Feasibility

of

an

in

situ

measurement

device

for

bubble

size

and

distribution

The

feasibility

of

in

situ

measurement

device

for

bubble

size

and

distribution

was

explored

A

novel

in

situ

probe

measurement

system

the

EnviroCam

was

developed

Where

possible

this

probe

incorporated

strengths

and

minimized

weaknesses

of

historical

and

currently

available

real-time

measurement

methods

for

bubbles

The

system

was

based

on

a

digital

high-speed

high

resolution

modular

camera

system

attached

to

a

stainless

steel

shroud

compatible

with

standard

Ingold

ports

on

fermenters

Still

frames

and/or

video

were

produced

capturing

bubbles

passing

through

the

notch

of

the

shroud

An

LED

light

source

was

integral

with

the

shroud

Bubbles

were

analyzed

using

customized

commercially

available

image

analysis

software

and

standard

statistical

methods

Using

this

system

bubble

sizes

were

measured

as

a

function

of

various

operating

parameters

-LRB-

e.g.

agitation

rate

aeration

rate

-RRB-

and

as

a

function

of

media

properties

-LRB-

e.g.

viscosity

antifoam

cottonseed

flour

and

microbial/animal

cell

broths

-RRB-

to

demonstrate

system

performance

and

its

limitations

For

selected

conditions

mean

bubble

size

changes

qualitatively

compared

favorably

with

published

relationships

Current

instrument

measurement

capabilities

were

limited

primarily

to

clear

solutions

that

did

not

contain

large

numbers

of

overlapping

bubbles

Introduction

Accurate

and

representative

bubble

sizes

and

distributions

are

used

to

characterize

biochemical

processes

containing

gas-in-liquid

dispersions

specifically

processes

for

industrially

important

fermentation

products

Quantification

of

bubble

sizes

and

distributions

during

fermentation

is

important

to

establish

mass

transfer

characteristics

-LRB-

based

on

gas

liquid

interfacial

area

-RRB-

when

oxygen

transport

to

cells

across

gas

liquid

interfaces

becomes

a

limiting

factor

In

these

situations

there

is

a

direct

influence

of

bioreactor

parameters

that

affect

bubble

size

such

as

agitation

on

culture

yields

Thus

measurements

of

bubble

sizes

and

distributions

are

useful

for

biochemical

process

optimizations

Direct

application

of

bubble

measurements

in

fermentation

is

based

on

linking

bubble

size

to

operational

parameters

such

as

agitation

and/or

airflow

rate

that

influence

gas

liquid

volumetric

mass

transfer

coefficients

-LSB-

-RSB-

Such

measurements

can

confirm

that

selected

agitation/aeration

conditions

do

not

lead

to

impeller

flooding

and

can

quantify

bubble

distribution

changes

for

-LRB-

-RRB-

geometrically

similar

fermenters

with

scale

up

and

-LRB-

-RRB-

different

fermenters

of

similar

scale

possessing

different

operating

conditions

geometry

or

bioreactor

internals

Owing

to

the

automated

and

fast

-LRB-

min

-RRB-

nature

of

the

Envirocam

bubble

measurement

and

analysis

it

is

a

potential

control

tool

for

maintaining

agitation/airflow

rate

set

points

to

obtain

desired

bubble

sizes

and

distributions

for

mass

transfer

for

gas

hold

up

or

for

minimizing

bubble

damage

to

cells

For

example

utility

costs

for

electricity

to

drive

agitators

or

air

compressors

might

be

optimized

further

for

large

production

scale

fermenters

Historical

and

currently

available

bubble

measurement

systems

using

photographic

methods

have

been

previously

summarized

and

the

references

cited

within

describe

several

applications

of

bubble

analysis

in

clear

solutions

-LSB-

-RSB-

The

accuracy

representation

and

simplicity

of

bubble

size

measurements

improve

when

these

measurements

are

performed

on-line

and

in

situ

rather

than

off-line

using

broth

samples

A

newly

developed

novel

on-line

and

in

situ

bubble

measurement

device

is

described

and

evaluated

Novel

in

situ

probe

measurement

system

EnviroCam

probe

The

EnviroCam

-LRB-

Enviroptics

Colmar

PA

-RRB-

probe

consisted

of

a

hermetically

sealed

shroud

constructed

of

l

stainless

steel

for

wetted

product-contact

parts

-LRB-

Fig.

-RRB-

A

silicone

o-ring

was

used

for

the

Ingold-style

tank

port

insertion

The

camera

module

itself

attached

to

this

shroud

and

thus

it

was

readily

relocatable

to

other

vessels

without

disturbing

process

integrity

There

were

no

cracks

or

crevices

since

the

shroud

was

entirely

welded

and

helium

leak-tested

The

overall

length

of

shroud

was

set

at

in

for

consistency

with

insertion

lengths

of

other

commercially

available

in

situ

probes

-LRB-

such

as

pH

dissolved

oxygen

or

optical

density

probes

-RRB-

This

length

avoided

interfering

with

fermenter

internals

such

as

larger

diameter

hydrofoil

impellers

but

extended

far

enough

into

the

well-mixed

fermenter

zone

Since

bubble

sizes

and

distributions

vary

according

to

their

distance

from

high

shear

impeller

zones

measurements

were

taken

at

a

constant

insertion

length

from

the

fermenter

sidewall

External

shroud

attachments

were

minimized

to

permit

location

in

tight

areas

in

some

cases

near

platform

structural

steel

supports

The

captive

retaining

nut

was

obtained

from

Mettler-Toledo

-LRB-

Ingold

Bedford

MA

-RRB-

to

match

existing

Ingold

ports

Fig.

shroud

diagram

Various

prototype

shrouds

and

camera

modules

were

developed

to

evaluate

different

hardware

options

A

high-strength

sapphire

window

was

integrated

into

the

l

stainless

steel

shroud

via

a

gold

brazed

joint

rather

than

epoxy

to

provide

improved

robustness

The

sapphire

window

was

constructed

at

a

angle

to

offset

the

angle

of

the

fermenter

's

Ingold

port

This

angle

resulted

in

the

window

being

parallel

to

the

vertical

tank

wall

upon

insertion

Owing

to

the

low

surface

tension

of

sapphire

some

small

bubbles

adhered

to

the

window

surface

particularly

at

the

low

agitation

speeds

used

for

animal

cell

cultivation

Using

the

vertical

orientation

as

well

as

raising

agitation

speeds

reduced

but

did

not

eliminate

this

accumulation

A

ceramic

disc

backscreen

provided

a

measured

path

length

Initially

light

from

a

Watt

halogen

lamp

was

reflected

internally

into

the

shroud

backscreen

via

a

high

performance

liquid

light

guide

but

this

arrangement

did

not

result

in

uniform

illumination

Next

LEDs

-LRB-

red

orange

nm

wavelength

-RRB-

were

incorporated

directly

into

the

shroud

backscreen

for

more

uniform

illumination

which

produced

bubble

images

with

improved

border

definition

To

minimize

the

impact

of

distortion

in

the

depth

and

width

of

the

measurement

volume

a

diffuser

was

installed

in

the

backscreen

A

calibrated

reticle

consisting

of

two

intrusion

lines

-LRB-

apart

with

a

gap

of

mm

-RRB-

emanating

from

the

circumference

and

heading

towards

its

center

became

the

standard

for

in

situ

calibration

This

reticle

was

located

on

the

sapphire

window

itself

rather

than

the

backscreen

to

avoid

interferences

in

opaque

media

With

a

backscreen

present

on

the

shroud

a

defined

path

length

or

notch

was

introduced

Prototype

shrouds

were

constructed

to

change

the

notch

size

between

and

in.

but

experiments

were

conducted

with

a

in

notch

unless

otherwise

noted

Due

to

the

small

width

of

the

support

-LRB-

in

-RRB-

the

orientation

of

the

notch

-LRB-

i.e.

up

down

sideways

with

the

opening

facing

with

or

against

agitator

rotation

-RRB-

was

deemed

not

to

prevent

larger

bubbles

from

entering

the

measurement

field

nor

influence

measured

bubble

sizes

or

distributions

The

externally

located

connector

for

the

LED

power

supply

provided

confirmation

of

the

internal

notch

position

Regardless

of

notch

size

the

bottom

of

the

notch

was

located

in

from

the

insertion

end

of

the

shroud

-LRB-

Fig.

