FluidFM as a tool to study adhesion forces of bacteria - Optimization of parameters and comparison to conventional bacterial probe Scanning Force Spectroscopy.

The FluidFM enables the immobilization of single cells on a hollow cantilever using relative underpressure. In this study, we systematically optimize versatile measurement parameters (setpoint, z-speed, z-length, pause time, and relative underpressure) to improve the quality of force-distance curves recorded with a FluidFM. Using single bacterial cells (here the gram negative seawater bacterium Paracoccus seriniphilus and the gram positive bacterium Lactococcus lactis), we show that Single Cell Force Spectroscopy experiments with the FluidFM lead to comparable results to a conventional Single Cell Force Spectroscopy approach using polydopamine for chemical fixation of a bacterial cell on a tipless cantilever. Even for the bacterium Lactococcus lactis, which is difficult to immobilze chemically (like seen in an earlier study), immobilization and the measurement of force-distance curves are possible by using the FluidFM technology.

Endomicrobial Community Profiles of Two Different Mealybugs: Paracoccus marginatus and Ferrisia virgata.

Mealybugs (Hemiptera: Coccomorpha: Pseudococcidae) harbour diverse microbial symbionts that play essential roles in host physiology, ecology, and evolution. In this study we aimed to reveal microbial communities associated with two different mealybugs, papaya mealybug (Paracoccus marginatus) and two-tailed mealybug (Ferrisia virgata) collected from the same host plant. Comparative analysis of microbial communities associated with these mealybugs revealed differences that appear to stem from phylogenetic associations and different nutritional requirements. This first report on both bacterial and fungal communities associated with these mealybugs provides a preliminary insight on factors affecting the endomicrobial communities. .

Paracoccus jeotgali sp. nov., isolated from Korean salted and fermented shrimp.

A Gram-stain-negative and facultatively aerobic bacterium, designated as strain CBA4604T, was isolated from a traditional Korean salted and fermented shrimp food (saeu-jeot). Phylogenetic analysis based on 16S rRNA gene sequences showed that strain CBA4604T formed a clearly distinct phyletic lineage from closely related species within the genus Paracoccus. Strain CBA4604T was the most closely related to P. koreensis Ch05T (97.5% 16S rRNA gene sequence similarity) and other type strains (≤ 97.0%). The genome comprised a chromosome and two plasmids of 3,299,166 bp with 66.5% G+C content. The DNA-DNA relatedness values between strain CBA4604T and P. koreensis Ch05T, P. alcaliphilus DSM 8512T, and P. stylophorae KTW-16T were 30.5%, 22.9%, and 16.7%, respectively. Cells of the strain were short rod-shaped and oxidase- and catalase-positive. The growth of strain CBA-4604T was observed at 10-40°C (optimum, 37°C), pH 6.0-10.0 (optimum, pH 7.0), and in the presence of 0-8.0% (w/v) NaCl (optimum, 0-2.0%). Strain CBA4604T contained ubiquinone 10 as the sole isoprenoid quinone and summed feature 8 (C18:1ω7c/C18:1ω6c) and C18:0 as the major cellular fatty acids. The polar lipids consisted of phosphatidylcholine, phosphatidylglycerol, diphosphatidylglycerol, phospholipid, an unidentified aminolipid, an unidentified glycolipid, and three unidentified lipids. Based on its phylogenetic, genomic, phenotypic, and chemotaxonomic features, we concluded that strain CBA-4604T represents a novel species in the genus Paracoccus and we propose the name Paracoccus jeotgali sp. nov. The type strain is CBA4604T (= KACC 19579T = JCM 32510T).

Paracoccus seriniphilus adhered on surfaces: Resistance of a seawater bacterium against shear forces under the influence of roughness, surface energy, and zeta potential of the surfaces.

