The use of soil biofilms for observing protozoan movement and feeding

Protozoan movement and feeding regimes in soil biofilms were observed with inverted microscopes and Utermohl plankton counting chambers (4 mm deep). In a new use for these counting chambers, the three-dimensional appearance of a soil pore network can be simulated using long working distance objectives (40x). Protozoa were often associated with soil surfaces and penetrated soil crevices of less than 3 microm in diameter by either distorting their body outlines or with slender pseudopodia of less than 2 microm at the tips. Many ciliates repeatedly collided with soil particles and released attached bacteria into the soil solution for subsequent predation.     

Seasonal and successional influences on bacterial community composition exceed that of protozoan grazing in river biofilms

The effects of protozoa (heterotrophic flagellates and ciliates) on the morphology and community composition of bacterial biofilms were tested under natural background conditions by applying size fractionation in a river bypass system. Confocal laser scanning microscopy (CLSM) was used to monitor the morphological structure of the biofilm, and fingerprinting methods (single-stranded conformation polymorphism [SSCP] and denaturing gradient gel electrophoresis [DGGE]) were utilized to assess changes in bacterial community composition. Season and internal population dynamics had a greater influence on the bacterial biofilm than the presence of protozoa. Within this general framework, bacterial area coverage and microcolony abundance were nevertheless enhanced by the presence of ciliates (but not by the presence of flagellates). We also found that the richness of bacterial operational taxonomic units was much higher in planktonic founder communities than in the ones establishing the biofilm. Within the first 2 h of colonization of an empty substrate by bacteria, the presence of flagellates additionally altered their biofilm community composition. As the biofilms matured, the number of bacterial operational taxonomic units increased when flagellates were present in high abundances. The additional presence of ciliates tended to at first reduce (days 2 to 7) and later increase (days 14 to 29) bacterial operational taxonomic unit richness. Altogether, the response of the bacterial community to protozoan grazing pressure was small compared to that reported in planktonic studies, but our findings contradict the assumption of a general grazing resistance of bacterial biofilms toward protozoa.     

Influence of nanophase titania topography on bacterial attachment and metabolism

Surfaces with nanophase compared to conventional (or nanometer smooth) topographies are known to have different properties of area, charge, and reactivity. Previously published research indicates that the attachment of certain bacteria (such as Pseudomonas fluorescens 5RL) is higher on surfaces with nanophase compared to conventional topographies, however, their effect on bacterial metabolism is unclear. Results presented here show that the adhesion of Pseudomonas fluorescens 5RL and Pseudomonas putida TVA8 was higher on nanophase than conventional titania. Importantly, in terms of metabolism, bacteria attached to the nanophase surfaces had higher bioluminescence rates than on the conventional surfaces under all nutrient conditions. Thus, the results from this study show greater select bacterial metabolism on nanometer than conventional topographies, critical results with strong consequences for the design of improved biosensors for bacteria detection.     

Microcolonies, quorum sensing and cytotoxicity determine the survival of Pseudomonas aeruginosa biofilms exposed to protozoan grazing

This study was based on the hypothesis that biofilms of the opportunistic pathogen Pseudomonas aeruginosa are successfully adapted to situations of protozoan grazing. We tested P. aeruginosa wild type and strains that were genetically altered, in structural and regulatory features of biofilm development, in response to the common surface-feeding flagellate Rhynchomonas nasuta. Early biofilms of the wild type showed the formation of grazing resistant microcolonies in the presence of the flagellate, whereas biofilms without the predator were undifferentiated. Grazing on biofilms of quorum sensing mutants (lasR and rhlR/lasR) also resulted in the formation of microcolonies, however, in lower numbers and size compared to the wild type. Considerably fewer microcolonies than the wild type were formed by mutant cells lacking type IV pili, whereas no microcolonies were formed by flagella-deficient cells. The alginate-overproducing strain PDO300 developed larger microcolonies in response to grazing. These observations suggest a role of quorum sensing in early biofilms and involvement of flagella, type IV pili, and alginate in microcolony formation in the presence of grazing. More mature biofilms of the wild type exhibited acute toxicity to the flagellate R. nasuta. Rapid growth of the flagellate on rhlR/lasR mutant biofilms indicated a key role of quorum sensing in the upregulation of lethal factors and in grazing protection of late biofilms. Both the formation of microcolonies and the production of toxins are effective mechanisms that may allow P. aeruginosa biofilms to resist protozoan grazing and to persist in the environment.     

