ESTRUTURA DE BIOREATORES NÁDIA S. PARACHIN

Aula 2

BIORREATORES OU REATORES BIOQUÍMICOS

São aqueles nos quais se dão várias reações químicas na presença de biocatalisadores que podem ser células vivas ou enzimas.

BIORREATORES

PRESENÇA DE CÉLULAS VIVAS REATORES ENZIMÁTICOS REATORES EM FASE NÃO-AQUOSA

•  Microorganismos: Naturais ou Recombinantes

•  Células Animais •  Células Vegetais

•  Fase Homogênea: Enzimas Livres

•  Fase Heterogênea: Enzimas imobilizadas

•  Cultivos em meios semi-sólidos

Tipos de Bioreatores

+

BIOPRODUTOS

•  ENZIMAS

•  VACINAS

•  VITAMINAS

•  ANTIBIÓTICOS

•  PROTEÍNAS

•  ETC...

MICRORGANISMOS

(Agitação mecânica)

defletor

Válvula e manômetro

alimentação

Technical illustration of an 18 ml working volume miniature stirred bioreactor (MSBR) prototype.

Fonte: Betts and Baganz Microbial Cell Factories 2006 5:21

Vista lateral do biorreator New Brunswick Scientific Bioflo 110.

Vista superior do biorreator New Brunswick Scientific Bioflo 110

19

New Brunswick Scientific User’s Guide

Whenever you assemble or disassemble the vessel components, if you need to lay the drive assembly aside while it is still attached to the headplate and the agitation impeller shaft, note that there is a correct and an incorrect way to position the assembly on a flat surface. The wrong way, which is resting the headplate and impeller shaft on a surface (see Figure 2a) puts the impeller shaft at risk for damage:

Figure 2a: WRONG Handling of Drive Assembly

The correct way, which is resting the drive assembly and headplate on the surface (see Figure 2b below), protects the impeller shaft from bearing weight. Naturally, you will have to take care not to hit the shaft as you work around it

Figure 2b: CORRECT Handling of Drive Assembly

4.7 Vessel Assembly: Non-Jacketed

The vessels are available in four sizes: 1.3 liters, 3.0 liters, 7.5 liters and 14.0 liters (total volume; for more detail, see Specifications).

DRIVE ASSEMBLY

HEADPLATE

IMPELLER SHAFT

DRIVE ASSEMBLY

HEADPLATE

IMPELLER SHAFT

Como lidar com a tampa do fermentador

19

New Brunswick Scientific User’s Guide

Whenever you assemble or disassemble the vessel components, if you need to lay the drive assembly aside while it is still attached to the headplate and the agitation impeller shaft, note that there is a correct and an incorrect way to position the assembly on a flat surface. The wrong way, which is resting the headplate and impeller shaft on a surface (see Figure 2a) puts the impeller shaft at risk for damage:

Figure 2a: WRONG Handling of Drive Assembly

The correct way, which is resting the drive assembly and headplate on the surface (see Figure 2b below), protects the impeller shaft from bearing weight. Naturally, you will have to take care not to hit the shaft as you work around it

Figure 2b: CORRECT Handling of Drive Assembly

4.7 Vessel Assembly: Non-Jacketed

The vessels are available in four sizes: 1.3 liters, 3.0 liters, 7.5 liters and 14.0 liters (total volume; for more detail, see Specifications).

DRIVE ASSEMBLY

HEADPLATE

IMPELLER SHAFT

DRIVE ASSEMBLY

HEADPLATE

IMPELLER SHAFT

Tanque de agitação Estrutura típica de um fermentador utilizado para fermentação microbiana

Fermentadores em escalas de até 10L são feitos em pirex. Acima disso são feitos de alumínio

Table 1 Typical dimension ratios among parameters in Figure 1

T/D H/D Li /T Wi /T Hb/T Wb/D

0.3–0.6 1–2 (until 4) 0.25 0.2 1 0.1

(a) (b) (c) (d)

Stirred Tank Bioreactors 181

Wb

H Li

Wi

Hb T

D

Figure 1 Basic stirred tank design with a radial impeller (Rushton turbine) and four equally spaced baffles.

