BIODEGRADATION OF LINEAR BIODEGRADATION OF LINEAR ALKYLBENZENE SULFONATE ALKYLBENZENE SULFONATE BY BACTERIAL CONSORTIUMBY BACTERIAL CONSORTIUM

PraswastiPraswasti PDK PDK WulanWulan, , MisriMisri GozanGozan, , AnondhoAnondho WijanarkoWijanarko, , DianursantiDianursanti, Marisa , Marisa TejoTejo, , MahmudMahmud SudibandriyoSudibandriyo

INTRODUCTIONINTRODUCTIONLAS was the first surfactant introduced to solve an LAS was the first surfactant introduced to solve an enviromental problemenviromental problem. . LAS is one of synthetic surfactants LAS is one of synthetic surfactants that is widely used.that is widely used. Synthetic type surfactants are often Synthetic type surfactants are often used because they perform better and more economical used because they perform better and more economical compared to natural detergentscompared to natural detergents

People assume that People assume that detergents detergents performing bulk of foam represent the performing bulk of foam represent the quality of detergents.quality of detergents.

Although LAS is Although LAS is biodegradable, its biodegradable, its introduction to the introduction to the

environment environment in big amount in big amount harms the water bodiesharms the water bodies ..

Using Using Bioremediation to degradate polutants in Bioremediation to degradate polutants in environment. The advantages are: no side environment. The advantages are: no side effect, more economic compare with other effect, more economic compare with other methodsmethods

Pusat Penelitian dan Pengembangan Teknologi Minyak dan Gas Pusat Penelitian dan Pengembangan Teknologi Minyak dan Gas Bumi (LEMIGAS) has foBumi (LEMIGAS) has fouund mixtures of microorganism (bacterial nd mixtures of microorganism (bacterial consortium) in sewage plant that degrade oil pollutantsconsortium) in sewage plant that degrade oil pollutants

In nature pseudomonas live contiguous with bacillus

Chemical structure of LAS : have linear chainChemical structure of LAS : have linear chain

Critical Micelle Concentration (CMC) :Critical Micelle Concentration (CMC) : Limiting concentration of surfactant for beginning a micelle formation which consist of several hundred collected surfactant monomers (Hart et al, 2003).

micelle from LAS in water (Hart, et. al, 2003) CMC visualized a minimum surface tension (Rosen, 1989)thethe surfactant solution consists monomer in surfactant solution consists monomer in

equilibrium condition and micelle form that equilibrium condition and micelle form that contribute to stop the decreasing of solution contribute to stop the decreasing of solution surface tension surface tension (Rosen, 1989)(Rosen, 1989)

AAerob degradaerob degradationtion MetabolismMetabolism of of Linear AlkylBenzene Sulfonate (LAS)Linear AlkylBenzene Sulfonate (LAS)(Schkeheck, 2003)(Schkeheck, 2003)

A. Aerob Biodegradation: LAS will be change to SPC (sulphophenyl Carboxilates) = intermediate product.

B. Aromatic chain will be total conversion of water, carbondioxideand sulphate

Pseudomonas Pseudomonas aeroginosaaeroginosa

Bacillus cereusBacillus cereus(Scimat , 2005) Bacillus Bacillus subtilissubtilis

METHODOLOGYMETHODOLOGYPure CultivationPure CultivationBacterial consortium which Bacterial consortium which was collected from LEMIGAS was collected from LEMIGAS had been cultivated purely had been cultivated purely with nutrient broth mediumwith nutrient broth medium

Enrichment of bacterial Enrichment of bacterial consortium in consortium in Lockhead Lockhead and Chaseand Chase (LC) medium(LC) mediumTTo adaptate the bacterial o adaptate the bacterial consortium toward LAS. consortium toward LAS.

Degradating Degradating implementation of bacteria implementation of bacteria consortium towards LASconsortium towards LASThis step took time in 264 hours This step took time in 264 hours (12 days) (12 days) in 250 ml in 250 ml erlenmeyererlenmeyer, , 22 rpm 22 rpm with with several several variation variation concentration of LAS: 100, 400, concentration of LAS: 100, 400, 700, 1000, 1500 ppm. These 700, 1000, 1500 ppm. These concentrations were chosen in concentrations were chosen in conconssideration of using amount of ideration of using amount of LAS in detergent. LAS in detergent.

