259

Biopolymers in wastewater treatment

Jennifer I Houghton* and Joanne Quarmbyt
Studies investigating the formation and manipulation of utilized in treatment processes, such as membrane biore-
biopolymers produced by the heterogeneous microbial actors, rotating biological contact units, and trickling
cultures found in wastewater treatment are not advancing at a filters. Biofilms consist of a gel-like matrix of microbial-
fast rate, reflecting the inherent complexity of the systems produced biopolymer attached to a solid substrate, within
involved. Adjustment of the nutrients present within the which microbial cells are immobilized. The structure of
treatment process currently appears to offer more potential the biopolymer network determines many of the physical
than bioaugmentation with genetically modified organisms for properties of the biofilm [Z].
improving certain aspects, such as sludge settling properties
and chlorinated organic pollutant removal. An area likely to be This review focuses on the research that has been pub-
of increasing importance in the future is the role of the lished within the past eighteen months in relation to
microbial biopolymer in retaining water within the sludge biopolymers produced by microorganisms in the waste-
matrix. Modification of the biopolymer to aid the removal of water treatment industry. The main topics covered are new
water from waste sludges could have immense implications for methods of investigating the presence and composition of
the treatment and minimization of the waste sludge produced. the biopolymer present, and the effect of biopolymer prop-
erties in relation to waste treatment processes. The volume
Addresses of work being carried out in this area at the present time is
School of Water Sciences, Cranfield University, Cranfield, Bedford, low, reflecting the slow introduction of new investigative
MK43 OAL, UK techniques into heterogeneous waste treatment systems.
*e-mail: j.houghton@cranfield.ac.uk
te-mail: j.quarmby@cranfield.ac.uk
Quantification of biopolymer
Current Opinion in Biotechnology 1999, 10:259-262 Investigations into the quantity and quality of the biopoly-
http://biomednet.com/elecref/0958166901000259
mer present have generally commenced with the
extraction of the biopolymer from the waste sludge. The
0 Elsevier Science Ltd ISSN 0958-l 669 aim of any extraction method is to remove as much of the
biopolymer as possible without damaging the integrity of
Introduction the bacterial cell. There have been numerous physical and
Microorganisms have a vital role to play in both the aerobic chemical methods, for example high-speed centrifugation,
and anaerobic treatment of wastewater. They degrade the boiling in acid or alkali and utilization of cation exchange
organic material present in the wastewater and convert the resins, applied to the extraction of biopolymer from differ-
resultant products into microbial biomass, humic material, ent types of sludges. Although these methods have been
and waste gases, such as carbon dioxide, nitrogen com- critically reviewed (for example see [A]), as yet no one
pounds and methane. The sllldge produced by these method has been adopted as a standard procedure. The
treatment processes contains a high proportion of bacterial use of non-standardized procedures makes comparison of
cells and cellular debris. results difficult. A different method for biopolymer extrac-
tion utilizing glutaraldehyde has recently been reported to
Riopolymers, (also referred to in the literature as extracel- give favourable results. Addition of glutaraldehyde to acti-
lular polysaccharides, exoccllular polysaccharides, vated sludge appears to solubilize the constituents of the
exopolymeric substances, exopolymers, or microbial solu- polymeric matrix, allowing the biopolymer to be separated
ble products) are produced by many different types of from the bacterial cells by centrifugation. When compared
bacteria. They are found either in a capsule-like structure with three alternative methods, the glutaraldehyde
associated with the outer cell wall, or in solution as loose method extracted greater quantities of organic matter and
slime polymers that are completely detached from the cell caused less cell disruption [4’].
wall. Bacterial capsules may contain up to 99% water and
are thought to aid in the survival of the bacterium in the Wastewater biofilms are complex systems because of their
natural environment by preventing desiccation and con- spatial organization and intrinsic heterogeneity created by
trolling the diffusion of nutrients to the cell wall [ 11. the establishment of diffusion gradients for gases and
nutrients. This determines the ecology of the microbial
Biopolymers are of major importance in wastewater treat- population within the biofilm. Any extractive technique
ment. Biopolymers act in conjunction with divalent metal leads to the destruction of the spatial organization of the
ions, such as calcium, to aid the formation and settlement biofilm itself, with a consequent loss of information [S].
of sludge floes in both aerobic and anaerobic treatment sys- The development of confocal scanning laser microscopy
tems. This enables the biomass to be separated from the and its use with fluorescent probes and markers has per-
liquid phase leaving a clean. treated effluent. Biopolymers mitted the detailed quantitative examination of the
are also one of the main components of biofilms? which are architecture of biofilms. The effect of different treatment
 260 Environmental biotechnology