Bacteria in flowing media are exposed to shear forces exerted by the fluid. Before a biofilm can be formed, the bacteria have to attach to a solid surface and have to resist these shear forces. Here, the authors determined dislodgement forces of single Paracoccus seriniphilus bacteria by means of lateral force microscopy. The first measurement set was performed on very flat glass and titanium (both as very hydrophilic samples with water contact angles below 20°) as well as highly oriented pyrolytic graphite (HOPG) and steel surfaces (both as more hydrophobic surfaces in the context of biological interaction with water contact angles above 50°). The different surfaces also show different zeta potentials in the range between -18 and -108 mV at the measurement pH of 7. The second set comprised titanium with different RMS (root mean square) roughness values from a few nanometers up to 22 nm. Lateral forces between 0.5 and 3 nN were applied. For Paracoccus seriniphilus, the authors found as a general trend that the surface energy of the substrate at comparable roughness determines the detachment process. The surface energy is inversely proportional to the initial adhesion forces of the bacterium with the surface. The higher the surface energy (and the lower the initial adhesion force) is, the easier the dislodgement of the bacteria happens. In contrast, electrostatics play only a secondary role in the lateral dislodgement of the bacteria and may come only into play if surface energies are the same. Furthermore, the surface chemistry (glass, titanium, and steel as oxidic surfaces and HOPG as a nonoxidic surface) seems to play an important role because HOPG does not completely follow the above mentioned general trend found for the oxide covered surfaces. In addition, the roughness of the substrates (made of the same material) is limiting the lateral dislodgement of the bacteria. All examined structures with RMS roughness of about 8-22 nm on titanium prevent the bacteria from the lateral dislodgement compared to polished titanium with an RMS roughness of about 3 nm.

Algicidal Activity of Novel Marine Bacterium Paracoccus sp. Strain Y42 against a Harmful Algal-Bloom-Causing Dinoflagellate, Prorocentrum donghaiense.

Prorocentrum donghaiense blooms occur frequently in the Yangtze River estuary and the adjacent East China Sea. These blooms have damaged marine ecosystems and caused enormous economic losses over the past 2 decades. Thus, highly efficient, low-cost, ecofriendly approaches must be developed to control P. donghaiense blooms. In this study, a bacterial strain (strain Y42) was identified as Paracoccus sp. and was used to lyse P. donghaiense The supernatant of the strain Y42 culture was able to lyse P. donghaiense, and the algicidal activity of this Y42 supernatant was stable with different temperatures and durations of light exposure and over a wide pH range. In addition to P. donghaiense, Y42 showed high algicidal activity against Alexandrium minutum, Scrippsiella trochoidea, and Skeletonema costatum, suggesting that it targets primarily Pyrrophyta. To clarify the algicidal effects of Y42, we assessed algal lysis and determined the chlorophyll a contents, photosynthetic activity, and malondialdehyde contents of P. donghaiense after exposure to the Y42 supernatant. Scanning electron microscopy and transmission electron microscopy analyses showed that the Y42 supernatant disrupted membrane integrity and caused algal cell breakage at the megacytic zone. Photosynthetic pigment loss and significant declines in both photosynthetic efficiency and the electron transport rate indicated that the Y42 supernatant damaged the photosynthetic system of P. donghaiense Malondialdehyde overproduction indicated that the Y42 supernatant caused lipid peroxidation and oxidative damage to membrane systems in the algal cell, ultimately leading to death. The findings of this study reveal the potential of Y42 to remove algal cells from P. donghaiense blooms.IMPORTANCEP. donghaiense is one of the most common dinoflagellate species that form harmful algal blooms, which frequently cause serious ecological pollution and pose health hazards to humans and other animals. Screening for bacteria with high algicidal activity against P. donghaiense and studying their algicidal processes and characteristics will contribute to an understanding of their algicidal effects and provide a theoretical basis for preventing algal blooms and reducing their harm to the environment. This study reports the algicidal activity and characteristics of Paracoccus against P. donghaiense The stability of the algicidal activity of Paracoccus in different environments (including different temperature, pH, and sunlight conditions) indicates its potential for use in the control of P. donghaiense blooms.

Adhesion forces of the sea-water bacterium Paracoccus seriniphilus on titanium: Influence of microstructures and environmental conditions.

The bacterial attachment to surfaces is the first step of biofilm formation. This attachment is governed by adhesion forces which act between the bacterium and the substrate. Such forces can be measured by single cell force spectroscopy, where a single bacterium is attached to a cantilever of a scanning force microscope, and force-distance curves are measured. For the productive sea-water bacterium Paracoccus seriniphilus, pH dependent measurements reveal the highest adhesion forces at pH 4. Adhesion forces measured at salinities between 0% and 4.5% NaCl are in general higher for higher salinity. However, there is an exception for 0.9% where a higher adhesion force was measured than expected. These results are in line with zeta potential measurements of the bacterium, which also show an exceptionally low zeta potential at 0.9% NaCl. In the absence of macromolecular interactions, the adhesion forces are thus governed by (unspecific) electrostatic interactions, which can be adjusted by pH and ionic strength. It is further shown that microstructures on the titanium surface increase the adhesion force. Growth medium reduces the interaction forces dramatically, most probably through macromolecular bridging.

