Quantified small-scale turbulence inhibits the growth of a green alga

1. Laboratory experiments were conducted to elucidate the effect of small-scale turbulent fluid motion on the growth of laboratory cultures of the freshwater algae Scenedesmus quadricauda . Turbulent flow was generated using an oscillating-grid apparatus. The experiments were performed under the range of fluid flow conditions similar to those occurring in nature. The only growth limiting factor was the effect of small-scale fluid motion; all other environmental factors, such as light, temperature and nutrients, were kept constant.
2. Growth of Scenedesmus quadricauda , measured in terms of chlorophyll a concentration, was inhibited when the level of turbulence in the water column was increased. Algal growth was maximum in a quiescent fluid. The inhibitory effect of fluid motion was observed independently of flow regime (laminar, transitional, turbulent) in the water column.
3. Cell destruction and aggregation of dead and living cells of algae were observed in a turbulent flow. High shear rates, estimated from the dissipation of turbulent kinetic energy, caused the cell destruction, algal collision and agglomeration of algae. Data on Scenedesmus responses to small-scale fluid motion will enhance and broaden our ability to develop predictive multispecies models for freshwater phytoplankton communities.     

Small-scale fluid motion mediates growth and nutrient uptake of Selenastrum capricornutum

1. A fluid‐flow reactor using submersible speakers was constructed to generate small‐scale fluid motion similar to conditions measured in open water environments; flow was quantified by particle image velocimetry. Additionally a Couette‐type rotating cylinder was used to generate shear flows; flow was quantified using an optical hotwire probe and torque measurements. Growth rates of the green alga Selenastrum capricornutum were determined from changes in cell counts and viability was tested using the fluorogenic probe fluoresceine diacetate. 2. Evidence that fluid motion directly affects growth rates was obtained as a significant difference between growth in a moving versus non‐moving fluid. A near 2‐fold increase in growth rate was achieved for an energy dissipation rate of ? = 10^?7 m² s^?3; a rate common in lakes and oceans. The onset of the viability equilibrium, identified as the day of the test period when the number of active cells equalled non‐active cells, was delayed by 2 days for moving fluid conditions as compared with a non‐moving fluid. 3. Nutrient uptake was determined by a decrease in the bulk fluid concentration and cellular phosphorus concentration was also estimated. The thickness of the diffusive sublayer surrounding a cell, a zone dominated by molecular diffusion, was estimated. Increasing fluid motion was found to decrease the thickness of this layer. The Sherwood number (ratio of total mass flux to molecular mass flux) showed that advective flux surrounding cells dominated molecular diffusion flux with regard to Péclet numbers (ratio of advective transport to molecular diffusion transport). Fluid motion facilitated uptake rates and resulted in increased growth rates, compared with no‐flow conditions. The rate‐of‐rotation and the rate‐of‐strain in a moving fluid equally mediated the diffusive sublayer thickness surrounding the cells. Our study demonstrates that small‐scale fluid motion mediates algal growth kinetics and therefore should be included in predictive models for algal blooms.     

Turbulent mixing mediates the photosynthetic activities and biochemical composition of <Emphasis Type="Italic">Anabaena</Emphasis>: implications for bioengineering

Cyanobacteria exhibit a variety of adaptive strategies that allow them to thrive in ever-changing aquatic environments. Here, successive steady states of continuous cultures were used to investigate the effects of quantified turbulence on the biochemical compounds and physiological processes of Anabaena flos-aquae in a photobioreactor under different dilution rates. A rapid increase in cell density was clearly observed following an increase in the turbulent dissipation rate at all growth rates of A. flos-aquae. The photosynthetic response to irradiance curves showed that the turbulence-treated strains exhibited lower photosynthetic oxygen evolution and saturating irradiance as well as higher respiration in rapidly growing young cells, indicating that they might not be very adaptable to high turbulent dissipation rates. Additionally, there was an increase in the protein levels of A. flos-aquae with increasing turbulence at all growth rates, whereas carbohydrate formation and lipid accumulation demonstrated the opposite trends. At a high growth rate, the level of carbohydrates decreased whereas that of lipids increased, which was interpreted as reflecting an adaptation to the turbulent environment. These findings suggest that turbulence sensitivity is shear regimen- and growth rate-dependent in A. flos-aquae. The high respiration capacity, low saturating irradiance, and conversion of carbohydrates to lipids represent effective measures for revealing the adaptive strategies of rapidly growing young cells under hydrodynamic regimes. The results regarding optimum lipid accumulation with specific growth traits have important implications for the design of cultivation methods of cyanobacteria resource utilization with respect to regulating turbulence.     