regimes on mixed-population biofilms can be evaluated by Increasing cell-surface hydrophobicities of bacterial cul-
using techniques such as multiple parameter imaging with tures isolated from wastewater correlate well with the
nucleic acid stains for bacterial cell detection, fluorescent increased adhesion of cells to sludge floes [ll]. Examina-
lectin probes for biopolymer staining, and autofluores- tion of the isolated exopolymeric substances from
cence for imaging algal biomass [6’]. activated sludge indicated that a significant proportion of
this fraction is hydrophobic, and consists of proteins but
Biopolymer composition not carbohydrates [ 12’1.
Microbial biopolymrrs are primarily composed of polysac-
charides and protein, with ntrcleic acids and lipids also ‘The architecture of activated sludge floes is also depen-
reported to be present. The biopolymer matrix in waste- dent on the interaction between the microbially produced
water treatment systems is extremely heterogeneous, and hiopolymer and the cations present in the sludge. Small
evidence is ncJ\v accumulating to support the concept that changes in ionic strength and ionic composition can alter
the composition and propertie: of the biopolymers may be the structural properties of activated sludge floes [13].
more important than the actual quantity of polymer present. Addition of the divalent cations calcium and magnesium to
Bench-scale studies have shown that the chemical oxygen the feed of laboratory grown activated sludge systems has
demand ((:OLI):N:P ratio of the wastewater can influence been shown to increase the hound protein content of the
hydrophobic+, surfiace charge and the biopolymer compo- microorganisms present, which in turn improved the set-
sition of microbial floes in the activated sludge floe matrix tling properties of the sludge. ‘I’he presence of a single
[7”]. (;rowth temperature. type of substrate, and N and P lectin-like protein in the hiopolymer fraction allowed a
concentrations have also been shown to affect the amount of new model of bioflocculation to he proposed. ‘I‘his model
biopotymer produced by a :Il~t~~rnolNlt.trrii~/?~ sp. involved in states that the hiopolymer network is stabilized by lectin-
the de\,elopment of compact anaerobic granular sludge [Xl. like proteins binding polysaccharides that are cross-linked
to adjacent proteins, and that divalent cations provide fur-
Spatial organization occurs within biofilms due to varia- ther structural stability by bridging negatively charged
tions in properties, such as dissolved oxygen and nutrient sites on the biopolymers [ 141.
concentration with depth relative to the biofilm surface.
Measurement of the distribution of proteins and polysac- Sorption properties of biopolymers
charides vertically through a biofilm degrading a toxic One of the major aims of wastewater treatment is the
organic azo dye gave a balanced ratio of protein conccntra- removal of organic and inorganic pollutants. ‘I’he distribu-
tion to polysuccharide concentr-ation, even though the total tion and fate of these pollutants is highly influenced by the
amount of biopolymcr \,aried with depth, implying that a sorption properties of the microbial biomass. ‘I‘he bacteria
balanced environment existelI within the biofilm [Y]. present in wastewater may either take up the pollutant
Kstablishment of a balanced en\,ironment equates to into the cell, or retain the pollutant externally. sorbed to
steady-state operating conditions, enabling more accurate either the cell wall or within the surrounding biopolymer.
monitoring of the treatment p~occss.
Roth sorption processes are controlled by the rate of diffu-
hlathcmatical modeling has found increasing applications sion of the pollutant into the sludge floe or hiofilm, the
within the biological sciences in recent years, and biopoly- diffusion coefficient determining the overall reaction rate.
mer studies are no exception. Nielsen P/ al. [ 101 suggested A new technique has been developed that employs video
a conceptual model for the cOl’Ilp~JSitiOn of exopotymers in imaging to determine the effective diffusion coefficient of
hiofilms that takes into account polymer production and 1,l.Strichloroethane through naturally formed biofilms.
transformation lvithin the hiofllm by considering the indi- 1,l ,Z-trichloroethane is a non-reactive tracer molecule that
vidual fractions of the hiopolvmer. Limitations to this is easily measured by gas chromatography. offering the
approach at present include the lack of good, reliable data potential to improve our limited understanding of pollu-
to enter into such models. As ne\v methods for quantifying tant diffusion rates within hiofilms [lS*].
and characterizing biopoiymer constituents emerge this
limitation should be overcome ‘I’he individual Fates of pollutants in wastewater treatment
systems are likely to vary depending on the pollutant type.
Biopolymer content and the effect on sludge ITractionation of the biomass from a sequencing batch
properties biofilm reactor illustrated that organic and inorganic sub-
One of the main arcas of interest in biopolymcr properties stances reacted in different manners, with 230% of the
is the role that the biopolymer plays in the flocculation of inorganic metals cadmium and Ant being found in the cel-
particulate matter and hacteri 11 cells during waste treat- lular fraction, and >hO% of the organic compounds used
ment processes. Hydrophobic interactions of the secreted (benzene, toluene, and m-xylenc) localized in the biopoly-
polymer (i.e. the bchaviour of particles and molecules that mer fraction [ 16’1. The apparent differences in sorption sites
are tlnable to interact with water, either via hydrogen for organic and inorganic substances lvere not as expected,
bonds or clectrostaticatly, to dl-aw together in an aqueo~~~ the biopolymer having less influence on the removal of
environment) h3L.e been receiving attention of late. metal ions than previously thought. ‘This may be due to the
 Biopolymers in wastewater treatment Houghton and Quarmby 261