-RRB-

As

notch

size

decreased

-LRB-

-RRB-

the

number

of

bubbles

viewed

per

frame

decreased

resulting

in

less

overlap

for

high

bubble

volume

broths

and

-LRB-

-RRB-

the

path

length

decreased

providing

more

light

for

opaque

solutions

In

contrast

larger

notch

gaps

may

be

required

for

lower

bubble

volume

broths

such

as

animal

cell

cultivations

EnviroCam

imaging

system

The

EnviroCam

imaging

system

is

shown

in

Fig.

One

imaging

system

containing

the

camera

module

-LRB-

about

tenfold

higher

cost

than

shroud

-RRB-

could

be

used

with

multiple

shrouds

Two

high

performance

megapixel

bit

monochrome

cameras

were

tested

each

of

which

attached

directly

to

the

shroud

-LRB-

-RRB-

a

monochrome

camera

-LRB-

Pixelink

Ottowa

ON

-RRB-

equipped

with

a

CMOS

image

sensor

which

operated

at

a

shutter

speed

of

s

-LRB-

s

μs

selected

-RRB-

and

-LRB-

-RRB-

a

monochrome

camera

-LRB-

Prosilica

Burnaby

BC

-RRB-

equipped

with

a

CCD

image

sensor

-LRB-

Exview

Sony

Tokyo

Japan

-RRB-

which

operated

at

a

shutter

speed

up

to

s

-LRB-

s

μs

selected

-RRB-

Primary

magnification

of

-LRB-

resulting

in

a

system

optical

magnification

of

-RRB-

was

implemented

using

one

additional

screw-on

lens

The

faster

shutter

speed

associated

with

the

CCD

camera

required

additional

light

which

was

sufficiently

provided

by

the

backlit

LED

geometry

of

the

shroud

A

partially

telecentric

mm

lens

-LRB-

Computar

CBC

Commack

New

York

-RRB-

was

selected

for

the

CCD

camera

which

reduced

the

viewing

angle

but

achieved

magnification

errors

of

while

providing

high

resolution

and

contrast

with

low

distortion

The

CCD

camera

became

the

preferred

configuration

consistent

with

the

designs

of

other

currently

available

photographic

systems

-LSB-

-RSB-

Fig.

system

diagram

The

number

of

pixels

-LSB-

horizontal

-LRB-

H

-RRB-

vertical

-LRB-

V

-RRB-

-RSB-

was

altered

using

user-defined

region

of

interest

-LRB-

ROI

-RRB-

controls

Higher

pixel

numbers

increased

resolution

but

reduced

the

number

of

frames

per

second

-LRB-

fps

-RRB-

For

the

selected

CCD

camera

the

hardware

was

set

at

fps

using

pixels

with

a

minimum

pixel

size

of

μm

and

a

pixel

depth

of

bits

-LRB-

without

additional

magnification

via

screw-on

lenses

-RRB-

This

fps

rate

of

resulted

in

a

time

scale

of

about

s

for

the

initial

image

scan

of

frames

which

was

small

relative

to

expected

changes

in

bubble

size

characteristics

during

the

time

course

of

a

typical

fermentation

Bubble

residence

time

through

the

measurement

field

-LRB-

in

mm

path

length

-RRB-

was

quantitatively

estimated

to

be

about

ms

near

the

impeller

blade

tip

-LRB-

impeller

tip

speed

of

m/s

-RRB-

and

likely

was

up

to

an

order

of

magnitude

slower

away

from

the

impeller

Consequently

it

was

not

necessary

to

increase

the

frame

speed

further

above

fps

which

corresponded

to

a

characteristic

measurement

time

of

ms/frame

The

bubble

residence

time

in

the

in

notch

measurement

field

was

qualitatively

determined

to

be

ms

by

comparing

common

features

of

subsequent

frames

Thus

it

was

necessary

to

skip

at

least

four

to

five

frames

to

ensure

bubbles

were

not

counted

more

than

once

For

the

CCD

camera

pixel

size

was

set

at

μm/pixel

With

the

magnification

added

and

other

geometric

and

positional

factors

considered

it

was

reduced

to

μm/pixel

A

small

annulus

of

the

viewing

range

-LRB-

pixels

wide

-RRB-

was

discarded

to

reduce

grey

scale

variations

near

the

edges

The

measurement

area

was

and

the

measurement

volume

was

-LRB-

mm

diameter

mm

height

-RRB-

A

number-based

hold-up

estimate

of

for

a

bubble

size

range

of

mm

was

calculated

-LSB-

-RSB-

Thus

for

the

calculated

measurement

volume

of

nearly

about

bubbles

were

expected

per

frame

image

The

actual

number

of

bubbles

per

image

ranged

up

to

or

more

when

larger

numbers

of

smaller

bubbles

less

than

mm

were

detected

In

comparison

with

these

values

selected

above

for

the

Environcam

system

prior

researchers

have

used

μm/pixel

and

pixels

for

gas/liquid

and

liquid/liquid

bubble/drop

dispersion

measurements

-LSB-

-RSB-

For

cell

morphology

measurements

μm/pixel

was

typical

-LSB-

-RSB-

and

pixels

was

the

most

common

for

recent

studies

-LSB-

-RSB-

The

remainder

of

the

Environcam

hardware

system

is

shown

in

Fig.