Taking data and analysisTaking data and analysis••Total Plate CountTotal Plate Count (TPC) which was (TPC) which was used for counting bacteria inoculumused for counting bacteria inoculum..•• MMeasuring LAS concentration with easuring LAS concentration with Standard Test Method for Methylene Standard Test Method for Methylene Blue Active Substances (ASTM D Blue Active Substances (ASTM D 2330)2330). . ••MMeasuring test for easuring test for Chemical Oxygen Chemical Oxygen DemandDemand (COD).(COD).: to represent : to represent degradation LAS in system because degradation LAS in system because LAS was the only organic chemistry LAS was the only organic chemistry in this system and to detect suitability in this system and to detect suitability of water.of water.

Extraction at Extraction at Methylene Blue Active SubstancesMethylene Blue Active Substancesstandard standard MethodeMethode :: to measure LAS concentrationto measure LAS concentration

LAS SOLUTION (LAS Solution +methylene blue)

Extraction result with chloroform: 2 phase in LAS solution + methylene blue

Methylene blue + LAS (anionic surfactant) → blue color

Callibration curve : absorbansi vs LAS concentration (ppm) has to be made before Turbidity was observed

with Spektrophotometerwith 650 nm wavelength

EXPERIMENT RESULTEXPERIMENT RESULTCultivation : to get growth of bacteria dataCultivation : to get growth of bacteria data

Biodegradation step can be running until 264 hour (12 days)Biodegradation step can be running until 264 hour (12 days)

1.E+001.E+011.E+021.E+031.E+041.E+051.E+061.E+071.E+081.E+09

0 24 120 144 168 192 264 288 312

hours

CFU

/mL

+ growth rate- growth rate = - 0,032/h

Optimum Growth Optimum Growth of Bacteria of Bacteria Consortium in Consortium in NutientNutient Both Both Medium = 0.039 Medium = 0.039 hourhour--11

It means growth rate of bacteria vanishes

Growth of Bacteria Consortium in Enrichment Step : Growth of Bacteria Consortium in Enrichment Step : tto adaptate the bacterial consortium toward LAS o adaptate the bacterial consortium toward LAS and using LAS and using LAS as a carbon sources for its metabolismas a carbon sources for its metabolism

The first enrichment

added 100 ppmperiodically:

• to ensure that the consortium could acclimatize with higher concentration of LAS.

• to definite there was no lack of carbon source.

•This addition increase amount of consortium.

It means that the consortium could acclimatize with LAS

Optimum Growth of Bacteria Optimum Growth of Bacteria Consortium = 0.041 hourConsortium = 0.041 hour--1 at 360 h1 at 360 h

Start to death phase

The first adaptation bacterial consortium toward LAS

•Leads to decrease amount of bacteria because their adapting phase in new environment which contains of LAS.

•Sudden contact of bacteria consortium with relatively high LAS could disturb their living

to maintain carbon source for bacteria growth.

Amount of Bacteria Consortium in Various Amount of Bacteria Consortium in Various LASLASConcentrationConcentration

1.00E+005.00E+071.00E+081.50E+082.00E+082.50E+083.00E+08

0 50 100 150 200 250 300

t (hours)

Am

ount

of B

acte

ria

Con

sort

ium

0 ppm 100 ppm 400 ppm 700 ppm1000 ppm 1500 ppm

Bacteria do not need long time period compare to the other concentrations for adapt well with new environment. It can reach peak condition and stationer condition. After reaching maximum amount of population, there was lack of nutrition, and then the consortium started to reach death phase.

Standard of comparison system

1. Bacteria consortium can live in LAS environment. The use of LC medium which contains adequate nutrition made the consortium could live and grow.

2. Bacteria decrease at the first time biodegradation process because they have to adapt well with new environment (enter to growth phase). Weak bacteria will be dead.

3. Bacteria that can adapt well will be growth until reach its peak. In this condition LAS biodegradation will increase until maximum condition.

4. Growing phase pattern of bacteria consortium relatively more unclear. After they had reached maximum population, they shortly entered death phase. The stationary phase relatively unclear at higher LAS concentration.