Figure 2 Some types of impellers employed in stirred tank bioreactor (STBR) and the predominant flow pattern they produce: (a) propeller and pitched blade (axial flow); (b) turbine (radial flow); (c) anchor (tangential flow); and (d) helical (axial flow).

Características geométricas

•  Mistura do meio de cultura é prioridade •  Estrutura cilindrica ou “abaulada” para facilitar mistura •  Dimensões pré-definidas.De acordo com padrões

internacionais.

•  Partes essenciais: motor , “sparger”, e pá rotatória

Geometria típica de um tanque de agitação

Volume de trabalho vs. “Head space”

•  Volume de trabalho: volume total do meio de cultura •  Head space: volume restante •  De um modo geral o volume de trabalho = 70-80% do

volume total do fermentador. Varia de acordo com o fermentador utilizado

47

New Brunswick Scientific User’s Guide

55 SSPPEECCIIFFIICCAATTIIOONNSS BioFlo/CelliGen 115 System

Total Volume 1.3 L 3.0L 7.5L 14.0L Working Volume

0.4-1.0L 0.8-2.2L 2.0-5.6L 4.0-10.5L Vessels

Design Heat-blanketed and Water-jacketed All vessels are borosilicate glass, autoclavable, with dished-bottom

Design Advanced compact controller with integrated utility station capable of supporting 2 additional utility stations and vessels

Display 8.4” industrial color touchscreen display is standard with control station but not included with for 2nd or 3rd utility station

Control Station/ Utility Station

Function Fermentation and cell culture monitoring and control Indication Digital display in 0.1°C increments Range 20°C above coolant temperature to 70°C max temperature

(65°C max temperature for 14.0L) Control PID for heating and cooling

Heat-blanketed Vessels: External heating blanket and immersed stainless steel coiling coil Water-jacketed Vessels: Water jacket heater and circulation loop

Temperature

Sensor Platinum RTD probe (Pt 100) Drive Magnetic Drive or Direct Drive. Indication Digital display in 1 RPM increments. Range Direct Drive: 50-1200 RPM for fermentation, 25-400 for cell culture

Magnetic Drive : 25-200 RPM Control PID control; manual, automatic, or cascade settings Impellers Rushton-style standard with fermentation system

Pitched blade standard with cell culture Optional: Spin filter

Agitation

Baffles Removable 316L stainless steel; fermentation only Gas Flow options

0-4 Rotameters x 0-150 mLpm x 250-2500 mLpm x 1-5 Lpm x 1-20 Lpm

1 Thermal Mass flow Controller (TMFC) x 0-20 SLPM

Gas Mixing options

Automatic 4-Gas Mixing (via 4 solenoids) Manual Gas mixing (via 4 gas manifold)

Sparger Standard: Ring sparger Optional: Microsparger

Inlet Filter 0.2ȝm interchangeable cartridge

Aeration

N2 Gas For calibration of DO probe Indication Digital display in 0.01 pH increments Range 2-14 pH Control PID, link to pumps or gases, adjustable deadband

pH

Sensor pH probe

...continued...

Volume de trabalho

Volume vazio

Componentes de um bioreator

•  Sistema de agitação •  Sistema de ar •  Controle de espuma •  Controle de temperatura •  Controle de pH •  Porte de retirada de

amostra/entrada de nutrientes

Sistema de Agitação •  O Sistema de agitação consiste no agitador e aletas. •  Aletas- quebra o fluxo de líquido para aumentar a eficiência

de mistura •  Principais funções do sistema de agitação:

1.  Proporcionar boas taxas de transferência de mistura e, assim, aumentar a massa através do líquido em volume e camadas limite de bolhas.

2.  Providenciar que as bolhas de ar sejam quebradas

Sistema de agitação •  Componentes do agitador

Motor

Selador mecânico Redutor de velocidade

Eixo de rotação

Impulsores

Pás rotatórias

Tipos de pás rotatórias

Table 1 Typical dimension ratios among parameters in Figure 1

T/D H/D Li /T Wi /T Hb/T Wb/D

0.3–0.6 1–2 (until 4) 0.25 0.2 1 0.1

(a) (b) (c) (d)

Stirred Tank Bioreactors 181

Wb

H Li

Wi

Hb T

D

Figure 1 Basic stirred tank design with a radial impeller (Rushton turbine) and four equally spaced baffles.