The

computer

itself

consisted

of

an

Intel

Pentium

class

notebook

PC

-LRB-

XP

operating

system

-RRB-

with

GB

RAM

-LRB-

NEMA

rating

as

required

by

the

facility

-RRB-

and

a

graphic

card

minimum

display

resolution

of

-LRB-

with

bit

color

-RRB-

The

video

output

was

the

Firewire/IEEE

interface

a

universal

interface

that

allowed

direct

connection

of

the

camera

to

the

PC

laptop

A

USB

port

was

used

for

computer

control

of

the

external

light

source

supplied

by

a

Nema

power

supply

which

was

powered

only

when

images

were

being

acquired

by

the

camera

The

lens

mount

was

a

standard

C-mount

adapter

used

for

camera

installation

to

lenses

and

to

standard

microscopes

A

CD/DVD

R/W

drive

USB

drive

and/or

some

other

type

of

network

interface

was

used

for

archiving

images

and/or

data

files

preferably

with

at

least

GB

of

storage

The

operating

temperature

range

was

limited

to

°C

for

the

camera

module

consequently

it

was

not

attached

to

the

shroud

during

vessel

sterilization

The

shroud

LED-operating

temperature

ranged

up

to

°C

but

its

non-operating

temperature

ranged

up

to

°C

All

other

shroud

components

were

steam-sterilizable

including

the

glass

diffuser

Thus

the

shroud

could

be

sterilized

with

the

vessel

if

the

LEDs

remained

unpowered

A

LED-power

supply

kill

switch

based

on

a

bimetallic

temperature

sensor

was

installed

with

a

trip

value

of

°C

and

reset

value

of

°C

When

it

was

powered

typically

intermittently

for

s

per

frame

measurement

cycle

or

continuously

for

video

stream

the

LED

was

required

to

be

submerged

in

liquid

as

a

heat

sink

for

adequate

cooling

LED

lifetime

was

h

assuming

the

non-operating

temperature

remained

less

than

°C

raising

it

a

few

degrees

above

this

level

for

effective

sterilization

might

sacrifice

some

lifetime

however

Shrouds

were

heat-tested

using

a

s

temperature

ramp

from

ambient

to

°C

held

for

h

then

returned

to

ambient

temperature

No

significant

degradation

as

measured

by

pixel

light

output

was

observed

after

temperature

cycles

In

addition

an

actual

sterilization

was

conducted

successfully

with

the

shroud

in

a

pilot

scale

fermenter

-LRB-

l

volume

°C

min

hold

time

-RRB-

Based

on

this

performance

shrouds

were

expected

to

withstand

about

sterilizations

of

min

hold

times

for

about

a

life

span

assuming

a

batch

length

The

USAF

resolution

target

was

used

to

evaluate

the

pure

video

resolution

of

the

computer

monitor

which

was

influenced

by

the

quality

of

the

video

graphics

card

Resolution

was

measured

at

line

pairs/mm

the

reciprocal

of

which

resulted

in

a

resolution

of

μm/pixel

significantly

higher

than

the

camera

resolution

of

μm/pixel

Thus

the

accuracy

of

the

image

display

did

not

diminish

the

accuracy

of

the

photographs

obtained

The

software

platform

was

customized

based

on

National

Instruments

-LRB-

Austen

TX

-RRB-

LabVIEW

Graphical

Development

Environment

-LRB-

version

-RRB-

The

system

's

main

screen

is

shown

in

Fig.

The

image

analysis

software

had

the

following

key

features

Measured

frames

were

included

in

analysis

until

the

number

of

desired

objects

to

be

measured

-LRB-

a

user

input

-RRB-

was

attained

Specifically

for

a

measurement

time

of

s

-LRB-

corresponding

to

fps

-RRB-

which

generated

frames

typically

every

tenth

frame

was

skipped

-LRB-

i.e.

measured

frames

were

taken

every

s

-RRB-

and

frames

were

measured

to

identify

at

least

bubbles

This

number

was

consistent

with

other

currently

available

measurement

systems

described

in

the

literature

-LSB-

-RSB-

This

approach

also

was

similar

to

skipping

every

frame

when

measuring

floatation

cell

moving

aggregate

sizes

-LSB-

-RSB-

The

background

was

subtracted

from

the

original

image

which

avoided

repeatedly

counting

bubbles

lodged

on

the

sapphire

window

and

to

remove

fixed

blemishes

in

the

backscreen

-LRB-

This

approach

is

similar

to

subtracting

a

background

image

without

cells

from

the

original

image

-LSB-

-RSB-

-RRB-

The

background

image

was

calculated

by

averaging

all

frames

obtained

during

the

measurement

period

so

that

moving

objects

were

filtered

out

to

obtain

a

clear

composite

background

image

This

approach

was

speedy

-LRB-

ms

for

frames

-RRB-

In

addition

the

background

image

was

readily

reconstructable

from

stored

images

if

necessary

A

user-selectable

alternative

algorithm

was

developed

to

construct

a

background

composed

of

the

brightest

pixel

of

each

of

the

frames

for

each

position

assuming

that

no

pixel

should

be

any

brighter

than

a

background

pixel

The

time

to

construct

the

background

using

either

of

these

methods

was

similar.Edge

enhancement

techniques

based

on

a

contrast

threshold

were

applied

to

convert

grey

images

to

binary

black

and

white

images

so

that

the

outside

perimeters

-LRB-

or

diameters

-RRB-

of

bubbles

were

readily

identifiable

via

object

recognition.The

analyzed

image

output

was

prepared

using

the

calibrated

reticle

-LRB-

measurement

shown

in

red

in

Fig.

-RRB-

located

on

the

shroud

backscreen

in

the

form

of

a

binary

mask

In

addition

a

blue

reference

grid

of

mm

squares

-LRB-

Fig.

-RRB-

was

overlayed

to

permit

the

user

to

approximate

bubble

sizes

on

the

computer

screen.Depending

on

the

application

bubbles

of

sizes

greater

than

a

designated

cut-off

-LRB-

e.g.

mm

in

diameter

-RRB-

between

and

this

cut-off

and

below

μm

were

identified

Both

visual

and

numerical

indications

of

the

classification

of

objects

were

developed

according

to

three

tiers

of

predetermined

rules

Green

circles

represented

the

first

level

of

filtering

Rules

in

this

first

tier

were

single

bubbles

of

sizes

within

the

target

measurement

range

circularity

cut-off

based

on

a

user-selected

tolerance

above

value

and

discard

of

bubbles

touching

the

border

The

green

circle

was

the

best

fit

circle

so

there

were

slight

inaccuracies

around

some

of

the

circumference

if

the

bubbles

were

not

uniformly

round

Larger

or

smaller

circular

bubbles

outside

the

target

measurement

range

were

excluded.Yellow

outlines

indicated

the

second

level

of

filtering

typically

using

similar

rules

as

the

first

level

but

with

more

relaxed

user-defined

cut-offs

Additional

rules

also

were

added

in

this

second

tier

Up

to

three

more

or

less

circular

bubbles

that

were

touching

but

still

individually

discernable

were

included

in

the

count

Specifically

the

ratio

cut-off

of

cluster

-LRB-

i.e.

more

than

one

adjacent

bubble

overlapping

-RRB-

area

to

the

calculated

equivalent

area

for

completely

separated

bubbles

of

the

cluster

was

based

on

a

user-selected

tolerance

below

Bubbles

in

this

category

were

further

differentiable

using

additional

capabilities

included

in

the

LabVIEW

software.Red

outlines

comprised

a

third

level

that

indicated

bubbles

located

by

the

system

that

did

not

pass

filtering

levels

or

White

outlines

comprised

a

fourth

level

of

classification

This

fourth

tier

contained

non-circular

blobs

and

large

irregularly

shaped

or

greatly

overlapping

bubbles

The

percentage

of

viewing

area

occupied

by

blobs

was

estimated

and

measured

frames

were

omitted

based

on

a

user-selected

cut-off

-LRB-

e.g.

if

greater

than

a

target

percentage

of

of

the

measurement

area

was

comprised

of

blobs

the

frame

was

discounted

-RRB-

The

relevant

statistical

quantities

were

calculated

-LRB-

e.g.

arithmetic

geometric

and/or

Sauter

mean

diameters

-RRB-

and

the

appropriate

histograms

were

displayed

-LSB-

-RSB-

A

customized

SQL

program

extracted

data

into

csv

files

-LRB-

containing

all

raw

data

and

analysis

calculations

-RRB-

These

csv

files

had

a

practical

limit

of

including

information

about

bubbles

typically

an

amount

greater

than

required

for

a

single

measurement

cycle

Data

was

imported

into

an

Excel

template

containing

graphs

and

some

statistical

quantities

as

part

of

the

measurement

cycle

-LRB-

typically

requiring

s

to

min

-RRB-

and

then

into

other

statistical

programs

-LRB-

e.g.