Optimum Growth Rate of Bacteria in Optimum Growth Rate of Bacteria in VariatedVariated LAS ConcentrationLAS Concentration

LAS Concentration (ppm) Optimum Growth Rate (per hours)0 0.041

100 0.042400 0.039700 0.041

1000 0.0421500 0.041

Several factors that may affect grow of bacteria in this research are :

• Agreement of bacterial consortium with LC medium.

• Growing time period which did not same for each bacteria in this consortium.

• LAS could be toxic to some bacteria.

• Competition with each bacteria.

Relation of LAS Concentration with Relation of LAS Concentration with Surface Tension :Surface Tension :

The first adaptation of bacteria with new environment:

•They had to do adaptation hardly.

•Their ability to do metabolism by explored LAS as carbon source and agreement with their living medium which might toxic to them

Addition of LAS with larger concentration influenced bacteria’s living environment.

Bacteria adapt well with environment that they can reproduce andutilize LAS as a carbon source

CMC LAS = 513,98 ppm. Below CMC : higher LAS concentration, lower surface tension will be happened. Surface tension has already reached minimum number and start to be steady at LAS concentration above CMC. 0 ppm >100 ppm >400 ppm >700 0 ppm >100 ppm >400 ppm >700 ppm, LAS 700 ppm = 1000 ppm = 1500 ppm.ppm, LAS 700 ppm = 1000 ppm = 1500 ppm.

COD in LAS Biodegradation ProcessCOD in LAS Biodegradation Process

0

1000

2000

3000

4000

5000

0 50 100 150 200 250 300t (hours)

CO

D (p

pm)

100 400 700 1000 1500

COD symbolizes oxygen which is needed for degradating organic chemical in water. This figure show decreasing pattern of COD in all system with variatedLAS concentration. This indicated that decreasing concentration of LAS or LAS biodegradation process by bacteria consortium was trully happened.

Result of Degradation COD Research in Various LAS ConcentrationResult of Degradation COD Research in Various LAS Concentration ::

LAS ConcentrationLAS Concentration

Total Total Degradation Degradation

COD (%)COD (%)100 100 ppmppm 45.4245.42400 400 ppmppm 27.1727.17700 700 ppmppm 35.1435.141000 1000 ppmppm 14.4614.461500 1500 ppmppm 17.5017.50

COD that measured : amount oxygen is needed by contaminant in system. Can not see contaminant in LAS form or intermediate product biodegradation by bacteria . COD = amount of COD = amount of oxigenoxigen needed that degradation of organic substances in water.needed that degradation of organic substances in water.

• Lower LAS concentration higher COD decreasing

• COD value was not greatly reduced. It means that biodegradation had happened but there still a lot of carbon source available whether in form of LAS or intermediate form. All this organic carbons was counted as COD value.

• Using this Bacteria consortium have advantages : when there were lot of bacteria died because of toxic material, there is still another bacteria could hold out and do the degradation.

CONCLUSIONCONCLUSION

The consorThe consorttium of bacteria (ium of bacteria (Pseudomonas aeroginosaPseudomonas aeroginosa, , Bacillus subtilisBacillus subtilis, , Bacillus aglomeransBacillus aglomerans, , Bacillus cereusBacillus cereus, , Bacillus alvaeBacillus alvae) could survive until 1500 ppm of LAS. ) could survive until 1500 ppm of LAS. While at higher LAS concentration the stationery phase While at higher LAS concentration the stationery phase was relatively unclear.was relatively unclear.LAS concentration above LAS concentration above CMCCMC, surface tension has , surface tension has already reached minimum number and start to be already reached minimum number and start to be steady. steady. Below CMC, higher LAS concentration lower Below CMC, higher LAS concentration lower surface tensionsurface tension will be happened will be happened ::0 ppm >100 ppm >400 ppm >700 ppm, 0 ppm >100 ppm >400 ppm >700 ppm, LAS 700 ppm = 1000 ppm = 1500 ppm.LAS 700 ppm = 1000 ppm = 1500 ppm.Bacteria consortium that was used in this research Bacteria consortium that was used in this research could degradate LAS but less efficient to reducing COD could degradate LAS but less efficient to reducing COD value in those systems. value in those systems.