Biofilm development at a surface is the net result of several physical, chemical, and microbial processes including the following: 1) transport of dissolved and particulate matter from the bulk fluid to the surface; 2) firm microbial cell attachment to the surface; 3) microbial transformations (growth, reproduction, etc.) within the biofilm resulting in production of organic matter; 4) partial detachment of the biofilm due primarily to fluid shear stress. This report presents a framework for analyzing the interrelated processes contributing to biofilm development. Some of the available rate and composition data are presented so that the relative process rates can be compared.

The kinetics and stoichiometry of Desulfovibrio desulfuricans attached to a polycarbonate surface were determined in RotoTorque(TM) reactors and compared with those of suspended cells. Biofilm specific cellular growth rate (mu(b)) and detachment rate (q(dx)) were determined from unsteady state biofilm experiments. In the initial biofilm accumulation phase, the specific cellular growth rate was the same as the maximum specific growth rate for D. desulfuricans in suspension (mu(max) = 0.37 . h(-1)); thereafter mu(b) decreased and approached a steady state value of about 0.1 . h(-1). The decrease in average cellular specific growth rate could be attributed to substrate (lactate) limitation in some experiments, but in others there was no evidence of this. Biofilm-specific cellular detachment rate decreased similarly to biofilm-specific cellular growth rate. In biofilms, cellular yield at mu(b) = 0.1 . h(-1) was approximately 18% of planktonic cellular yield partly due to the production of extensive extracellular polymeric substance. A linear relationship between mu(b) and specific lactate utilization rate (q(s)) in the biofilm did not exist. During the steady state biofilm accumulation phase (mu(b) = .0.1 . h(-1)), specific lactate utilization by biofilm cells was about 2-3 times greater than by planktonic cells, whereas it was essentially the same during the initial biofilm accumulation phase. These results suggest that kinetic and stoichiometric data derived from suspended cells must be cautiously incorporated into biofilm accumulation models.

Scientists and engineers have realized the industrial and environmental significance of biofilm accumulation and activity. The ability to predict and control biofilm formation has led to less fouling and corrosion in industrial systems and a better understanding of biofilm importance in natural aquatic systems. Understanding the fundamental processes contributing to biofilm formation is beneficial to anyone involved with natural or industrial systems where biofilms may play a significant role in determining variables such as bulk water quality, toxic compound biodegradation, or product quality.

Sulfide product inhibition kinetics for growth and activity of Desulfovibrio desulfuricans was investigated in batch and continuous cultures at pH = 7.0. A non-competitive inhibition model adequately described sulfide product inhibition kinetics. Inhibition coefficient (Ki) for maximum specific growth rate (μinhmax) was 251 mg l−1 S in a batch experiment. Cell yield determined in a chemostat was reduced in half by a sulfide concentration of about 250 mg l−1 S, which was very close to the Ki value for the batch growth. Maximum specific growth rate (μinhmax) and cell yield (YcLac) were strongly inhibited by high levels of sulfide concentrations, whereas specific lactate utilization rate increased with increasing sulfide concentrations. The results indicated an increase in the relative energy needed for maintenance to overcome sulfide inhibition and uncoupling growth from energy production. However, D. desulfuricans to some extent could recover from the shock of high sulfide concentrations. Stoichiometry for catabolic reactions (energy producing) did not change at high sulfide concentrations, while anabolic reactions (cellular synthesis) were strongly inhibited by high sulfide concentrations. These results suggested that separation of sulfide product inhibition into growth (cell yield) and activity (substrate utilization rate) was important to incorporate the sulfide product inhibition kinetics in a variety of applications.

Packed-bed bioreactors filled with diatomaceous earth (D.E.) pellets were used to evaluate the effects of competition between inoculated and invading microbial species on the spatial and temporal distribution of microorganisms within an individual pellet. The (D.E.) pellets were cylinders 6 mm in diameter and 5–10 mm long with a mean pore diameter of 20 μm. Bench-scale experiments evaluated competition between two distinct microbial species: Pseudomonas aeruginosa, a motile, obligate aerobe (μmax = 0.4 h−1) and Klebsiella pneumoniae, a non-motile, facultative organism (μmax = 2.0 h−1). Organism growth rate appeared to be more important than motility or order of introduction in determining organism spatial and temporal distribution within the pellets. Pilot-scale experiments used pellets colonized with a pseudomonad growing on chlorobenzene as the sole carbon and energy source. Organic-rich ground water containing benzene, chlorobenzene and a population of indigenous microorganisms was used as feed. Pellet concentrations of the inoculated pseudomonad dropped from 109 to 106 colony forming units (cfu) ml−1 pellet volume over 15 days. These experiments demonstrate that inoculated organisms within porous packing media may undergo significant loss in colonization numbers when faced with competition from faster growing organisms.

Measurement of cellular and extracellular carbon contents of sulfate-reducing bacteria (SRB) is essential and important in studies of the role of SRB in corrosion and biofouling. An epifluorescence (EPI) microscopic technique and a lipopolysaccharide (LPS) assay were used to quantify cellular and extracellular carbon contents in Desalfovibrio desulfuricans biofilms. The average contents of lipopolysaccharide (LPS) and cellular carbon were 7.3 +/- 2.8 (fg LPS) cell(-1) and 39.9 +/- 9.9 (fg cellular-C) cell(-1) respectively, in a D. desulfuricans chemostat culture. A ratio of cellular carbon content to LPS content was 6.5 +/- 2.8, and was used to estimate cellular carbon contents in a D. desulfuricans biofilm. The LPS and EPI methods gave comparable results for suspended samples, but not for biofilm samples.