choice of organic compounds and inorganic ions used. More Wastewater treatment itself has been carried out, to vary-
detailed information about the accumulation and location of ing degrees, for over one hundred years in industrialized
chlorinated organics in biofilm exopolymers was obtained countries. As we enter the twenty-first century we are
using image analysis and dual channel imaging in conjunc- having to contend with increasing legislative demands
tion with confocal scanning laser microscopy and for cleaner water. This results in increased levels of
lectin-specific fluorescent probes [ 17**]. The distribution of treatment being carried out on all wastewater treated,
lectin binding sites was found ro vary depending on the which in turn leads to an increase in the amount of waste
characteristics of the substrate supplied to the biofilm. sludge being produced. The cost of disposing of this
Where biofilms were grown using the herbicide diclofop ever growing amount of waste is likely to direct research
methyl as a sole carbon source, the lectin binding sites were into ways of producing an end product that is easier to
nonuniformly distributed throughout the biofilm. Areas of dewater. reducing the bulk volume and making the
biopolymer containing high numbers of lectin binding sites sludge easier to handle. The role of the microbial
accumulated diclofop and its metabolites. Increased under- binpolymer in retaining water makes it an ideal target for
standing and control of the location and distribution of such future research in this area.
binding sites may offer the potential to alter the level of pol-
lutant removal in full-scale systems, utilizing the indigenous Acknowledgements
microbial population without genetically manipulating the
microorganisms involved.

Potential for biopolymer manipulation in References and recommended reading

Papers of particular interest, published within the annual period of review,
wastewater treatment have been highlighted as:
Biopolymers produced by pathogenic bacteria and bacteria
l of special interest
that arc of industrial importance have been investigated in **of outstanding interest
pure cultures. The structural genes involved in biopolymer
1. Costerton JW, Irvin RT: The bacterial glycocalyx in nature and
production in bacterial species \uch as At;etohnt;t~r- X$~SHII~N~. disease. Annu Rev Microbial 1981, 35:299-324.
Xarrrho~~~~~s c.~~~~prsf~lsand Kh&/l/i~t~ “pp., along with the 2. Christensen BE: The role of extracellular polysaccharides in
inherent difficulties in obtaining this information, have biofilms. I Biofechnoll989, IO:1 81-202.
recently been reviewed [18]. ‘I’llis basic knowledge is nec- 3. Gehr R, Henry JG: Removal of extracellular material. Techniques
essary before attempts can be made tC) improve the and pitfalls. Water Res 1983, 17:1743-l 748.

biopolymer production of selected bacterial strains by 4. Azeredo J, Oliveira R, Lazarova V: A new method for extraction of
. exopolymers from activated sludges. Water Sci Jechnol 1998,
genetic manipulation, thereby increasing the absorption 37:367-370.
properties or flocculation potential of the overall treatment The first report of the use of glutaraldehyde to extract biopolymers from actt-
system. ‘I’hc potenCals and limitations of utilizing such vated sludge. This method appears to increase the amount of blopolymer
removed while minimizing cell lys~s.
strains for the hioaugmentatioll of activated sludge sys-
5. Lazarova V, Manem J: Biofilm characterization and activity analysis
tems, with examples of currcnr research, is discussed by- in water and wastewater treatment. Water Res 1995, 29:2227-
Van I,imbergen er nl. [ 19’1. ‘I‘hc small number of success- 2245.
ful bioaugmentation studies carried out so far have focused 6. Lawrence JR, Neu TR, Swerhone GDW: Application of multiple
on the introduction of bacteria, namely f?~fu~orno?r~.s sp., . parameter imaging for the quantification of algal, bacterial and
exopolymer components of microbial biofilms. J Microbid
capable of enhancing pollurion degradation. Kesults Methods 1998, 32:253-261.
obtained highlight the need to USC’ strains that are well The method described offers the potential for detailed study of blofilms in
situ, allowing the effects of different treatments to be assessed. The authors
adapted to the wascewater treatment environment. Such Illustrate how advances in microscopy can be applied to increase our level
techniclues offer the potential to improve the efficiency of of understanding of naturally produced biofilms.
wastewater treatment, but. as with any application involT-- 7. Bura R, Cheung M, Liao B, Finlayson J, Lee BC, Droppo IG,
.. Leppard GG, LISS SN: Composition of extracellular polymeric
ing genetically modified orgallisms, they arc likely to
substances in the activated sludge floe matrix. Water Sci Jechnol
cncountcr legislative opposition to their implementation. 1998, 37:325-333.
This paper increases the information avallable on btopolymers In activated
sludge, introducmg the concept that biopolymer quality, rather than quantity,
Conclusions may be more important. An interesting study into the effect of nutrient con-
Our knowledge of the role played by microorganisms and centrations on sludge floe properties, including biopolymer composition.