Molecular ecological network analysis reveals the effects of probiotics and florfenicol on intestinal microbiota homeostasis: An example of sea cucumber.

Animal gut harbors diverse microbes that play crucial roles in the nutrition uptake, metabolism, and the regulation of host immune responses. The intestinal microbiota homeostasis is critical for health but poorly understood. Probiotics Paracoccus marcusii DB11 and Bacillus cereus G19, and antibiotics florfenicol did not significantly impact species richness and the diversity of intestinal microbiota of sea cucumber, in comparison with those in the control group by high-throughput sequencing. Molecular ecological network analysis indicated that P. marcusii DB11 supplementation may lead to sub-module integration and the formation of a large, new sub-module, and enhance species-species interactions and connecter and module hub numbers. B. cereus G19 supplementation decreased sub-module numbers, and increased the number of species-species interactions and module hubs. Sea cucumber treated with florfenicol were shown to have only one connecter and the lowest number of operational taxonomic units (OTUs) and species-species interactions within the ecological network. These results suggested that P. marcusii DB11 or B. cereus G19 may promote intestinal microbiota homeostasis by improving modularity, enhancing species-species interactions and increasing the number of connecters and/or module hubs within the network. In contrast, the use of florfenicol can lead to homeostatic collapse through the deterioration of the ecological network.

Removing biofilms from stainless steel without changing surface properties relevant for bacterial attachment.

The influence of oxygen (and argon) plasma cleaning and a base-acid cleaning procedure on stainless steel surfaces was studied. The main aim was to clean stainless steel samples from Paracoccus seriniphilus biofilms without changing the surface properties which are relevant for bacterial attachment to allow reuse in a biofilm reactor. It is shown that oxygen plasma cleaning, which very successfully removes the same kind of biofilm from titanium surfaces, is not suitable for stainless steel. It largely influences the surface chemistry by producing thick metal oxide layers of varying compositions and changing phenomenological surface properties such as wettability. A promising method without changing surface properties while cleaning satisfactorily is a combination of base and acid reagents at elevated temperature.

Membrane vesicle-mediated bacterial communication.

The classical quorum-sensing (QS) model is based on the assumption that diffusible signaling molecules accumulate in the culture medium until they reach a critical concentration upon which expression of target genes is triggered. Here we demonstrate that the hydrophobic signal N-hexadecanoyl-L-homoserine lactone, which is produced by Paracoccus sp., is released from cells by the aid of membrane vesicles (MVs). Packed into MVs, the signal is not only solubilized in an aqueous environment but is also delivered with varying propensities to different bacteria. We propose a novel MV-based mechanism for binary trafficking of hydrophobic signal molecules, which may be particularly relevant for bacteria that live in open aqueous environments.

Screening of bromotyramine analogues as antifouling compounds against marine bacteria.

Rapid and efficient synthesis of 23 analogues inspired by bromotyramine derivatives, marine natural products, by means of CuSO4-catalysed [3+2] alkyne-azide cycloaddition is described. The final target was then assayed for anti-biofilm activity against three Gram-negative marine bacteria, Pseudoalteromonas ulvae (TC14), Pseudoalteromonas lipolytica (TC8) and Paracoccus sp. (4M6). Most of the synthesised bromotyramine/triazole derivatives are more active than the parent natural products Moloka'iamine (A) and 3,5-dibromo-4-methoxy-ß-phenethylamine (B) against biofilm formation by the three bacterial strains. Some of these compounds were shown to act as non-toxic inhibitors of biofilm development with EC50 < 200 µM without any effect on bacterial growth even at high concentrations (200 µM).

Paracoccus aquimaris sp. nov., isolated from seawater.