氨溴索对粘液型铜绿假单胞菌生物膜藻酸盐作用的体外研究

目的探讨氨溴索对铜绿假单胞菌临床分离株形成的生物膜（biofilm,BF）主要成分藻酸盐的干预作用,研究其对藻酸盐合成过程中起重要作用的基因表达和合成过程中限速酶活性的影响,以及其对藻酸盐降解的影响。方法建立铜绿似单胞菌临床分离株BF体外模型,培养7d后得到成熟BF。将BF内的细菌振荡下来后,用疏酸-苯酚法检测氨溴索对藻酸盐含量的影响;RT-PCR检测藻酸盐合成过程中重要基因algD、algU、algR和mucA的mRNA表达;分光光度计检测合成过程中限速酶——GDP-甘露糖脱氢酶（guanosine diphospho-D-mannose dehydrogenase,GMD）的活性,并检测藻酸盐的降解情况。结果在氨溴索3．75mg／ml作用下,藻酸盐含量（mg／g）由86．4024±0．8588下降到59．9199±0．5803（F=66．2,P〈0．01）;其合成重要基因algD、algU、algR和mucA的mRNA的表达分别由1．2994±0．0173、1．0488±0．0457、0．9888±0．0267和0．8731±0．0336变化为1．0253±0．0265、0．9594±0．0106、0．8536±0．0179和1．0770±0．0503（F=91．9,41．1,88．4和56,9,P均〈0．05）;其合成限速酶GMD活性由0．0989±0．0055下降到0．0558±0．0016（F=121．2,P〈0．01）;藻酸盐的降解量（△mg／g）由1．4122±0．0073变化为1．4175±0．0019（F=21．81,P〉0．05）。1．875mg／ml氨溴索作用下,有同样的趋势但效应不如高浓度明显。结论氨溴索可以降低铜绿假单胞菌BF藻酸盐的含量,影响藻酸盐合成过程中重要基因algD、algU、algR和mucA的mRNA的表达,降低藻酸盐合成限速酶GMD活性,但对藻酸盐的降解无影响。     

Potential importance of protozoan grazing on the accumulation of polychlorinated biphenyls (PCBs) in the pelagic food web

Experiments were conducted to study the distribution of three selectedpolychlorinated biphenyl (PCB) congeners within the microbial food web attwo different nutrient levels; control and nutrient enriched. The objectivewas to quantify the uptake of PCBs through grazing by protozoa. The¹⁴C-PCBs tested were 4-chlorobiphenyl (IUPAC # 3),2,2,5,5-tetrachlorobiphenyl (IUPAC # 52), and2,2,4,4,5,5-hexachlorobiphenyl (IUPAC # 153). EachPCB was incubated in triplicate seawater samples at 20 idref;Cover one week. Daily, samples were separated into four fractions; <0.2µm (dissolved), 0.2-2 µm (bacteria), 2-10 µm(flagellate), and > 10 µm (microplankton; phytoplankton andprotozoa) by selective filtration. Of the PCB fraction that initiallyadsorbed to particles, 60–100% was associated to the bacterialfraction and 0–5% to the microplankton fraction. The totaluptake was highest in the nutrient enriched samples, but when normalized tothe carbon biomass the concentration was lower or equal to the control inall particle fractions. The recovery of the PCBs in the particulatefractions depended on the degree of chlorination, as the highest values wereobserved for the 2,2,4,4,5,5-hexachlorobiphenyl and thelowest for the 4-chlorobiphenyl. The concentrations in the bacterial andflagellate fractions decreased over the first 48–96 hours whilst theconcentration increased in the highest trophic level (>10 µmfraction). Approximately 75% of the increase in concentration of the2,2,4,4,5,5-hexachlorobiphenyl in the > 10 µmfraction was estimated to be the result of bacterivory. Our results indicatethe microbial food web can contribute to a rapid uptake of higherchlorinated PCBs, particularly in oligotrophic ecosystems where thebacterial biomass dominates.     