Sigmaplot

LabView

modules

-RRB-

for

more

advanced

analysis

should

the

user

desire.The

front

panel

image

containing

the

configuration

and

user-adjustable

controls

was

archived

so

that

an

identical

set

up

could

be

reproduced

in

the

future

if

desired.Tiff

files

-LRB-

still

frames

-RRB-

and/or

avi

-LRB-

video

stream

-RRB-

files

were

archived

but

avi

files

were

reconstructable

from

Tiff

files

to

save

storage

space

A

video

playback

rate

of

fps

appeared

appropriate

The

actual

measurement

frames

utilized

for

analysis

were

saved

separately

for

ease

of

review

Minimum

storage

requirements

for

these

unanalyzed

frames

were

estimated

at

about

mB

for

each

frame

measurement

cycle

-LRB-

versus

GB

for

analyzed

frames

-RRB-

with

the

entire

analysis

reconstructable

from

saved

data

Thus

the

ability

to

retrieve

images

and

recheck/reanalyze

results

readily

existed.For

measurement

frames

a

review

panel

of

the

analysis

sequence

was

created

from

archived

files

-LRB-

Fig.

-RRB-

The

analysis

progression

panels

were

reviewed

manually

to

confirm

accuracy

To

obtain

an

image

of

acceptable

contrast

the

user

varied

the

camera

shutter

speed

-LRB-

length

of

exposure

to

light

-RRB-

brightness

level

-LRB-

luminescence

of

image

on

LCD

monitor

-RRB-

aperture

-LRB-

amount

of

light

reaching

camera

lens

-RRB-

and

gain

-LRB-

amplification

of

signal

strength

-RRB-

Several

parameters

in

the

software

were

adjustable

to

ensure

optimal

image

analysis

and

the

values

selected

could

be

archived

As

a

first

step

threshold

was

varied

automatically

by

the

software

to

determine

the

value

at

which

the

maximum

number

of

bubbles

was

identified

Subsequently

various

filtering

strategies

available

in

the

software

were

examined

FFT

-LRB-

fast

Fourier

transform

-RRB-

was

found

to

potentially

improve

accuracy

for

measurements

in

water

but

it

was

not

implemented

for

the

present

work

Localized

thresholding

-LSB-

-RSB-

was

found

to

improve

accuracy

for

bubble

measurements

in

broths

and

it

was

incorporated

The

largest

inscribable

diameter

was

taken

to

calculate

the

bubble

diameter

-LSB-

-RSB-

Fig.

main

screen

displayFig

composite

for

a

bubble

frame

-LRB-

l

DI

water

with

agitation

rpm

air

flowrate

rpm

-RRB-

showing

a

original

raw

camera

image

b

binary

image

output

with

edge

enhancement

c

binary

mask

prior

to

application

to

image

d

image

after

binary

mask

e

analysis

output

image

containing

colored

circles

identifying

objects

according

to

the

three

tiers

of

rules

and

f

instrument

screen

including

histogram

Key

features

of

the

EnviroCam

gas

bubble

measurement

system

are

summarized

in

Table

in

a

format

readily

comparable

to

those

assembled

for

prior

and

currently

available

optically

based

measurement

systems

-LSB-

-RSB-

Table

characteristicsCamera

and

mode

of

attachmentMagnification/calibrationIllumination/shutter

or

frame

speedImage

measurement

and

analysis

methodCCD

monochrome

camera

-LRB-

Prosilica

-RRB-

probe

inserted

into

an

in

situ

Ingold-fitting

shroud

line

internal

reticleLEDs

in

back

screen

-LRB-

back

lit

-RRB-

fps

s

-LRB-

ms

selected

-RRB-

National

Instruments

-LRB-

Austin

TX

-RRB-

LabVIEW

Graphical

Development

Environment

as

basisMeasurement

time

per

conditionNumber

of

objects

per

measurementMeasurement

error/size

rangeNumber

of

images

-LRB-

pictures/frames

-RRB-

per

s

data

acquisition

min

data

analysis

-LRB-

typically

up

to

-RRB-

for

monodisperse

-LRB-

bubbles/picture

-RRB-

Bead

calibration

A

calibration

was

conducted

using

beads

of

a

known

size

distribution

in

applicable

size

ranges

for

bubbles

Calibration

beads

were

measured

individually

by

manually

moving

the

bead

past

the

notch

or

in

a

small

volume

mixed

solution

contained

in

a

magnetically

stirred

beaker

Vortexing

was

minimized

by

reducing

the

stirring

speed

Settling

of

the

larger

glass

microsphere

beads

-LRB-

e.g.