A Gram-stain negative, motile and short rod-shaped bacterial strain, designated KF89(T), was isolated from seawater of Tong-Yeong, South of Korea. Growth occurred between 10 and 35 °C (optimum 32 °C) and at pH 6.0-9.0 (optimum pH 7.0) and at 0-7 % NaCl (optimum at 2 % NaCl). The 16S rRNA gene sequence identified it as a member of the genus Paracoccus that belongs to the phylum Proteobacteria. Based on the 16S rRNA gene sequence, the highest degree of gene sequence similarities were shared with Paracoccus homiensis DD-R11(T) (97.3 %), P. zeaxanthinifaciens ATCC21588(T) (97.1 %), P. rhizosphaerae CC-CCM15-8(T) (96.9 %), P. beibuensis JLT1284 (96.9 %) and P. aestuarii B7(T) (96.6 %). Strain KF89(T) contained Ubiquinone-10 as the major respiratory quinone, and the polar lipid profile included phosphatidylglycerol, phosphatidylcholine, diphosphatidylglycerol and an unidentified aminolipid. The major fatty acid of strain KF89(T) is C18:1 ω7c and the DNA G+C content is 57 mol%. Strain KF89(T) showed a DNA-DNA relatedness with P. homiensis KACC 11518(T) of 49 %. Based on phylogenetic and phenotypic analyses, strain KF89(T) is clearly shown to be a novel member of the genus Paracoccus. The type strain is Paracoccus aquimaris KF89(T) (=KEMB 3-508(T) = JCM 19892(T)).

Effects of dietary Bacillus cereus G19, B. cereus BC-01, and Paracoccus marcusii DB11 supplementation on the growth, immune response, and expression of immune-related genes in coelomocytes and intestine of the sea cucumber (Apostichopus japonicus Selenka).

Probiotics have positive effects on the nutrient digestibility and absorption, immune responses, and growth of aquatic animals, including the sea cucumber (Apostichopus japonicus Selenka). A 60-day feeding trial was conducted to evaluate the effects of Bacillus cereus G19, B. cereus BC-01 and Paracoccus marcusii DB11 supplementation on the growth, immune response, and expression level of four immune-related genes (Aj-p105, Aj-p50, Aj-rel, and Aj-lys) in coelomocytes and the intestine of juvenile sea cucumbers. One group was fed the basal diet (control group), while three other groups were fed the basal diet supplemented with B. cereus G19 (G19 group), B. cereus BC-01 (BC group), or P. marcusii DB11 (PM group). The growth rate of sea cucumbers fed diets with probiotics supplementation was significantly higher than that of the control group (P < 0.05). Sea cucumbers in the G19 and PM groups had a significantly greater phagocytic activity of coelomocytes compared to the control group (P < 0.05), while those in the G19 and BC groups had a greater respiratory burst activity (P < 0.05). The alkaline phosphatase (AKP) activity of coelomocytes in sea cucumbers fed diets with probiotics supplementation was significantly higher than the control group (P < 0.05). Comparatively, superoxide dismutase (SOD) activity of coelomocytes for sea cucumber in the PM group was significantly greater (P < 0.05). As for the immune-related genes, B. cereus G19 supplementation significantly increased the expression level of the Aj-rel gene in coelomocytes (P < 0.05), while B. cereus BC-01 supplementation significantly increased that of the Aj-p50 gene as compared to the control group (P < 0.05). In the intestine, the relative expression level of Aj-p105, Aj-p50, and Aj-lys genes in the PM group was significantly higher than that in the control group (P < 0.05). These results suggested that B. cereus G19 and B. cereus BC-01 supplementation could improve the growth performance and the immune response in coelomocytes, while P. marcusii DB11 supplementation could have a positive effect on the growth performance and immune response in coelomocytes and the intestine of sea cucumbers.

Inhibition or promotion of biodegradation of nitrate by Paracoccus sp. in the presence of nanoscale zero-valent iron.

To investigate the effect of nanoscale zero-valent iron (nZVI) on the growth of Paracoccus sp. strain and biodenitrification under aerobic conditions, specific factors were studied, pH, concentration of nitrate, Fe (II) and carbon dioxide. Low concentration of nZVI (50mg/L) promoted both cell growth and biodegradation of nitrate which rose from 69.91% to 76.16%, while nitrate removal fell to 67.10% in the presence of high nZVI concentration (1000 mg/L). This may be attributed to the ions produced in nZVI corrosion being used as an electron source for the biodegradation of nitrate. However, the excess uptake of Fe (II) causes oxidative damage to the cells. To confirm this, nitrate was completely removed after 20 h when 100mg/L Fe (II) was added to the solution, which is much faster than the control (86.05%, without adding Fe (II)). However, nitrate removal reached only 45.64% after 20 h, with low cell density (OD 600=0.62) in the presence of 300 mg/L Fe (II). Characterization techniques indicated that nZVI adhered to microorganism cell membranes. These findings confirmed that nZVI could affect the activity of the strain and consequently change the biodenitrification.