The effect of shear on the desorption of liposomes adsorbed to bacterial biofilms

With the aid of a flow cell assembly the desorption of cationic liposomes prepared from mixtures of dipalmitoylphoshatidylcholine (DDPC), cholesterol, and either dimethyldioctadecylammonium bromide (DDAB) or 3,beta[N-(N1,N-dimethylethylenediamine)-carbamoyl]cholesterol (DC-chol) from immobilized biofilms of Staphylococcus aureus has been studied as a function of shear stress by confocal microscopy. A shear stress theory has been adapted from fluid mechanics of laminar flow between parallel plates and used to determine the critical shear stress for liposome desorption. The critical shear stress for both DDAB and DC-chol liposomes has been determined as a function of cationic lipid content and hence surface charge as reflected in their zeta potentials. The critical shear stress has been used to obtain the potential energy of liposome-biofilm interaction which together with the electrostatic interaction energy has enabled estimates of the London-Hamaker constants to be made. The values of the London-Hamaker constants at small liposome-bacterial cell separation were found to be independent of liposome composition.     

Nitrification in activated sludge batch reactors is linked to protozoan grazing of the bacterial population

Protozoa feed upon free-swimming bacteria and suspended particles inducing flocculation and increasing the turnover rate of nutrients in complex mixed communities. In this study, the effect of protozoan grazing on nitrification was examined in activated sludge in batch cultures maintained over a 14-day period. A reduction in the protozoan grazing pressure was accomplished by using either a dilution series or the protozoan inhibitor cycloheximide. As the dilutions increased, the nitrification rate showed a decline, suggesting that a reduction in protozoan or bacterial concentration may cause a decrease in nitrification potential. In the presence of cycloheximide, where the bacterial concentration was not altered, the rates of production of ammonia, nitrite, and nitrate all were significantly lower in the absence of active protozoans. These results suggest that a reduction in the number or activity of the protozoans reduces nitrification, possibly by limiting the availability of nutrients for slow-growing ammonia and nitrite oxidizers through excretion products. Furthermore, the ability of protozoans to groom the heterotrophic bacterial population in such systems may also play a role in reducing interspecies competition for nitrification substrates and thereby augment nitrification rates.     

The role of quorum sensing mediated developmental traits in the resistance of Serratia marcescens biofilms against protozoan grazing

Resistance against protozoan grazers is a crucial factor that is important for the survival of many bacteria in their natural environment. However, the basis of resistance to protozoans and how resistance factors are regulated is poorly understood. In part, resistance may be due to biofilm formation, which is known to protect bacteria from environmental stress conditions. The ubiquitous organism Serratia marcescens uses quorum sensing (QS) control to regulate virulence factor expression and biofilm formation. We hypothesized that the QS system of S. marcescens also regulates mechanisms that protect biofilms against protozoan grazing. To investigate this hypothesis, we compared the interactions of wild-type and QS mutant strains of S. marcescens biofilms with two protozoans having different feeding types under batch and flow conditions. Under batch conditions, S. marcescens forms microcolony biofilms, and filamentous biofilms are formed under flow conditions. The microcolony-type biofilms were protected from grazing by the suspension feeder, flagellate Bodo saltans, but were not protected from the surface feeder, Acanthamoeba polyphaga. In contrast, the filamentous biofilm provided protection against A. polyphaga. The main findings presented in this study suggest that (i) the QS system is not involved in grazing resistance of S. marcescens microcolony-type biofilms; (ii) QS in S. marcescens regulates antiprotozoan factor(s) that do not interfere with the grazing efficiency of the protozoans; and (iii) QS-controlled, biofilm-specific differentiation of filaments and cell chains in biofilms of S. marcescens provides an efficient mechanism against protozoan grazing.     