Corrosion of mild steel under completely anaerobic conditions in the presence of a mixed population biofilm, including sulfate-reducing bacteria (SRB), has been studied in a continuous flow system. The closed channel flow reactor was continuously fed with low concentration substrate at different dilution rates that influenced biofilm accumulation. No direct correlation was observed between corrosion and SRB activity in the absence of ferrous iron. Furthermore, corrosion of mild steel in the SRB environment was determined by the nature of the metal and environmental conditions such as dissolved iron concentration. When formation of an iron sulfide film on mild steel was prevented before the biofilm accumulated, the metal surface retained its scratch lines after a 21-day experiment (SRB at 2.6 x 10(9)/cm2). However, when the iron sulfide film was formed before the accumulation of biofilm, visible localized corrosion appeared after 14 days and increased up to 21 days. Intergranular and pitting attack was found in the localized corrosion area. Inclusions (Al, Mn, and Fe) and grain boundary triple points were also found in the localized corrosion area. At high iron concentration (approximately 60 mg/L in the bulk water), all biogenic sulfide was precipitated and corrosion had significantly enhanced. Intergranular attack was found over the entire metal surface.

Fluorescently labelled latex microbeads were used to study the interaction of particles with Pseudomonas aeruginosa biofilms in a continuous flow annular reactor. Beads were readily distinguished and enumerated in both intact and disaggregated biofilm samples. The fraction of beads that attached to biofilm during a 24 h period ranged from 0.001 to 0.01 and was proportional to biofilm cell carbon and to the standard deviation of biofilm thickness. Microbeads added to biofilm of steady state thickness (30 μm) were observed to be located throughout the entire biofilm depth in 24 h. Many of the microbeads that attached to biofilm shortly after bacterial inoculation (thickness of 2 μm) remained near the substratum as cells grew past and covered them. Microbeads were observed near the biofilm-substratum interface for up to 5 days after bead addition. Beads formed aggregates on biofilms, but not in bulk water. Beads captured by biofilm remained in the reactor system longer than beads that never attached to biofilm.

Fluorescent latex microbeads added to a Pseudomonas aeruginosa biofilm as tracers of particle movement penetrated the biofilm and remained in it much longer than predicted by a model of advective displacement due to cell growth. Beads with a nominal diameter of 1 mum that were added in the bulk fluid became distributed throughout the biofilm depth. Some microbeads penetrated to the substratum within the 24-h bead addition period. The biofilms had a mean thickness of approximately 34 mum but have been previously shown to be quite rough. Measured rates of bead release from the biofilm corresponded to first order time coefficients of 0.01-0.03 h(-1). These bead release rates were approximately an order of magnitude less than the predicted time scale of advective transport, which is just the experimentally measured specific cellular growth rate of 0.15 h(-1). Computer simulations of bead transport using the biofilm model BIOSIM were compared with bead release rate data and with bead position distributions within the biofilm as determined by microscopic examination of thin cross sections of embedded biofilm. The model predicted much faster release of beads from the biofilm than actually occurred. It is hypothesized that both the ability of beads to penetrate the biofilm and the unexpectedly low advective displacement velocity of particles in the biofilm were due to the rough nature of the biofilm.

One of the least understood processes affecting biofilm accumulation is detachment. Detachment is the removal of cells and cell products from an established biofilm and subsequent entrainment in the bulk liquid. The goal of this research was to determine the effects of shear stress and substrate loading rate on the rate of biofilm detachment. Monopopulation Pseudomonas aeruginosa and undefined mixed population biofilms were grown on glucose in a RotoTorque biofilm reactor. Three levels of shear stress and substrate loading rate were used to determine their effects on the rate of detachment. Suspended cell concentrations were monitored to determine detachment rates, while other variables were measured to determine their influence on the detachment rate. Results indicate that detachment rate is directly related to biofilm growth rate and that factors which limit growth rate will also limit detachment rate. No significant influence of shear on detachment rate was observed.A new kinetic expression that incorporates substrate utilization rate, yield, and biofilm thickness was compared to published detachment expressions and gives a better correlation of data obtained both in this research and from previous research projects, for both mono- and mixed-population biofilms.

The effects of temperature and phosphorous concentration on the rate and the extent of microbial sulfate reduction with lactate as carbon and energy source were investigated for Desulfovibrio desulfuricans. The continuous culture experiments (chemostat) were conducted at pH 7.0 from 12 to 48 degrees C. The maximum specific growth rate (micro(max)) was relatively constant in the range 25 degrees C-43 degrees C and dramatically decreased outside this temperature range. The half-saturation coefficient was minimum at 25 degrees C. Cell yield was highest in the optimum temperature range (35 degrees C-43 degrees C) for growth. Maintenance energy requirements for D. desulfuricans were not significant. Two moles of lactate is consumed for every mole of sulfate reduced, and this stoichiometric ratio is not temperature dependent. Steady state rate and stoichiometric coefficients accurately predicted transient behavior during temperature shifts. The extent of extracellular polymeric substance (EPS) is related to the concentration of phosphorous in the medium. EPS production rate increased with decreased phosphorous loading rate. Failure to discriminate between cell and EPS formation by D. desulfuricans leads to significant overestimates of the cell yield. The limiting C:P ratio for D. desulfuricans was in the range of 400:1 to 800:1.