specific

gravity

of

-RRB-

was

reduced

by

performing

the

measurements

in

a

perfluoropolyether

-LRB-

Da

specific

gravity

of

Fomblin

Solvay

Solexis

Bollate

Italy

-RRB-

In

measurement

frames

beads

had

a

softer

lighter

less-contrasted

outline

when

compared

with

bubbles

which

slightly

reduced

the

effectiveness

of

the

optical

imaging

system

without

additional

filters

Thus

measurement

of

beads

was

limited

to

sizes

μm

slightly

higher

than

the

lower

limit

for

bubbles

First

monodisperse

spherical

polyethylene

calibration

beads

-LRB-

Baltec

Balzers

Lichtenstein

-RRB-

in

-LRB-

rsd

-RRB-

or

mm

in

diameter

were

measured

to

evaluate

the

higher

end

of

the

expected

Environcam

measurement

range

-LRB-

Other

reported

options

for

calibration

beads

were

expanded

polystyrene

beads

with

mean

diameters

of

and

mm

and

a

density

of

-LSB-

-RSB-

mm

ball

bearings

-LSB-

-RSB-

and

red

spherical

particles

of

diameters

and

μm

-LSB-

-RSB-

-RRB-

Using

the

external

halogen

lamp

light

source

and

the

CMOS

camera

system

a

value

of

μm

-LRB-

single

measurement

-RRB-

was

obtained

which

was

higher

than

the

standard

's

value

Using

the

LED

light

source

installed

in

the

shroud

backscreen

and

the

CCD

camera

system

a

value

of

μm

was

obtained

which

was

lower

than

the

standard

's

value

Next

high

precision

NIST-tracable

glass

micro-spheres

-LRB-

Whitehouse

Scientific

Chester

UK

-RRB-

were

selected

in

various

monodisperse

and

polydisperse

sizes

Distributions

-LRB-

in

the

format

of

count

versus

bin

size

-RRB-

were

fit

to

Sigmoidal

or

modified

Gaussian

distributions

using

Sigmaplot

-LRB-

Systat

San

Jose

CA

-RRB-

-LRB-

Table

-RRB-

These

distribution

equations

better

fit

the

bead

measurement

data

than

distribution

equations

used

for

bubbles

-LSB-

-RSB-

Monodisperse

bead

sizes

ranging

from

to

μm

in

increments

of

at

least

μm

-LRB-

corresponding

to

the

instrument

resolution

-RRB-

were

tested

-LRB-

Table

-RRB-

Measured

size

values

agreed

reasonably

well

with

the

manufacturer

's

data

values

Measured

ranges

for

of

the

bead

size

range

typically

enveloped

the

manufacturer

's

data

with

differences

most

likely

owing

to

the

nature

of

the

fitted

distributions

The

distribution

of

polydisperse

beads

over

most

of

the

Environcam

measurement

range

-LRB-

μm

-RRB-

also

was

measured

and

compared

with

the

analyzed

manufacturer

's

distributions

for

three

size

ranges

-LRB-

Table

-RRB-

Reasonable

agreements

in

the

distribution

shape

were

obtained

The

accuracy

of

these

Environcam

measurements

showed

that

differences

in

means

were

observed

and

standard

deviations

were

lower

In

contrast

other

researchers

using

calibration

beads

for

optically

based

instrument

systems

reported

that

means

were

identical

to

the

standard

's

values

but

standard

deviations

were

higher

-LRB-

manufacturer

μm

measured

μm

manufacturer

μm

measured

μm

-RRB-

-LSB-

-RSB-

Table

used

to

fit

calibration

bead

measurement

data

-LRB-

from

Sigma

plot

software

-RRB-

DistributionProbability

density

functionModified

Gaussian

-LRB-

G

-RRB-

Y

Yo

a

exp

-LSB-

-LRB-

abs

-LRB-

X

Xo

-RRB-

b

-RRB-

-RSB-

Sigmoidal

-LRB-

G

-RRB-

Y

Yo

a

-LRB-

exp

-LSB-

-LRB-

X

Xo

-RRB-

b

-RSB-

-RRB-

Table

of

manufacturer

size

-LRB-

Whitehouse

Scientific

Chester

UK

-RRB-

with

measured

size

range

for

monodisperse

beadsManufacturer

's

size

data

-LRB-

μm

-RRB-

Manufacturer

's

data

for

of

beads

within

given

range

-LRB-

μm

-RRB-

EnviroCam

measured

size

-LRB-

μm

-RRB-

EnviroCam

data

for

of

beads

within

given

range

-LRB-

μm

-RRB-

Not

able

to

be

Not

able

to

be

-LRB-

G

-RRB-

-LRB-

S

-RRB-

-LRB-

S

-RRB-

-LRB-

G

-RRB-

-LRB-

S

-RRB-

-LRB-

S

-RRB-

-LRB-

G

-RRB-

-LRB-

S

-RRB-

-LRB-

S

-RRB-

-LRB-

G

-RRB-

-LRB-

S

-RRB-

-LRB-

S

-RRB-

-LRB-

G

-RRB-

-LRB-

S

-RRB-

-LRB-

S

-RRB-

-LRB-

G

-RRB-

-LRB-

S

-RRB-

-LRB-

S

-RRB-

-LRB-

G

-RRB-

-LRB-

S

-RRB-

-LRB-

S

-RRB-

-LRB-

G

-RRB-

-LRB-

S

-RRB-

-LRB-

S

-RRB-

-LRB-

G

-RRB-

-LRB-

S

-RRB-

-LRB-

S

-RRB-

-LRB-

G

-RRB-

-LRB-

S

-RRB-

-LRB-

S

-RRB-

Envirocam

measurements

fitted

to

modified

Gaussian

equation

-LRB-

G

-RRB-

and

sigmoidal

equations

-LRB-

S

-RRB-

using

Sigmaplot

software

All

values

unless

otherwise

noted

Beads

of

sizes

and

μm

were

not

measurable

owing

to

blurry

edgesTable

of

manufacturer

size

-LRB-

Whitehouse

Scientific

Chester

UK

-RRB-

distribution

with

measured

size

distribution

for

polydisperse

beadsBead

distribution

-LRB-

μm

-RRB-

Size

at

fixed

percentiles

-LRB-

μm

-RRB-

StandardEnviroCam

StandardEnviroCam

StandardEnviroCam

StandardEnvirocam

Envirocam

measurements

fitted

to

sigmoidal

equation

using

Signmaplot

software

Duplicate

runs

executed

for

μm

distribution

Gas

bubble

data

analysis

The

smallest

bubble

measurable

was

μm

in

diameter

based

on

an

expected

maximum

system

resolution

of

μm/pixel

A

minimum

of

pixels

were

needed

to

quantify

the

radii

and

radii

were

used

for

diameter

calculations

based

on

initial

ease

of

programming

Since

the

radius

accuracy

was

pixel

the

relative

standard

error

for

a

μm

bubble

was

dropping

to

for

a

μm

bubble

If

required

for

other

applications

smaller

objects

down

to

μm

might

be

measured

using

-LRB-

-RRB-

two

pixels

to

determine

diameter

or

-LRB-

-RRB-

using

a

back-calculated

diameter

based

on

equivalent

surface

area

since

only

pixels

were

required

to

define

surface

area

Bubble

diameter

measurements

generated

using

the

image

analysis

software

were

compared

with

those

generated

manually

for

objects

from

a

single

frame

image

taken

using

the

CCD

camera

system

and

a

in

gap

shroud

Results

demonstrated

that

the

greatest

percentage

error

was

observed

with

smaller

bubbles

in

the

diameter

range

of

μm

This

error

generally

decreased

with

larger

bubble

diameters

Manually

measured

diameters

were

slightly

longer

than

image

analysis

measurements

greater

than

of

the

time

most

likely

due

to

a

small

amount

of

shadowing

around

bubble

edges

The

measurement

error

was

for

the

calibration

line

itself

Coincidentally

for

some

actual

bubble

measurement

conditions

the

number

of

objects

rose

considerably

for

bubbles

μm

in

diameter

This

rise

may

be

caused

by

the

greater

measurement

inaccuracy

at

this

size

thus

small

changes

in

size

were

not

detectable

Since

the

minimum

hardware

resolution

was

μm

based

on

at

least

a

pixel

change

in

radius

buckets

in

the

distribution

possessed

μm

increments

This

distribution

resolution

was

consistent

with

the

μm

value

reported

for

an

optical

bubble

measurement

system

with

a

magnification

-LSB-

-RSB-

and

similar

to

the

μm

value

reported

for

a

stationary

flatbed

scanner

set

up

-LSB-

-RSB-

The

system

's

ability

to

measure

bubble

size

changes

due

to

typical

differences

in

fermenter

operating

conditions

was

estimated

based

on

this

resolution

Specifically

an

agitation

rate

increase

from

to

rpm

decreased

the

size

of

μm

bubbles

to

μm

and

an

agitation

rate

increase

from

to

rpm

decreased

the

size

of

μm

bubbles

to

μm

-LRB-

D

α

N

-LSB-

-RSB-

-RRB-

Both

of

these

changes

were

substantially

greater

than

the

μm

resolution

and

thus

bubble

size

differences

caused

by

agitation

rate

changes

were

believed

detectable

using

this

measurement

system

For

other

operating

conditions

where

the

effect

on

bubble

change

was

less

pronounced

-LSB-

-RSB-

these

differences

were

not

expected

to

be

as

readily

detectable

Incremental

and

cumulative

distributions

were

plotted

typically

as

number

or

cumulative

percentage

versus

bubble

size

respectively

Arithmetic

Sauter

and

geometric

means

and

standard

deviations

-LRB-

as

applicable

-RRB-

then

were

calculated

according

to

published

methods

-LSB-

-RSB-

In

addition

the

largest

bubbles

and

smallest

bubbles

were

discarded

and

the

arithmetic

and

Sauter

means

recalculated

Using

these

cut-offs

significantly

reduced

the

skewing

and

variability

impact

of

smaller

numbers

of

larger

bubbles

in

the

size

distributions

on

the

arithmetic

and

Sauter

mean

diameter

calculations

-LRB-

Fig.

b

-RRB-

Owing

to

the

nature

of

the

geometric

mean

calculation

there

was

little

effect

when

the

cut-off

was

used

-LRB-

Fig.