Enhanced uptake of dissolved oxygen and glucose by <Emphasis Type="Italic">Escherichia coli</Emphasis> in a turbulent flow

Laboratory experiments were conducted to study the effect of turbulence on Escherichia coli cells in an oscillating grid reactor under conditions of no oxygen transfer to the liquid phase. Fluid flow was quantified at a submillimeter resolution using a particle image velocimetry measuring technique. The root-mean-square estimates of the velocity gradient tensor components indicated the dominance of shear rate deformation in the fluid surrounding E. coli. The E. coli growth rate, dissolved oxygen (DO), and glucose uptake rates were facilitated by fluid-flow energy dissipation in the turbulent fluid. The Kolmogorov length scale (eta(K)) and velocity (u( K )) underlined characteristic scales at which enhanced DO and glucose uptake by E. coli were determined in a turbulent flow in comparison to still-water controls. A first-order power-law relation between the mass transport to the cells and the moving fluid is developed. The combined effects of the enhanced rate of strain at eta(K) scale and uniform velocity at u(K) determined the facilitated DO and glucose fluxes to E. coli. The mass transport to the E. coli was modeled by the Sherwood (Sh)-Péclet (Pe) number relationship by Sh = 1 + 1.08Pe(uK)(0.62) where Pe(uK) is the Péclet number defined by the u(K) velocity scale. The proposed first-order model described experimental data fairly well.     

Respiratory activity of Candida tropicalis during growth on hexadecane and on glucose

Summary Rates of oxygen uptake and the oxygen demand during growth of Candida tropicalis on hexadecane and glucose were determined in batch experiments. Oxygen demand was 2.5 fold higher for the synthesis of one unit of cell mass from hydrocarbon than from glucose. On the other hand specific respiration is of the same order of magnitude for both substrates, e.g. 12 mmoles O₂xh^-1xg^-1 (dry weight) and seems to be a constant of this organism. Higher rates of oxygen supply into the medium had no effect on the specific rates of respiration. Specific growth rates on hexadecane were 2.4 times lower than on glucose. It is concluded, that rates of synthesis of cell components are controlled by the overall capacity of the respiratory pathways.     

Effects of dilution on dissolved oxygen depletion and microbial populations in the biochemical oxygen demand determination

The biochemical oxygen demand (BOD) value is still a key parameter that can determine the level of organics, particularly the content of biodegradable organics in water. In this work, the effects of sample dilution, which should be done inevitably to get appropriate dissolved oxygen (DO) depletion, on the measurement of 5-day BOD (BOD₅), was investigated with and without seeding using natural and synthetic water. The dilution effects were also evaluated for water samples taken in different seasons such as summer and winter because water temperature can cause a change in the types of microbial species, thus leading to different oxygen depletion profiles during BOD testing. The predation phenomenon between microbial cells was found to be dependent on the inorganic nutrients and carbon sources, showing a change in cell populations according to cell size after 5-day incubation. The dilution of water samples for BOD determination was linked to changes in the environment for microbial growth such as nutrition. The predation phenomenon between microbial cells was more important with less dilution. BOD₅ increased with the specific amount of inorganic nutrient per microbial mass when the natural water was diluted. When seeding was done for synthetic water samples, the seed volume also affected BOD due to the rate of organic uptake by microbes. BOD₅ increased with the specific bacterial population per organic source supplied at the beginning of BOD measurement. For more accurate BOD measurements, specific guidelines on dilution should be established.     

Distribution and movement of toxaphene in anaerobic saline marsh soils

The biological oxygen demand (BOD) of filtered water from Lake Wingra, Wisconsin is significantly higher in the littoral zone than in the pelagial zone. Laboratory experiments indicate that BOD is not influenced by water temperature at the time of sampling or by enrichment with nitrate or ammonia. Rather, enrichment with macrophyte leachate sharply increases BOD, and enrichment with phosphate produces a small but significant increase in BOD. We conclude that high BOD in littoral waters of the lake is an indication of production of labile organic matter in the water by dense beds of the macrophyte Myriophyllum spicatum.     