Figure 2 Some types of impellers employed in stirred tank bioreactor (STBR) and the predominant flow pattern they produce: (a) propeller and pitched blade (axial flow); (b) turbine (radial flow); (c) anchor (tangential flow); and (d) helical (axial flow).

Sistema de entrada de ar Consite nas seguintes partes: •  Compressor/ Balas de gás •  Sistema interno de controle •  Sistema de filtro para esterelizar •  “Sparger” •  Saída de ar pelo condenssador

Fonte: http://www.dasgip.com/catalog/SR0200SS/

O Sistema de saída de ar de fermentadores pequenos (< 10L) contém um condensador

Condensador

Condensador

•  Troca de calor por onde há passagem de água •  Compostos voláteis e vapor de água se

condensam na superfície interna do condensador.

•  Reduz a perda de compostos voláteis na fermentação ex: etanol.

“Sparger”- Espalhador de ar •  Quebra o ar que está entrando no fermentador em

pequenas bolhas

•  Modelo mais frequente consiste em um anel cilindrico com pequenos círculos.

Sparger-localizado abaixo das pás rotatórias

Quando o fermentador é esvaziado o ar deve estar desligado para evitar contamização com a linha de gás.

Impacto na oxigenação do fermentador e velocidade da pá giratória

As bolhas de ar não são quebradas e tendem a subir

rapidamente. Baixa diluição no líquido

Quebra das bolhas de ar que de difundem mais facilmente

Baixa velocidade Alta velocidade

Importante- compromisso ar, velocidade, tipo de cultura

Cascata Ar/Agitação •  Permite aumentar a quantidade de ar no

fermentador de acordo com a necessidade do microrganismo

Modo padrão é nenhuma cascata. Os modos de cascata disponíveis aparecem em preto

Selecione a cascata desejada. Nesse ex. agitação e ar

Digite os valores desejados/apropriados

Unidades comuns de fluxo de ar •  Volume por volume por minuto ou VVM

definida por : taxa de fluxo de ar volumétrico volume líquido

VL

FA

É importante que o fluxo de ar seja expresso em volume/ minuto para que seja compatível com o volume do reator

Controle de espuma •  O controle de formação de espuma é essencial para fermentações

aeradas. Dependendo do microrganismo utilizado a espuma formada irá interferir com o procesos de fermentação

Controle de espuma no fermentador

Problemas associados a formação de espuma: 1.  Entupimento de filtros 2.  Aumento da pressão

interna no bioreator 3.  Perda de meio de

cultura

Soluções: 1.  Detecção da espuma 2.  Adição de

antiespumantes

A Necessidade de antiespumante vai depender de:

•  composição do meio. Meio rico em proteínas gera mais espuma

•  produtos da fermentação. Produtos com características de detergentes geram mais espuma • Taxa de aeração e velocidade de agitação. • Uso de controladores mecanicos Equipamentos de ultrasom reduzem a formação de espuma

•  Volume vazio ou “head space” Fermentações que tendem a formar muita espuma requerem um maior volume de “head space” •  Temperatura do condensador Condensadores resfriados reduzem a formação de espuma

Sonda para detecção de espuma no fermentador

Quando a sonda superior não estiver sendo molhada pela espuma a bomba de antiespumante fica desligada.

Quando a sonda superior estiver imersa na espuma a bomba será acionada permitindo a entrada de antiespumante no fermentador.

Nível das sondas

Antiespumante

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Controle de Temperatura O Sistema de controle de temperatura consiste em:

• Sondas de temperatura • Sistema de transferência de calor O Sistema de transferência de calor consiste em um sitema de jaqueta que envolve o fermentador.

Sistema de aquecimento/refrigeração

Jaqueta de aquecimento

182 Bioreactors – Design

(a)

(b) (c)

Figure 3 Heat transfer ways in a stirred tank bioreactor (STBR): (a) jacket; (b) internal coils; and (c) external heat exchanger.