-RRB-

Fig.

-LRB-

a

-RRB-

Sauter

-LRB-

b

-RRB-

and

geometric

-LRB-

c

-RRB-

means

and

standard

deviations

-LRB-

as

applicable

-RRB-

calculated

using

all

the

bubbles

and

for

the

cutoff

Calculations

conducted

on

frames

using

every

tenth

frame

for

frames

and

each

calculation

point

covering

different

frames

starting

from

successive

initial

frames

The

total

number

of

bubbles

required

to

be

analyzed

per

measurement

greatly

affected

the

measurement

and

data

acquisition

times

-LRB-

MAT

and

DAT

respectively

-RRB-

plus

the

data

storage

requirements

Based

on

a

survey

of

published

techniques

-LSB-

-RSB-

the

target

number

was

bubbles

per

measurement

condition

This

value

was

confirmed

based

on

determining

that

means

and

standard

deviations

changed

only

minimally

-LRB-

less

than

-RRB-

when

bubbles

numbers

below

and

beyond

were

analyzed

-LRB-

specifically

and

-RRB-

also

suggesting

that

as

few

as

bubbles

were

sufficient

The

desired

total

bubble

number

target

may

be

achieved

based

on

a

higher

number

of

bubbles

per

frame

-LRB-

-RRB-

and

a

smaller

number

of

frames

-LRB-

-RRB-

or

a

lower

number

of

bubbles

per

frame

-LRB-

-RRB-

and

a

larger

number

of

frames

-LRB-

-RRB-

with

care

taken

not

to

count

bubbles

more

than

once

in

successive

frames

in

either

case

Measurement

reproducibility

was

evaluated

using

bubble

measurement

data

from

a

l

fermenter

by

analyzing

every

tenth

frame

for

frames

starting

at

the

first

frame

until

bubbles

were

obtained

then

re-analyzing

every

tenth

frame

starting

from

the

second

frame

then

again

starting

from

the

third

through

tenth

frames

The

relative

standard

deviations

of

the

averages

typically

were

under

for

the

cut-off

Sauter

mean

diameter

and

under

for

the

cut-off

arithmetic

and

geometric

means

Thus

the

sampling

of

frames

used

for

analysis

was

representative

of

the

total

number

of

frames

collected

Discarding

the

blob

area

from

the

measurement

was

not

felt

to

significantly

influence

bubble

diameter

means

and

distributions

nor

impact

subsequent

predictions

of

volumetric

mass

transfer

coefficients

The

overall

blob

interfacial

area

per

unit

volume

a

was

relatively

low

compared

to

that

of

the

selected

measured

bubbles

-LRB-

e.g.

for

cm

blobs

a

μm

for

mm

bubbles

a

μm

-RRB-

owing

to

the

large

diameters

of

the

blobs

In

addition

the

percentage

of

larger

bubbles

-LRB-

i.e.

mm

diameter

-RRB-

was

relatively

low

-LRB-

-RRB-

for

most

typical

agitator/sparger

system

set

points

and

configurations

Their

overall

contribution

to

interfacial

area

also

was

low

Similarly

although

bubbles

below

the

μm

limit

of

detection

had

a

large

collective

interfacial

area

their

small

individual

volume

resulted

in

fast

oxygen

depletion

by

the

broth

making

them

a

less

substantial

source

of

oxygen

supply

-LSB-

-RSB-

Thus

accuracy

for

bubble

sizes

outside

the

target

range

of

μm

mm

was

considered

less

critical

Consequently

the

maximum

bubble

size

cut-off

of

mm

used

for

measurement

appeared

acceptable

since

larger

bubbles

tended

to

be

non-spherical

as

previously

described

-LSB-

-RSB-

and

smaller

bubbles

-LRB-

mm

diameter

-RRB-

in

aqueous

solutions

previously

were

shown

to

behave

as

particles

with

rigid

interfaces

-LSB-

-RSB-

Consequently

significant

numbers

of

irregularly

shaped

bubbles

were

not

obtained

within

the

target

measurement

range

and

the

use

of

bubble

diameter

to

approximate

size

was

reasonably

accurate

Test

systems

Water/media/broth

Liquid

test

media

consisted

of

deionized

water

-LRB-

DIW

-RRB-

and

the

following

solutions

all

prepared

using

DIW

vol

glycerol

-LRB-

viscosity

of

Pa

s

-LSB-

-RSB-

vs.

Pa

s

for

water

-LSB-

-RSB-

-RRB-

ml/l

-LRB-

vol

-RRB-

-LRB-

polypropylene

glycol

Dow

Freeport

TX

-RRB-

ml/l

-LRB-

vol

-RRB-

antifoam

C

-LRB-

Sigma

-RRB-

mono/di

potassium

phosphate

-LRB-

effect

of

pH

at

constant

ionic

strength

of

M

g/l

monopotassium

phosphate

at

pH

versus

g/l

dipotassium

phosphate

at

pH

effect

of

ionic

strength

at

constant

pH

of

M

-LRB-

g/l

-RRB-

versus

M

-LRB-

g/l

-RRB-

monopotassium

phosphate

-RRB-

g/l

-LRB-

wt/vol

-RRB-

cottonseed

flour

-LRB-

size

of

of

particles

μm

Pharmamedia

Traders

Protein

Memphis

TN

-RRB-

sterility

medium

-LSB-

g/l

yeast

extract

-LRB-

Biospringer

Milwaukee

WI

-RRB-

g/l

cerelose

-LRB-

glucose

monohydrate

-RRB-

ml/l

-RSB-

and

low-protein

animal

cell

culture

media

-LRB-

LPKM

JRH

Biosciences

Lenexa

KS

-RRB-

Varying

viscosity

by

using

a

glycerol

solution

varied

surface

tension

by

only

a

few

dynes/cm

-LSB-

-RSB-

but

antifoam

addition

varied

surface

tension

without

appreciably

changing

viscosity

These

solutions

were

selected

to

vary

the

physical

and

optical

properties

of

the

liquid

phase

sufficiently

to

detect

changes

in

bubble

size

measurement

ability

Temperature

was

°C

except

for

when

its

effect

on

bubble

size

was

examined

for

set

points

of

and

°C

-LRB-

range

of

°C

-RRB-

Back-pressure

was

zero

except

when

its

effect

on

bubble

size

was

examined

-LRB-

-RRB-

To

ensure

that

contamination

did

not

cause

changes

in

surface

active

properties

-LSB-

-RSB-

test

media

that

supported

growth

were

sterilized

if

the

testing

period

was

expected

to

be

greater

than

h.