Nitrate levels modulate the abundance of Paracoccus sp. in a biofilm community.

Conditions required to enhance a particular species efficient in degradative capabilities is very useful in wastewater treatment processes. Paracoccus sp. is known to efficiently reduce nitrogen oxides (NOx) due to the branched denitrification pathway. Individual-based simulations showed that the relative fitness of Paracoccus sp. to Pseudomonas sp. increased significantly with nitrate levels above 5 mM. Spatial structure of the biofilm showed substantially less nitrite levels in the areas of Paracoccus sp. dominance. The simulation was validated in a laboratory reactor harboring biofilm community by fluorescent in situ hybridization, which showed that increasing nitrate levels enhanced the abundance of Paracoccus sp. Different levels of NOx did not display any significant effect on biofilm formation of Paracoccus sp., unlike several other bacteria. This study shows that the attribute of Paracoccus sp. to tolerate and efficiently reduce NOx is conferring a fitness payoff to the organism at high concentrations of nitrate in a multispecies biofilm community.

Nanomechanical properties of the sea-water bacterium Paracoccus seriniphilus--a scanning force microscopy approach.

The measurement of force-distance curves on a single bacterium provides a unique opportunity to detect properties such as the turgor pressure under various environmental conditions. Marine bacteria are very interesting candidates for the production of pharmaceuticals, but are only little studied so far. Therefore, the elastic behavior of Paracoccus seriniphilus, an enzyme producing marine organism, is presented in this study. After a careful evaluation of the optimal measurement conditions, the spring constant and the turgor pressure are determined as a function of ionic strength and pH. Whereas the ionic strength changes the turgor pressure passively, the results give a hint that the change to acidic pH increases the turgor pressure by an active mechanism. Furthermore, it could be shown, that P. seriniphilus has adhesive protrusions outside its cell wall.

Cleaning of titanium substrates after application in a bioreactor.

Plain and microstructured cp-titanium samples were studied as possible biofilm reactor substrates. The biofilms were grown by exposition of the titanium samples to bacteria in a flow cell. As bacteria the rod shaped gram negative Pseudomonas fluorescens and the spherical gram negative Paracoccus seriniphilus were chosen. Afterward, the samples were cleaned in subsequent steps: First, with a standard solvent based cleaning procedure with acetone, isopropanol, and ultrapure water and second by oxygen plasma sputtering. It will be demonstrated by means of x-ray photoelectron spectroscopy, fluorescence microscopy, and confocal laser scanning microscopy that oxygen plasma cleaning is a necessary and reliant tool to fully clean and restore titanium surfaces contaminated with a biofilm. The microstructured surfaces act beneficial to biofilm growth, while still being fully restorable after biofilm contamination. Scanning electron microscopy images additionally show, that the plasma process does not affect the microstructures. The presented data show the importance of the cleaning procedure. Just using solvents does not remove the biofilm and all its components reliably while a cleaning process by oxygen plasma regenerates the surfaces.

Paracoccus pacificus sp. nov., isolated from the Western Pacific Ocean.

A Gram-stain negative, short rod-shaped, non-motile, catalase- and oxidase-positive, aerobic bacterium, designated F14(T), was isolated from the Western Pacific Ocean. Phylogenetic and phenotypic properties of the organism supported that it belongs to the genus Paracoccus. The levels of 16S rRNA gene sequences similarity between strain F14(T) and other type strains of recognized members of the genus Paracoccus were 93.6-96.5%. Growth of strain F14(T) was observed at 4-40 °C (optimum, 28-30 °C), pH 6.0-10.0 (optimum, pH 7.0-8.0) and in the presence of 0-7% (w/v) NaCl (optimum, 1-2%). The major cellular fatty acid was summed feature 8 (C(18:1)ω6c and/or C(18:1)ω7c). The major respiratory quinone was ubiquinone-10. The polar lipid pattern indicated the presence of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine and three unknown lipids. The DNA G+C content was 61.4 mol%. On the basis of polyphasic characterization, strain F14(T) represents a novel species, for which the name Paracoccus pacificus sp. nov. is proposed. The type strain is F14(T) (=CGMCC 1.12755(T)=LMG 28106(T)=MCCC 1A09947(T)).

Determining the ecological impacts of organic contaminants in biosolids using a high-throughput colorimetric denitrification assay: a case study with antimicrobial agents.