A Desktop Apparatus for Studying Interactions Between Microorganisms and Small-Scale Fluid Motion

Low levels of kinetic energy dissipation were successfully generated in a reactor using two submersible speakers. A software programme controlled the amplitude and frequency of the signal fed to each speaker and achieved good repeatability of flow conditions. The flow reactor had a near isotropic flow regime with a low mean flow, values were calculated from particle image velocimetry measurements. The flow characteristics compared well with grid turbulence reactors, though as no moving parts are present in this reactor design the strain rates were lower compared to oscillating grid set-ups. The low range of Reynolds numbers based on Taylor microscales (Re_λ~0.5–5.9) covered both turbulent and non-turbulent flow regimes. The small-scale fluid motion produced over the entire volume of this reactor makes it suitable for experiments examining the physiological responses of fluid motion on microorganisms.     

On-line monitoring of a two-stage anaerobic digestion process using a BOD analyzer

A computer-controlled biochemical oxygen demand (BOD) analyzer has been developed for fast estimation of biochemical oxygen demand (BODst) automatically with the purpose of on-line monitoring of a process for conversion of biomass under field conditions. The instrument was tested by on-line monitoring of the connecting stream between two stages of a two-stage anaerobic process in laboratory scale. In the first stage, hydrolysis of sugar beet leaves and its conversion into volatile fatty acids and other low molecular weight substrates took place. The effluent from the first reactor was used as a feed stream to the second stage, i.e. an anaerobic contact reactor. The feed stream was sampled intermittently, diluted and analyzed by the BOD analyzer automatically in order to estimate the organic loading rate to the reactor. The results from this study demonstrated that the BOD analyzer could be a stand-alone and promising sensor device for rapid on-line monitoring of easily biodegradable organic substances in biological treatment processes.     

Integrated algal engineering for bioenergy generation,effluent remediation,and production of high-value bioactive compounds

Increased demand for energy worldwide has resulted in increasing interest in alternative renewable sources of biofuels. Demand for improved systems of bioenergy generation, environmental remediation, and coproduction of high value bioactive compounds has led to the potential use of algae in biomass utilization. In Malaysia, palm oil industries generate high amount of solid wastes. Palm Oil Mill Effluent (POME) is estimated to be three times of the amount of crude palm oil produced. POME is a heavily polluting wastewater due to its high chemical oxygen demand (COD), high biochemical oxygen demand (BOD), and high contents of minerals such as nitrogen and phosphorus that can cause severe pollution to the environment and water resources. A combination of wastewater treatment and renewable bioenergy co-generation with recovery of high-value biochemicals would benefit the palm oil industry.     

Sequential Substrate Removal in a Dilute System by Heterogeneous Microbial Populations

The sequential metabolism of substrates by heterogeneous bacterial populations has been previously reported from this laboratory in studies with high substrate concentrations. This phenomenon has now been shown to occur at very dilute substrate concentrations, i.e., 5 mg/liter of glucose plus 5 mg/liter of sorbitol, in studies conducted under the conditions of the standard biochemical oxygen demand (BOD) test. Sequential metabolism of these substrates resulted in a diphasic curve of accumulated oxygen uptake wherein the two phases were separated by a discernible plateau. These findings illustrate one possible explanation for the generation of discontinuity in the kinetic course of carbonaceous BOD exertion.     

Biochemical mediator demand – a novel rapid alternative for measuring biochemical oxygen demand

The biochemical oxygen demand (BOD) test (BOD₅) is a crucial environmental index for monitoring organic pollutants in waste water but is limited by the 5-day requirement for completing the test. We have optimised a rapid microbial technique for measuring the BOD of a standard BOD₅ substrate (150 mg glucose/l, 150 mg glutamic acid/l) by quantifying an equivalent biochemical mediator demand in the absence of oxygen. Elevated concentrations of Escherichia coli were incubated with an excess of redox mediator, potassium hexacyanoferrate(III), and a known substrate for 1 h at 37 °C without oxygen. The addition of substrate increased the respiratory activity of the microorganisms and the accumulation of reduced mediator; the mediator was subsequently re-oxidised at a working electrode generating a current quantifiable by a coulometric transducer. Catabolic conversion efficiencies exceeding 75% were observed for the oxidation of the standard substrate. The inclusion of a mediator allowed a higher co-substrate concentration compared to oxygen and substantially reduced the incubation time from 5 days to 1 h. The technique replicates the traditional BOD₅ method, except that a mediator is substituted for oxygen, and we aim to apply the principle to measure the BOD of real waste streams in future work. Received: 2 August 1999 / Received revision: 6 December 1999 / Accepted: 12 December 1999     