Contribution of virus-induced lysis and protozoan grazing to benthic bacterial mortality estimated simultaneously in microcosms

In contrast to the water column, the fate of bacterial production in freshwater sediments is still a matter of debate. Thus, the importance of virus-induced lysis and protozoan grazing of bacteria was investigated for the first time simultaneously in a silty sediment layer of a mesotrophic oxbow lake. Microcosms were installed in the laboratory in order to study the dynamics of these processes over 15 days. All microbial and physicochemical parameters showed acceptable resemblance to field data observed during a concomitant in situ study, and similar conclusions can be drawn with respect to the quantitative impact of viruses and protozoa on the bacterial compartment. Viral decay rates ranged from undetectable to 0.078 h⁻¹ (average, 0.033 h⁻¹), and the control of bacterial production from below the detection limit to 36% (average, 12%). The contribution of virus-induced lysis of bacteria to the dissolved organic matter pool as well as to benthic bacterial nutrition was low. Ingestion rates of protozoan grazers ranged from undetectable to 24.7 bacteria per heterotrophic nanoflagellate (HNF) per hour (average, 4.8 bacteria HNF⁻¹ h⁻¹) and from undetectable to 73.3 bacteria per ciliate per hour (average, 11.2 bacteria ciliate⁻¹ h⁻¹). Heterotrophic nanoflagellate and ciliates together cropped up to 5% (average, 1%) of bacterial production. The viral impact on bacteria prevailed over protozoan grazing by a factor of 2.5–19.9 (average, 9.5). In sum, these factors together removed up to 36% (average, 12%) of bacterial production. The high number of correlations between viral and protozoan parameters is discussed in view of a possible relationship between virus removal and the presence of protozoan grazers.     

Impact of protozoan grazing on nitrification and the ammonia- and nitrite-oxidizing bacterial communities in activated sludge

In activated sludge, protozoa feed on free-swimming bacteria and suspended particles, inducing flocculation and increasing the turnover rate of nutrients. In this study, the effect of protozoan grazing on nitrification rates under various conditions in municipal activated sludge batch reactors was examined, as was the spatial distribution of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) within the activated sludge. The reactors were monitored for ammonia, nitrite, nitrate, and total nitrogen concentrations, and bacterial numbers in the presence and absence of cycloheximide (a protozoan inhibitor), allylthiourea (an inhibitor of ammonia oxidation), and EDTA (a deflocculating agent). The accumulations of nitrate, nitrite, and ammonia were lower in batches without than with protozoa grazing. Inhibition of ammonia oxidation also decreased the amount of nitrite and nitrate accumulation. Inhibiting protozoan grazing along with ammonia oxidation further decreased the amounts of nitrite and nitrate accumulated. Induction of deflocculation led to high nitrate accumulation, indicating high levels of nitrification; this effect was lessened in the absence of protozoan grazing. Using fluorescent in situ hybridization and confocal laser scanning microscopy, AOB and NOB were found clustered within the floc, and inhibiting the protozoa, inhibiting ammonia oxidation, or inducing flocculation did not appear to lower the number of AOB and NOB present or affect their position within the floc. These results suggest that the AOB and NOB are present but less active in the absence of protozoa.     