their associated biopolymers in wastewater treatment is 8. Velga MC, Jain MK, Wu W-M, Holllngsworth RI. Zeikus JG:
slowly being elucidated. Cross-application of techniques Composition and role of extracellular polymers in methanogenic
granules. Appl Environ Microbid 1997, 63:403-407.
perfected in other areas of microbiology are allowing LIS to
9. Zhang X, Bishop PL, Kupferle MJ: Measurement of polysaccharides
establish the composition of biopolymers and the role they .. and proteins in biofilm extracellular polymers. Water SC; Techno/
play in sludge properties. biofilm formation and the sorp- 1998, 371345348.
The authors utilize a micro-splicing technique to study the spatial distribution
tion of pollurants. Within the open. heterogeneous systems of the biofilm, providing useful information on changes in blopolymer com-
discussed here. there appears to be little future at present position under different conditions. An Insight into the spatial distribution of
for the application of genetic manipulation to improve biopolymer in biofilms.

such processes, the future appearing to lie in the LIX of 10. Nielsen PH, Jahn A, Palmgren R: Conceptual model for production
and composition of exopolymers in biofilms. Water Sci 72dmol
techniques such as nutrient regtdation. 1997, 36:l l-1 9.
262 Environmental biotechnology

11. Zita A, Hermansson M: Effects of bacterial cell surface structures 16. Sp%th R, Flemming H-C, Wuertz S: Sorption properties of biofilms.
and hydrophobicity on attachment to activated sludge floes. Appl . Water Sci Technoll998, 37:207-210.
Environ Microbial 1997,63:1168-l 170. This paper illustrates that the biopolymer and the biomass should be ana-
lyzed when evaluating sorption processes within biofilms. This paper illus-
12. Jorand F, Bou&Bigne F, Block JC, Urbain V: Hydrophobic/hydrophilic
trates that sorption of pollutants is dependent on pollutant type.
. properties of activated sludge exopolymeric substances. Water
Sci Technoll998, 37:307-315. 17. Wolfaardt GM, Lawrence JR, Robarts RD, Caldwell DE: In situ
This paper presents evidence to support the hypothesis that the hydropho- l * characterization of biofilm exopolymers involved in the
bic fraction of the biopolymer aids in the organization of sludge floes. This accumulation of chlorinated organics. Microb Ecol 1998,
paper states a case for the role of hydrophobic biopolymers in flocculation. 35:213-223.
This paper describes the application of imaging techniques in conjunction
13. Keiding K, Nielsen PH: Desorption of organic macromolecules
from activated sludge: effect of ionic composition. Water Res with microscopy that offers the potential to determine biopolymer-pollutant
1997, 31 :1665-l 672. interactions in situ.

14. Higgins MJ, Novak JT: Characterization of exocellular protein and 18. Griffin AM, Edwards KJ, Gasson MJ, Morris VJ: Identification of
its role in bioflocculation. J Envion Engineer 1997, 123:485-497. structural genes involved in bacterial exopolysaccharide
production. Biotechnol Genet fng Rev 1996, 13:1-l 8.
15. Zhang S-F, Splendiani A, Freitas dos Santos LM, Livingston AG:
. DetemGnation of pollutant diffusion coefficients in naturally 19. Van Limbergen H, Top EM, Verstraete W: Bioaugmentation in
formed biofilms using a single tube extractive membrane . activated sludge: current features and future perspectives. Appl
bioreactor. Biofechnol Bioeng 1998, 59:80-89. Microbial Biotechnoll998, 50:16-23.
A novel technique, using non-reacting tracer molecules, that may provide A general, up-to-date review on the limited amount of work reported in
more accurate data for use in mathematical models on substrate transport. this area.