Heat transfer is necessary for temperature control during bioreactor operation because the heat of reaction and the metabolic activity of cells absorb or generate heat. The heat transfer in these devices is simple because most of the microorganisms grow and the enzymes work at mild temperature and inside the bioreactor there are small variations due to the mixing. Nevertheless, some microorganisms or enzymes work at extreme temperature conditions (e.g., thermophilic or psyhrophilic microorganisms or their enzymes). Usually, isothermal operations are carried out in these bioreactors; uniform and constant temperature is easily achieved by exchanging heat with different apparatus. Temperature control can be maintained by enclosure in a controlled incubator, a jacketed vessel, an external coil, an internal coil (helical and baffle type), and an external heat exchanger (of double pipe, shell and tube, and plate and spiral heat exchanger). Figure 3 shows a summary of the heat exchange possibilities in this type of apparatus at pilot and industrial scales, while incubators, with air or water circulation, are used in small scale (with shake flask, mainly). Jacket and external coil give low heat transfer area and are inadequate for cooling from sterilization temperature to operating temperature in a reasonable time; they are thus rarely used at an industrial scale. Internal coils can be operated under different liquid-exchange velocity and are frequently used in large tanks because they provide a relatively large heat transfer area. But the coils interfere with the mixing in the vessel and make cleaning of the inside of the reactor difficult. Another problem can be film growth of cells on the heat transfer surface. External heat exchanger units are independent of the reactor, easy to scale-up, and provide best heat transfer capability. However, conditions of sterility must often be kept; the cells must be able to withstand the shear forces imposed during pumping; and, in aerobic bioprocesses, residence time in the heat exchanger must be small enough to ensure the medium does not become depleted of oxygen.

The STBRs are mainly used for small to medium productions; most processes with microorganisms are carried out in batch or fed-batch operations, due to the usually low or medium production capacity needed, but also due to the difficulty of continuous production in case of industrial microbial processes.

Activities to be taken into account in the bioreactor design include G-L-S contacting, online sensing of concentrations, mixing, heat transfer, foam control, and feed of nutrients or reagents, as well as those for temperature, oxygen concentration, and pH control. The STBR technical design for a bioprocess needs to determine the required volume, which depends on the production to achieve and the process rate to be carried out. The rate of the process, usually a multiphase process and a network of reactions, is difficult to describe. For this purpose, it is necessary to know the rates of the different phenomena involved (mass transport, heat transfer, complex network of reactions, phase segregation, etc.) and combine the typical variables and parameters of each one of these phenomena into a kinetic model that adequately describes the complex evolution of the system. With this purpose, the balances of mass, energy, and momentum have to be formulated, but usually it is enough with only the first two balances, and often only the mass balance is necessary, because the temperature remains constant along the process evolution time.

Mass balance is different for each phase involved in the system (usually a liquid phase, according to a pseudo-homo-geneous approach, including the enzyme or the microorganism, and a gas phase, usually air, in the aerobic process, or CO2

and CH4 in anaerobic processes). This balance will depend on how the phase is treated: batch, semi-batch, or continuous. When the temperature or sequence of temperatures to follow is known, because it is predetermined, the heat balance to design heat transfer equipment able to maintain that temperature or set temperature programming is necessary. In the unusual case that the temperature is not programmed (e.g., in adiabatic operations), both mass and heat balances must be simultaneously solved.

Sistema de aquecimento

Jaqueta de água

Alternativas para la transferencia de calor

en los bioreactores tipo tanque agitado

a)  Vaso enchaquetado

b)   Enrrollado externo

c)  Enrrollado interno

d)   Deflector enrrollado

e)  Intercambiador de calor externo

Controle de pH

A sonda de pH é esterelizada com o fermentador

Sistema de controle de pH consiste em: •  Sonda •  Base •  Ácido

Controle de pH

A sonda de pH é esterelizada com o fermentador

Bases comumente utilizadas: •  NaOH •  KOH Ácidos comumente utilizados: •  H2SO4 < 10%

Controle de pH: Setpoint and deadband

Setpoint: pH desejado Deadband: intervalo no qual não será adicionado ácido ou base no fermentador

deadband

•  Objetivo: evitar excesso de ácido ou base no fermentador

•  Ajustado da seguinte forma:

Limite superior

Setpoint

Limite inferior

A bomba de ácido é ligada quando o pH ultrapassa esse limite

A bomba de ácido é ligada quando o pH ultrapassa esse limite

Ligar o controle de pH

Acesso a tela de controle

Deadband