A study of the efficacy of ultrasonic waves in removing biofilms

doi:10.1111/j.1741‐2358.2009.00325.x
A study of the efficacy of ultrasonic waves in removing biofilms Objective: The removal of adherent biofilms was assessed using ultrasonic waves in a non‐contact mode. Materials and Methods: In in vitro experiments, Streptococcus mutans (S. mutans) biofilms were exposed to ultrasonic waves at various frequencies (280 kHz, 1 MHz, or 2 MHz), duty ratios (0–90%), and exposure times (1–3 minutes), and the optimal conditions for biofilm removal were identified. Furthermore, the effect of adding a contrast medium, such as micro bubbles (Sonazoid^®), was examined. The spatial distribution and architecture of S. mutans biofilms before and after ultrasonic wave exposure were examined via scanning electron microscopy. The biofilm removal effect was also examined in in vivo experiments, using a custom‐made oral cleaning device. Results: When a 280 kHz probe was used, the biofilm‐removing effect increased significantly compared to 1 and 2 MHz probes; more than 80% of the adherent biofilm was removed with a duty cycle of 50–90% and a 3 minutes exposure time. The maximum biofilm‐removing effect was observed with a duty cycle of 80%. Furthermore, the addition of micro bubbles enhanced this biofilm‐removing effect. In in vivo experiments, moderate biofilm removal was observed when a 280 kHz probe was used for 5 minutes. Conclusions: This study demonstrated that ultrasonic wave exposure in a non‐contact mode effectively removed adherent biofilms composed of S. mutans in vitro.     

The interaction of phage and biofilms

Biofilms present complex assemblies of micro-organisms attached to surfaces. they are dynamic structures in which various metabolic activities and interactions between the component cells occur. When phage come in contact with biofilms, further interactions occur dependent on the susceptibility of the biofilm bacteria to phage and to the availability of receptor sites. If the phage also possess polysaccharide-degrading enzymes, or if considerable cell lysis is effected by the phage, the integrity of the biofilm may rapidly be destroyed. Alternatively, coexistence between phage and host bacteria within the biofilm may develop. Although phage have been proposed as a means of destroying or controlling biofilms, the technology for this has not yet been successfully developed.     

Comparative analysis of hydrogen-producing bacterial biofilms and granular sludge formed in continuous cultures of fermentative bacteria

A system for biohydrogen production was developed based on long-term continuous cultures grown on sugar beet molasses in packed bed reactors. In two separate cultures, consortia of fermentative bacteria developed as biofilms on granitic stones. In one of the cultures, a granular sludge was also formed. Metagenomic analysis of the microbial communities by 454-pyrosequencing of amplified 16S rDNA fragments revealed that the overall biodiversity of the hydrogen-producing cultures was quite small. The stone biofilm from the culture without granular sludge was dominated by Clostridiaceae and heterolactic fermentation bacteria, mainly Leuconostocaeae. Representatives of the Leuconostocaeae and Enterobacteriaceae were dominant in both the granules and the stone biofilm formed in the granular sludge culture. The culture containing granular sludge produced hydrogen significantly more effectively than that containing only the stone biofilm: 5.43 vs. 2.8 mol H₂/mol sucrose from molasses, respectively. The speculations that lactic acid bacteria may favor hydrogen production are discussed.     

The effect of bacterial and diatom biofilms on the settlement of the bryozoan Bugula neritina

Biofilms of marine bacteria and diatoms and their combinations were examined in laboratory choice assays to determine their effects on the attachment and successful metamorphosis of the larvae of the bryozoan Bugula neritina (Linnéus). The larval settlement in response to unfilmed surfaces, a natural biofilm (NBF) and adsorbed cells of three strains of bacteria, five strains of pennate diatoms and combinations of the two at different densities. Bacterial and diatom strains showed different effects on the larval settlement of B. neritina. Bacterial monospecific strains of an unidentified α-Proteobacterium and Vibrio sp. mediated the same percentage of settlement as a filtered seawater control. Biofilms of Pseudoalteromonas sp. caused significantly lower larval settlement. Larval settlement of B. neritina was negatively correlated with increasing densities of Pseudoalteromonas sp. The highest percentages of settlement were mediated by the biofilms of the diatom species Achnanthes sp., Amphora cofeaeformis, Amphora tenerrima, Nitzschia constricta and a 5-day-old natural biofilm, while the lowest settlement was found on a N. frustulum film. A three-way analysis of variance demonstrated that the density of bacteria and the presence of particular species of diatoms and bacteria in combined biofilms, significantly affected the settlement of B. neritina larvae. High settlement of larvae (50-90%) at all treatments indicated that B. neritina larvae are much more indiscriminate settlers than previously expected. Hence, using this species as a monitoring organism to trace ecologically relevant subtle changes of settlement cues in the natural environment should be carefully re-examined.     