Also

since

vessel

cleanliness

-LRB-

i.e.

residual

surfactant

concentrations

-RRB-

affected

bubble

density

and

size

all

vessels

were

first

rinsed

thoroughly

with

hot

water

and

then

a

known

amount

of

antifoam

-LRB-

vol

-RRB-

was

added

to

DIW

These

model

systems

formed

the

basis

for

initial

tests

of

actual

fermentation

broths

of

microbial

-LRB-

filamentous

bacterial

culture

Amycolatopsis

fastidiosa

-RRB-

and

animal

cell

-LRB-

suspension-adapted

CHO

-RRB-

cultivations

Fermenters

equipped

with

open

pipe/jet

spargers

When

performing

measurements

in

agitated

fermenters

equipped

with

open

pipe/jet

spargers

-LRB-

in

opening

at

and

l

scales

in

opening

at

l

scale

-RRB-

it

was

apparent

that

under

certain

conditions

the

bubble

distribution

was

bimodal

Some

images

consisted

of

smaller

spherical

bubbles

along

with

very

large

irregularly

shaped

blob

bubbles

presumably

owing

to

gas

entrainment

from

vortexing

and

possible

impeller

flooding

at

higher

aeration

rates

relative

to

agitation

rates

Quantification

of

the

discarded

blob

area

from

each

of

these

conditions

assisted

in

identifying

the

onset

of

flooding

conditions

and

possibly

was

directly

related

to

the

gas

hold

up

-LRB-

even

under

conditions

in

which

individual

bubble

diameters

can

not

be

discerned

-RRB-

Bubble

size

measurements

were

conducted

at

the

l

scales

spanning

an

range

in

scale

all

in

geometrically

similar

fermenters

For

some

combinations

of

agitation

and

airflow

rates

the

bubble

density

was

too

high

to

clearly

obtain

individual

bubble

diameter

measurements

This

limitation

to

low

agitation

and

airflow

rates

and

void

fractions

under

was

similar

to

that

observed

for

other

optically

based

systems

-LSB-

-RSB-

Qualitative

pictures

of

the

effect

of

agitation

and

airflow

rate

at

the

l

scale

for

a

fermenter

equipped

with

a

Rushton

impeller

are

shown

in

Fig.

As

expected

higher

agitation

rates

and

thus

power

inputs

-LRB-

estimated

at

and

l

-LSB-

-RSB-

-RRB-

created

larger

numbers

of

smaller

bubbles

and

higher

airflow

rates

-LRB-

and

vvm

-RRB-

increased

the

number

and

size

of

bubbles

Increases

in

both

agitation

and

airflow

rates

increased

bubble

density

often

to

the

degree

that

measurement

of

individual

bubbles

was

precluded

Fig.

bubble

size

as

function

of

agitation

and

airflow

rate

for

open

pipe

sparger

-LRB-

in

-RRB-

at

the

l

scale

-LRB-

in

DIW

-RRB-

Qualitative

pictures

of

the

effect

of

agitation

and

airflow

rate

at

the

l

scale

for

a

fermenter

equipped

with

a

Rushton

impeller

are

shown

in

Fig.

Trends

were

similar

to

those

obtained

at

the

smaller

scale

considering

vvms

-LRB-

and

vvm

-RRB-

and

power

inputs

-LRB-

estimated

at

and

l

-LSB-

-RSB-

-RRB-

were

considerably

lower

The

cut-off

Sauter

mean

diameter

is

shown

by

Fig.

following

expected

trends

with

agitation

and

airflow

rate

A

comparison

of

the

arithmetic

Sauter

and

geometric

mean

diameters

both

with

and

without

the

cut-off

is

shown

by

Fig.

with

expected

trends

generally

observed

The

relationship

between

bubble

size

and

agitation

rate

at

the

l

scale

was

quantified

for

the

cut-off

of

the

Sauter

mean

diameter

as

D

α

N

and

compared

to

literature

results

D

α

N

-LSB-

-RSB-

The

lower

dependence

observed

on

agitation

rate

likely

was

due

to

the

fact

that

the

measured

power

inputs

were

lower

than

those

used

for

the

published

correlation

Fig.

of

bubble

data

at

the

l

scale

containing

in

DIW

with

an

open

pipe

sparger

-LRB-

round

in

ring

with

four

jets

of

id

in

each

-RRB-

a

Qualitative

bubble

size

as

a

function

of

agitation

and

airflow

rates

b

Bubble

Sauter

mean

diameter

-LRB-

cutoff

-RRB-

as

a

function

of

agitation

and

airflow

rates

c

Arithmetic

Sauter

and

geometric

mean

comparisons

as

function

of

agitation

rate

at

airflow

rate

of

lpm

At

the

l

scale

vol

glycerol

solution

was

tested

using

a

fermenter

equipped

with

Rushton

impellers

at

constant

agitation

and

airflow

rates

The

effect

of

agitation

and

airflow

rate

increases

on

bubble

size

and

distribution

was

qualitatively

similar

between

DIW

-LRB-

Fig.

-RRB-

and

vol

glycerol

-LRB-

Fig.

-RRB-

The

number

of

smaller

bubbles

qualitatively

was

somewhat

greater

for

vol

glycerol

particularly

at

lower

agitation

rates

which

was

inconsistent

with

the

expectation

that

bubble

size

increase

with

solution

viscosity

However

at

the

same

rpm

agitation

rate

the

power

per

unit

volume

was

likely

higher

for

the

glycerol

solution

since

the

impeller

Reynold

's

number

NRe

was

in

the

laminar

flow

range

for

vol

glycerol

and

in

the

turbulent

range

for

DIW

Fig.

of

bubble

sizes

in

DIW

and

vol

glycerol

containing

as

a

function

of

agitation

and

airflow

rate

at

the

l

scale

At

the

l

scale

various

solutions

-LRB-

differing

in

ionic

strength

pH

temperature

pressure

-RRB-

were

tested

using

a

fermenter

equipped

with

Rushton

impellers

at

constant

agitation

and

airflow

rates

but

notable

qualitative

changes

in

bubble

distribution

were

not

apparent

As

greater

amounts

of

-LRB-

beyond

-RRB-

were

added

to

DIW

observations

became

progressively

more

limited

to

lower

agitation

and

airflow

rates

before

becoming

too

dark

due

to

overlapping

bubbles

Gas

hold

up

was

indirectly

measured

for

aeration

and

agitation

conditions

at

the

and

l

scales

for

conditions

which

had

high

discarded

area

percentages

-LRB-

Fig.

b

-RRB-

Hold

up

was

estimated

using

the

discarded

area

for

each

frame

of

the

measurement

and

then

averaging

the

results

Discarded

areas

were

not

calculated

precisely

as

individual

bubble

areas

however

and

standard

deviations

of

average

discard

areas

were

around

at

lower

values

dropping

to

at

higher

values

Trends

in

the

values

of

discarded

areas

indicated

that

hold

up

as

well

as

bubble

residence

time

increased

with

greater

agitation

rates

and

airflow

rates

as

observed

by

others

-LSB-

-RSB-

In

addition

as

airflow

rate

increased

the

impact

of

higher

agitation

rates

on

discard

areas

decreased

suggesting

impeller

flooding

which

at

vvm

occurred

for

the

l

scale

but

was

not

evident

at

the

l

scale

owing

to

lower

gassed

power

draws

Specifically

higher

discard

areas

were

evident

at

the

l

scale

for

hydrofoil

versus

Rushton

impellers

consistent

with

greater

measured

gassed

power

decreases

and

gas

hold

ups

-LSB-

-RSB-

Fig.