Land application accounts for ∼ 50% of wastewater solid disposal in the United States. Still, little is known regarding the ecological impacts of nonregulated contaminants found in biosolids. Because of the myriad of contaminants, there is a need for a rapid, high-throughput method to evaluate their ecotoxicity. Herein, we developed a novel assay that measures denitrification inhibition in a model denitrifier, Paracoccus denitrificans Pd1222. Two common (triclosan and triclocarban) and four emerging (2,4,5 trichlorophenol, 2-benzyl-4-chlorophenol, 2-chloro-4-phenylphenol, and bis(5-chloro-2-hydroxyphenyl)methane) antimicrobial agents found in biosolids were analyzed. Overall, the assay was reproducible and measured impacts on denitrification over 3 orders of magnitude exposure. The lowest observable adverse effect concentrations (LOAECs) were 1.04 µM for triclosan, 3.17 µM for triclocarban, 0.372 µM for bis-(5-chloro-2-hydroxyphenyl)methane, 4.89 µM for 2-chloro-4-phenyl phenol, 45.7 µM for 2-benzyl-4-chorophenol, and 50.6 µM for 2,4,5-trichlorophenol. Compared with gene expression and cell viability based methods, the denitrification assay was more sensitive and resulted in lower LOAECs. The increased sensitivity, low cost, and high-throughput adaptability make this method an attractive alternative for meeting the initial testing regulatory framework for the Federal Insecticide, Fungicide, and Rodenticide Act, and recommended for the Toxic Substances Control Act, in determining the ecotoxicity of biosolids-derived emerging contaminants.

Paracoccus siganidrum sp. nov., isolated from fish gastrointestinal tract.

A bacterial strain, designated M26(T), was isolated from a fish gastrointestinal tract, collected from Zhanjiang Port, South China. 16S rRNA gene sequence analysis indicated that strain M26(T) belongs to the subclass α-Proteobacteria, being related to the genus Paracoccus, and sharing highest sequence similarity with Paracoccus alcaliphilus JCM 7364(T) (98.1 %), Paracoccus huijuniae FLN-7(T) (97.3 %), Paracoccus stylophorae KTW-16(T) (97.1 %) and Paracoccus seriniphilus DSM 14827(T) (96.9 %). The major quinone was determined to be ubiquinone Q-10, with Q-9 and Q-8 as minor components. The major fatty acid was identified as C18:1ω7c, with smaller amounts of C18:0 and C16:0. The G+C content of the genomic DNA was determined to be 64.3 mol%. The DNA hybridization value between strain M26(T) and the most closely related type strain, P. alcaliphilus, was 29.0 ± 1.0 %. The results of physiological and biochemical tests and low DNA-DNA relatedness showed that the strain could be readily distinguished from closely related species. On the basis of these phenotypic and genotypic data, strain M26(T) is concluded to represent a novel species of the genus Paracoccus, for which the name Paracoccus siganidrum sp. nov. is proposed. The type strain is M26(T) (=CCTCC AB 2012865(T) = DSM 26381(T)).

Methyl-beta-cyclodextrin enhanced biodegradation of polycyclic aromatic hydrocarbons and associated microbial activity in contaminated soil.

The contamination of soils by polycyclic aromatic hydrocarbons (PAHs) is a widespread environmental problem and the remediation of PAHs from these areas has been a major concern. The effectiveness of many in situ bioremediation systems may be constrained by low contaminant bioavailability due to limited aqueous solubility or a large magnitude of sorption. The objective of this research was to evaluate the effect of methyl-beta-cyclodextrin (MCD) on bioaugmentation by Paracoccus sp. strain HPD-2 of an aged PAH-contaminated soil. When 10% (W/W) MCD amendment was combined with bioaugmentation by the PAH-degrading bacterium Paracoccus sp. strain HPD-2, the percentage degradation of total PAHs was significantly enhanced up to 34.8%. Higher counts of culturable PAH-degrading bacteria and higher soil dehydrogenase and soil polyphenol oxidase activities were observed in 10% (W/W) MCD-assisted bioaugmentation soil. This MCD-assisted bioaugmentation strategy showed significant increases (p < 0.05) in the average well color development (AWCD) obtained by the BIOLOG Eco plate assay, Shannon-Weaver index (H) and Simpson index (lambda) compared with the controls, implying that this strategy at least partially restored the microbiological functioning of the PAH-contaminated soil. The results suggest that MCD-aided bioaugmentation by Paracoccus sp. strain HPD-2 may be a promising practical bioremediation strategy for aged PAH-contaminated soils.