper mixing conditions in large vessels. However, limitation of using tmix as scale up criteria is that it increases markedly with the size of the vessel and it is difficult to maintain to constant in both vessels at reasonable power expenditure.

In mixing a fermentation broth two vital questions to be answered are:

(1) What size of motor is needed for a given impeller rotation speed? and

(2) how long the broth must be mixed to achieve a required degree of homogeneity?

From the present knowledge the first question can be answered using Np – NRe relations of Rushton and Bates et al. The answer of the second question has been provided8 by introducing a new dimensionless term mixing rate number [(N/K) (D/Dt)a], NMR in the NP – NRe profile to characterize the rate of approach to uniformity in mixing vessels. This profiles as shown in Fig. 8.1 a and b indicate that for large impeller Reynolds number (NRe > 104) the following correlations will be applicable depending on the type of mixer used.

For turbine impeller mixer

(2) Scale Up of Filamentous Cell Fermentation:

Aerobic fermentative bioprocessing using filamentous microorganisms cover a wide range of industrially important bio-product formation systems. Various Streptomyces sp. antibiotic fermentations fall in this group. Such bioprocessing exhibit viscous and non Newtonian behavior following Power law fluid characters.

These processes are typical and present considerable problems in mixing and mass transfer for scale up purpose. Thus, agitator power number (NP) and impeller diameter (Di) are important case factors towards implications of Power law fluid on scale up along with liquid volume (VL) and (HL /Dt) ratio.

Case 1:

Agitator power (Pt) number influence

a. Provided with same geometry, vs, (Pt/Vt), VL and Di manipulation of volumetric mass transfer equations show

(3) Mammalian Cell Processing Scale Up:

In scaling up bioreactors of mammalian cell bioprocess most of the correlations are based on laboratory models alone and still awaits confirmation of their utility in industrial production concern. For mammalian cell bioprocessing current industrial scales typically involve bulk liquid volume of 25/ per day per bioreactor for continuous or semi-continuous systems.

In batch processing this bioreactor volume ranges between 500-5000 I capacities, operating over 1-3 weeks. There are no established scales up criteria for scaling up mammalian cell culture process. Cell culture engineers therefore, still largely depend on concepts and scale up procedures developed for chemical or microbial/biochemical processes to begin with.

However, an engineering body of experience and data will increasingly enable more specific design engineering and scale up procedures for mammalian cell culture bioprocesses in which kinetics is likely to be influenced by many factors and associated with many problems and limitations.

In scaling up procedures specific to animal cell culture, effect of scale on oxygen mass transfer through micro-porous membranes like silicone tubes for supplying bubble free oxygenation has been investigated. In order to aerate cell cultures, the membrane is immersed in the medium and the gas mixture (e.g. air, CO2 and O2) is passed through the tube under pressure.

Depending on the difference in pressure between the gas and the medium, gas flows through the membrane tube. Bubble free aeration is achieved if the internal gas pressure does not exceed the pressure at which bubbles will form. Correlation for bubble free oxygenation through membrane tubes has been developed.

For this, novel bioreactors have been designed for cell culture engineering in large quantities of the fragile complex mammalian cells that synthesize commercially and medically important proteins such as interferon and monoclonal antibodies. The correlation for bubble free oxygen using silicone micro-porous tube for oxygen mass transfer in this novel bioreactor has been given by the following correlations.

In these correlations, Sh is Sherwood number, K is film mass transfer coefficient (m s_1), d is tube diameters, D0 is molecular diffusivity of oxygen (m2 s-1), e, f, g are specific constants, Dt is bioreactor diameter (m) and NRe is impeller Reynolds number.

The provision of an adequate oxygen supply to large volumes of mammalian cells is the most crucial barrier to scale up, especially in suspension systems. Oxygen is only sparingly solube in cell culture medium (0.2 m mole O2I-1). Oxygen demand of a culture (106 cells per ml) ranges between 0.053 m mole O2 l-1 h-1 and 0.59 m mole O2I-1 h-1 depending on the type of cell. Scale up of mammalian cell cultures are, therefore, bound by several barriers as shown in Table 8.1.

As d, D0, NRe and Dt are given and e, f and g are determined experimentally, then equation 8.48 enables calculation of the oxygen mass transfer coefficient from impeller speed. With the assumption that gas velocity and composition within the silicone tube are constant, one may calculate the power required for this form of aeration at the large scale using the correlation given below.

In this correlation P, is the power input per unit mass of liquid (W.kg-1), V is the reactor volume (m3), D; is impeller diameter (m), p is the kinematic liquid viscosity (m2 s-1) and NP is the power number. Thus, scale up might be carried out on the basis of impeller speed to yield a constant oxygen mass transfer rate, but the power input per unit mass will vary with scale. It will need to check the power against an upper limit which will be determined by the shear sensitivity of the mammalian cells.

Lavery and Ninow in their study of the conventional power input versus mass transfer relation in sparged mammalian cell culture indicated the necessity of consideration of both the effect of head space aeration and the effect of added antifoam. This could account for the deviations from the expected values of KLa in their result.

A number of serious problems associated with the design and scale up of high density perfusion cultures of mammalian cell bioprocessing have been pointed out. For overcoming the associated problems in cultivation of animal cells and their scale up the following usage has been suggested.

1. Micro bubble technology and soluble surfactants for meeting cellular oxygen uptake rate (OUR).

2. Turbine or low shear impellers for achieving well mix cultures, such that it provides independent requirements of mixing and bubble aeration.

3. Minimization of ammonia accumulation by low glutamine supplemented media design.

4. Appropriately sized microfiltration hollow fibre cartridges which are steam sterilizable and easily cleaned after repeated uses.

(4) Plant Cell Processing Scale Up:

A typical plant cell culture manufacturing process is shown in Fig. 8.2. The great upsurge of interest in process biotechnology in the 1970’s carried plant cell culture with it. This is primarily because of centuries plants have been an important source of drugs and chemicals.

The extensive effort in development of plant cell culture is evident from the literatures on the subject. As plant cell cultures have many differences in comparison to microbes the design and scale up of suspension cultures bioreactors for plant cells also differ to a great extent. This becomes more pronounced as plant cell cultures can achieve high cell densities and viscosities. In such systems need for good mixing must have a compromise to shear sensitivity of plant cells.

Thus, in plant cell culture scale up system information is very important. Useful system information’s for scale up of plant cell culture as proposed by Goldstein are presented in Table 8.2. In addition to these quantization of entities as effected through bioprocess engineering studies at laboratory scale needs information on operational variables as listed in Table 8.3.

The first consideration in generating these information’s is the selection of a suitable bioreactor type. Continuous stirred tank bioreactor (CSTBR), air lift bioreactor (ALB), immobilized plant cell bioreactor etc. have been used in plant cell cultivations either in batch, plug flow or in continuous modes. In these three modes culture residence time is analyzed based on the relations given in Table 8.4. Assuming autocatalytic growth these relations emerge from classical catalysis.

The process parameters for scale up of plant cell cultures as listed in Table 8.5 demand low shear bioreactor for plant cell aggregates. As reported in literature plant cell cultures often form aggregates in suspension cultures in bioreactor. It can make the provision of oxygen difficult: the high aeration or mechanical agitation necessary can both cause shear damage to the cells.

Table 8.5 Process parameters for scale up of plant cell culture:

For overcoming the above difficulty and to facilitate scale up of plant cell culture bioreactor a design of low shear bioreactor has been reported. This design is stated to create separate cell growth and aeration zones. The bioreactor design is analogous to fluidized bed columns except that the cells are immobilized naturally (by aggregation).