Organic and nitrogen removal in a two-stage rotating biological contactor treating municipal wastewater

A laboratory scale rotating biological contactor (RBC) predenitrification system incorporating anoxic and aerobic units was evaluated for the treatment of settled high-strength municipal wastewater. The system was operated under four recycle ratios (1, 2, 3 and 4) and loading rates of 38-182 gCOD/m(2)d and 0.22-14 gOxid-N/m(2)d on the anoxic unit and 3.4-18 gCOD/m(2)d and 0.24-1.8 gNH(4)-N/m(2)d on the aerobic. The average removal efficiency in terms of chemical oxygen demand (COD), biochemical oxygen demand (BOD(5)), total suspended solids (TSS) and total nitrogen (Total-N) was 82%, 86%, 63% and 54%; settling of the RBC effluent increased COD and TSS removal to 94% and 97%. An increase in hydraulic loading resulting from higher recirculation, had limited negative effect on organic removal but improved nitrogen removal, and in terms of Total-N removal efficiency increased up to a ratio of 3 and then decreased.     

A rapid BOD sensing system using luminescent recombinants of Escherichia coli

A biochemical oxygen demand (BOD) sensing system based on bacterial luminescence from recombinant Escherichia coli containing lux A-E genes from Vibrio fischeri has been developed. It was possible to use frozen cells of luminescent recombinants of E. coli as the bacterial reagents for measurement. Steady bioluminescence was observed during the incubation time between 90 and 150 min in the presence of a sole carbon source such as glucose, acetate, L-glutamate and BOD standard solution (GGA solution). This disposable bacterial reagent was applied to measure and detect organic pollution due to biodegradable substances in various wastewaters. The obtained values of this study showed a similar correlation with that of the conventional method for BOD determination (BOD5). Bacterial luminescence that was visualized with an imaging system using a charge coupled device (CCD) camera and a photomulti-counter demonstrated that this method could also be used for multi-sample detection of organic pollution due to biodegradable substances by using a microtiter plate. These results suggested for successful achievement of high-though-put detection of BOD in practical.     

On-line load monitoring of wastewaters with a respirographic microbial sensor

This paper demonstrates the functionality, laboratory testing and field application of a microbial sensor that is capable of monitoring the organic pollution extent of wastewaters both off-line in a laboratory and on-line in a wastewater treatment plant. The biosensor was first developed in the laboratory using synthetic wastewater and then applied to monitor the effluent of the unit. The basic working principle of the biosensor is based on the on-line measurement of CO2 concentration in the off gas produced during carbon compound degradation by microbial respiration activities. CO2 concentration under operation conditions (constant oxygen flow rate, residence time and pH) is closely related to the extent of organic pollution (biochemical oxygen demand, chemical oxygen demand). CO2 monitoring is carried out by an infrared spectrometer, whereas current organic pollution is determined off-line according to the conventional 5-day lasting BOD analysis. Off gas analysis of CO2 concentration strongly correlates with off-line biochemical oxygen demand measurements allowing continuous on-line monitoring of the organic load within a wastewater treatment plant. Thus, real time process control and operation become feasible.     

Development and evaluation of an online CO(2) evolution test and a multicomponent biodegradation test system

Well-established biodegradation tests use biogenously evolved carbon dioxide (CO(2)) as an analytical parameter to determine the ultimate biodegradability of substances. A newly developed analytical technique based on the continuous online measurement of conductivity showed its suitability over other techniques. It could be demonstrated that the method met all criteria of established biodegradation tests, gave continuous biodegradation curves, and was more reliable than other tests. In parallel experiments, only small variations in the biodegradation pattern occurred. When comparing the new online CO(2) method with existing CO(2) evolution tests, growth rates and lag periods were similar and only the final degree of biodegradation of aniline was slightly lower. A further test development was the unification and parallel measurement of all three important summary parameters for biodegradation--i.e., CO(2) evolution, determination of the biochemical oxygen demand (BOD), and removal of dissolved organic carbon (DOC)--in a multicomponent biodegradation test system (MCBTS). The practicability of this test method was demonstrated with aniline. This test system had advantages for poorly water-soluble and highly volatile compounds and allowed the determination of the carbon fraction integrated into biomass (heterotrophic yield). The integrated online measurements of CO(2) and BOD systems produced continuous degradation curves, which better met the stringent criteria of ready biodegradability (60% biodegradation in a 10-day window). Furthermore the data could be used to calculate maximal growth rates for the modeling of biodegradation processes.     