bubble

area

as

a

function

of

a

agitation

rate

at

constant

airflow

rate

and

b

airflow

rate

at

constant

agitation

rate

for

l

fermenter

containing

in

DIW

Fermenters

equipped

with

ring

spargers

Bubble

measurements

in

fermenters

with

open

pipe

spargers

exhibited

several

bubbles

in

each

frame

often

in

swarms

In

contrast

bubble

measurements

in

fermenters

with

ring

spargers

represented

the

opposite

extreme

in

which

only

a

few

bubbles

were

present

in

each

frame

These

latter

tests

were

conducted

using

purified

water

in

and

l

geometrically

similar

bioreactors

spanning

a

tenfold

size

range

Fermenters

were

equipped

with

impellers

and

a

ring

sparger

with

holes

drilled

in

in

diameter

on

the

top

surface

of

the

sparger

ring

Only

a

few

bubbles

were

observed

regardless

of

notch

orientation

-LRB-

left

right

down

top

-RRB-

It

was

believed

that

the

fewer

bubbles

observed

per

frame

were

due

partially

to

lower

gas

hold

ups

typically

vvm

but

also

the

relative

spatial

placement

of

the

probe

at

the

level

of

the

sparger

ring

As

the

airflow

rate

was

increased

to

its

higher

range

values

more

similarly

sized

bubbles

were

observed

In

contrast

higher

agitation

rates

caused

more

surface

air

entrainment

thus

greater

numbers

of

large

irregularly

shaped

bubbles

were

present

in

the

frames

Higher

airflow

rates

resulted

in

bubbles

similar

in

nature

to

those

observed

at

lower

airflow

rates

all

governed

by

the

holes

in

the

ring

sparger

In

some

cases

owing

to

the

slower

agitation

rates

smaller-sized

-LRB-

geometric

mean

of

vs.

μm

-RRB-

bubbles

collected

on

surface

of

the

shroud

's

sapphire

window

and

these

bubbles

needed

to

be

distinguished

from

the

freely

moving

bubbles

Increases

in

silicone

antifoam

from

to

did

not

increase

the

low

number

of

bubbles

observed

Use

of

an

in

situ

bubble

measurement

system

in

fermentation

The

ability

of

the

Envirocam

to

measure

in

opaque

solutions

was

examined

using

several

model

systems

For

the

l

fermenter

equipped

with

an

open

pipe

sparger

the

effect

of

Pharmamedia

on

bubble

images

was

investigated

When

g/l

Pharmamedia

and

ml/l

was

added

to

DIW

-LRB-

μs

shutter

speed

gain

brightness

and

aperture

-RRB-

the

contrast

of

bubble

edges

decreased

to

an

unacceptable

level

The

particles

caused

granularity

on

the

screen

and

blurred

bubble

edges

making

detection

difficult

using

the

in

gap

shroud

When

g/l

Pharamedia

and

ml/l

was

added

to

DIW

using

the

in

gap

shroud

to

decrease

path

length

-LRB-

μs

shutter

speed

brightness

gain

aperture

-RRB-

bubble

contrast

was

improved

but

the

resulting

opaque

solution

appeared

still

too

high

for

reliable

analysis

In

addition

there

was

limited

ability

to

obtain

bubbles

with

edges

sufficiently

sharp

enough

for

accurate

measurements

when

diluted

Amycolatopsis

broth

-LRB-

initial

dcw

of

g/l

-RRB-

was

tested

despite

raising

settings

to

maximum

values

The

use

of

localized

thresholding

was

attempted

to

sufficiently

sharpen

the

bubble

edges

by

reducing

fuzziness

for

analysis

but

it

was

only

partially

successful

-LRB-

Fig.

-RRB-

Note

that

the

bubble

density

is

considerably

lower

for

this

image

taken

at

rpm

and

lpm

compared

with

images

taken

in

at

rpm

and

lpm

-LRB-

Fig.

-RRB-

This

comparison

demonstrated

that

the

presence

of

broth

potentially

improves

the

agitation

and

aeration

range

over

which

the

Envirocam

can

measure

bubbles

should

the

optical

limitations

of

the

broth

on

the

measurement

be

mitigated

Fig.

photographs

in

fermentation

broth

aAmycolatopsis

fastidiosa

broth

with

g/l

dry

cell

weight

diluted

with

DIW

l

scale

and

open

pipe

sparger

rpm

lpm

b

LPKM

medium

with

antifoam

C

l

scale

and

ring

sparger

rpm

lpm

For

the

l

fermenter

with

the

ring

sparger

images

taken

in

cell-free

LPKM

medium

-LRB-

Fig.

-RRB-

were

compared

with

those

taken

in

a

CHO

cell

culture

containing

animal

protein-free

medium

-LRB-

containing

g/l

Pluronic

and

no

antifoam

-RRB-

with

about

cells/ml

at

viability

Despite

the

low

cell

density

of

this

broth

bubble

edges

remained

fuzzy

preventing

accurate

size

analysis

Summary

and

future

considerations

A

novel

in

situ

bubble

size

and

distribution

measurement

device

was

developed

The

bubble

measurement

instrument

design

strategy

permitted

one

camera

module

to

be

attached

to

a

shroud

and

thus

one

sensor

-LRB-

the

camera

-RRB-

was

able

to

be

moved

to

multiple

locations

without

disturbing

the

fermentation

process

The

small

size

and

flexibility

of

the

camera

attachment

permitted

it

to

be

readily

relocatable

An

Ethernet

connection

for

the

camera

can

further

reduce

the

extent

of

the

field

hardware

and

thus

permit

one

camera

system

to

be

even

more

transportable

The

measurement

system

was

tested

at

the

pilot

scale

both

in

clear

and

opaque

model

systems

which

included

fermentation

broth

Experimental

data

for

mean

bubble

size

changes

versus

expected

behavior

qualitatively

compared

favorably

with

published

relationships

for

selected

conditions

-LSB-

-RSB-

Quantitative

comparisons

were

more

difficult

to

establish

owing

to

limitations

in

bubble

size

measurement

capability

at

the

higher

agitation

and

airflow

rate

ranges

at

which

these

published

correlations

often

were

established

At

this

time

application

of

the

EnviroCam

bubble

measurement

system

appears

limited

to

clear

solutions

that

do

not

contain

large

numbers

of

overlapping

bubbles

Different

liquids

-LRB-

e.g.

water

cottonseed

flour

microbial

broth

animal

cell

broth

-RRB-

possess

different

UV

spectrum

and

light

scattering

properties

as

well

as

varying

surfactant

properties

which

influence

bubble

size

and

hold

up

These

differences

suggest

that

some

adaptation

of

the

measurement

system

is

necessary

when

moving

from

system

to

system

Specifically

agitation

and

aeration

rate

combinations

which

produce

too

many

bubbles

for

measurement

in

a

model

DIW

system

may

produce

acceptable

amounts

of

bubbles

in

a

fermentation

broth

owing

to

changes

in

surfactant

levels

but

the

ability

to

distinguish

these

bubble

edges

is

diminished

Further

expansion

of

the

versatility

and

range

of

this

instrument

is

the

subject

of

future

efforts

but

key

approaches

being

considered

involve

the

further

examination

of

available

LabView

filtering

techniques

to

process

bubble

images

and

the

use

of

smaller

notch

sizes

O O O B I B O O O B O B O O O B I B O O O B O B O O O O O B I O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B I O O O O O O O O O O O O O B I O B O O O O O O O B I O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O B B O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O B O O O B O O O O O B O O O 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