靛玉红、蛇床子素抗外阴阴道念珠菌病混合菌生物膜作用的研究

目的研究中药有效成分靛玉红、蛇床子素抗外阴阴道念珠菌病混合菌生物膜的作用。方法体外建立白念珠菌（Candidaalbicans）铜绿假单胞菌（Pseudomonasaeruginosa,P．a）混合菌生物膜（Biofilm,BF）,XTT减低法及形态学观察白念珠菌混合茵生物膜的形成过程;形态学观察、活菌计数法评价中药有效成分靛玉红（indirubin）、蛇床子素（Ostho）对白念珠菌混合菌生物膜的最小抑膜浓度（SMIC）,并经扫描电镜确认。结果白念珠菌混合菌48h能形成成熟的生物膜;62．5mg／L浓度的靛玉红能抑制白念珠菌混合菌生物膜的形成。500mg／L浓度的蛇床子素未见有抑制白念珠菌混合菌生物膜的作用。结论靛玉红由于具有抗生物膜的作用,可用于预防外阴阴道念珠菌病的复发。     

Estimation of amoeba cell volume from nuclear diameter and its application to studies in protozoan ecology

To facilitate the estimation of cell volume in uninucleate, naked amoebae (gymnamoebae) the relationship, log cell volume (µm³) = 0.882 + 3.117log nuclear diameter (µm³), is presented. This links mean cell volume to mean nuclear diameter and provides a useful tool for protozoan ecologists interested in estimating the biovolume of amoebae in laboratory or field samples. While it is virtually impossible to measure rigid axes from which volume can be calculated in these amorphous cells, it is relatively easy to measure the diameter of the nucleus in living or fixed material. This relationship has shown that most uninucleate amoebae surveyed have volumes ranging between only 188 µm³ and 2860 µm³; this range reflects the volumes of the majority of amoebae in the field. These small volumes are unexpected since many amoebae have locomotive forms greater than 20 µm in length giving the impression that their cell volumes should be correspondingly large. This is not the case, however, because most amoebae are extremely flat when viewed in profile. The small cell volume of most amoeba species has ecological implications when numerical data is transformed to biovolume and biomass units.     

Effects of flow rate and substrate concentration on the formation and H2 production of photosynthetic bacterial biofilms

Photosynthetic bacteria (PSB), Rhodopseudomonas palustris CQK 01, were immobilized on the surface of a thin glass slide in a lab-scale flat panel photobioreactor under different flow rates and substrate concentrations. The morphology, dry weight and thickness of the mature PSB biofilms were determined to reveal the relationship between biofilm formation and hydrogen production performance. The mature biofilm formed at a low flow rate and a high substrate concentration showed a looser structure, these structures of the mature biofilm then affected the H₂ production performance of the bioreactor during mature stage. The biofilm formed at a flow rate of 228 ml/h and a substrate concentration of 60 mmol/l exhibited the highest dry weight and optimally porous structure, which is beneficial not only for hydrogen removal from the biofilm but also glucose diffusion into the biofilm, thus significantly boosting the photo-hydrogen production performance.     

Limitations on the use of immobilized bacterial biofilms for enzymically-mediated uranyl ion accumulation

Summary A phosphatase-overproducing mutant of a Citrobacter sp. was evaluated in terms of its ability to self-immobilize as a biofilm on reticulated foam supports and to accumulate uranium when retained in a flow-through column. Parallel studies using polyacrylamide gel-immobilized cells suggested decreased substrate was available to cells of the mutant.