Biosensor analyzer for BOD index express control on the basis of the yeast microorganisms Candida maltosa, Candida blankii, and Debaryomyces hansenii

The parameters of biosensors based on the yeast strains Candida maltosa VKM Y-2359, Candida blankii VKM Y-2675, and Debaryomyces hansenii VKM Y-2482 for biochemical oxygen demand (BOD) detection are compared. The catalytic activity of the strains was analyzed in relation to the growth phase. The possibility of using D. hansenii as a basis for receptor element of a biosensor for BOD detection in municipal and biotechnological wastewaters was shown.     

Effect of drying and rewetting on bacterial growth rates in soil

The effect of soil moisture on bacterial growth was investigated, and the effects of rewetting were compared with glucose addition because both treatments increase substrate availability. Bacterial growth was estimated as thymidine and leucine incorporation, and was compared with respiration. Low growth rates were found in air-dried soil, increasing rapidly to high stable values in moist soils. Respiration and bacterial growth at different soil moisture contents were correlated. Rewetting air-dried soil resulted in a linear increase in bacterial growth with time, reaching the levels in moist soil (10 times higher) after about 7 h. Respiration rates increased within 1 h to a level >10 times higher than that in moist soil. After the initial flush, there was a gradual decrease in respiration rate, while bacterial growth increased to levels twice that of moist soil 24 h after rewetting, and decreased to levels similar to those in moist soil after 2 days. Adding glucose resulted in no positive effect on bacterial growth during the first 9 h, despite resulting in more than five times higher respiration. This indicated that the initial increase in bacterial growth after rewetting was not due to increased substrate availability.     

Computational fluid dynamics analysis of mixing and gas–liquid mass transfer in wave bag bioreactor

Single use bioreactors provide an attractive alternative to traditional deep-tank stainless steel bioreactors in process development and more recently manufacturing process. Wave bag bioreactors, in particular, have shown potential applications for cultivation of shear sensitive human and animal cells. However, the lack of knowledge about the complex fluid flow environment prevailing in wave bag bioreactors has so far hampered the development of a scientific rationale for their scale up. In this study, we use computational fluid dynamics (CFD) to investigate the details of the flow field in a 20-L wave bag bioreactor as a function of rocking angle and rocking speed. The results are presented in terms of local and mean velocities, mixing, and energy dissipation rates, which are used to create a process engineering framework for the scale-up of wave bag bioreactors. Proof-of-concept analysis of mixing and fluid flow in the 20-L wave bag bioreactor demonstrates the applicability of the CFD methodology and the temporal and spatial energy dissipation rates integrated and averaged over the liquid volume in the bag provide the means to correlate experimental volumetric oxygen transfer rates (k_La) data with power per unit volume. This correlation could be used as a rule of thumb for scaling up and down the wave bag bioreactors.     

Development and Evaluation of an Online CO2 Evolution Test and a Multicomponent Biodegradation Test System

Well-established biodegradation tests use biogenously evolved carbon dioxide (CO₂) as an analytical parameter to determine the ultimate biodegradability of substances. A newly developed analytical technique based on the continuous online measurement of conductivity showed its suitability over other techniques. It could be demonstrated that the method met all criteria of established biodegradation tests, gave continuous biodegradation curves, and was more reliable than other tests. In parallel experiments, only small variations in the biodegradation pattern occurred. When comparing the new online CO₂ method with existing CO₂ evolution tests, growth rates and lag periods were similar and only the final degree of biodegradation of aniline was slightly lower. A further test development was the unification and parallel measurement of all three important summary parameters for biodegradation—i.e., CO₂ evolution, determination of the biochemical oxygen demand (BOD), and removal of dissolved organic carbon (DOC)—in a multicomponent biodegradation test system (MCBTS). The practicability of this test method was demonstrated with aniline. This test system had advantages for poorly water-soluble and highly volatile compounds and allowed the determination of the carbon fraction integrated into biomass (heterotrophic yield). The integrated online measurements of CO₂ and BOD systems produced continuous degradation curves, which better met the stringent criteria of ready biodegradability (60% biodegradation in a 10-day window). Furthermore the data could be used to calculate maximal growth rates for the modeling of biodegradation processes.