Ana R. Lopes

Wastewater reuse in irrigation: A microbiological perspective on implications in soil fertility and human and environmental health

The reuse of treated wastewater, in particular for irrigation, is an increasingly common practice, encouraged by governments and official entities worldwide. Irrigation with wastewater may have implications at two different levels: alter the physicochemical and microbiological properties of the soil and/or introduce and contribute to the accumulation of chemical and biological contaminants in soil. The first may affect soil productivity and fertility; the second may pose serious risks to the human and environmental health. The sustainable wastewater reuse in agriculture should prevent both types of effects, requiring a holistic and integrated risk assessment. In this article we critically review possible effects of irrigation with treated wastewater, with special emphasis on soil microbiota. The maintenance of a rich and diversified autochthonous soil microbiota and the use of treated wastewater with minimal levels of potential soil contaminants are proposed as sine qua non conditions to achieve a sustainable wastewater reuse for irrigation.

Acinetobacter rudis sp. nov., isolated from raw milk and raw wastewater.

Two bacterial strains, G30(T) and A1PC16, isolated respectively from raw milk and raw wastewater, were characterized using a polyphasic approach. Chemotaxonomic characterization supported the inclusion of these strains in the genus Acinetobacter, with Q-8 and Q-9 as the major respiratory quinones, genomic DNA G+C contents within the range observed for this genus (38-47 mol%) and C(16:0), C(18:1)ω9c and C(16:1)ω7c/iso-C(15:0) 2-OH as the predominant fatty acids. The observation of 16S rRNA gene sequence similarity lower than 97% with other Acinetobacter species with validly published names led to the hypothesis that these isolates could represent a novel species. This hypothesis was supported by comparative analysis of partial sequences of the genes rpoB and gyrB, which showed that strains G30(T) and A1PC16 did not cluster with any species with validly published names, forming a distinct lineage. DNA-DNA hybridizations confirmed that the two strains were members of the same species, which could be distinguished from their congeners by several phenotypic characteristics. On the basis of these arguments, it is proposed that strains G30(T) and A1PC16 represent a novel species, for which the name Acinetobacter rudis sp. nov. is proposed. The type strain is strain G30(T) (=LMG 26107(T) =CCUG 57889(T) =DSM 24031(T) =CECT 7818(T)).

Responses of the alga Pseudokirchneriella subcapitata to long-term exposure to metal stress

The green alga Pseudokirchneriella subcapitata has been widely used in ecological risk assessment, usually based on the impact of the toxicants in the alga growth. However, the physiological causes that lead algal growth inhibition are not completely understood. This work aimed to evaluate the biochemical and structural modifications in P. subcapitata after exposure, for 72 h, to three nominal concentrations of Cd(II), Cr(VI), Cu(II) and Zn(II), corresponding approximately to 72 h-EC10 and 72 h-EC50 values and a high concentration (above 72 h-EC90 values). The incubation of algal cells with the highest concentration of Cd(II), Cr(VI) or Cu(II) resulted in a loss of membrane integrity of ~16, 38 and 55%, respectively. For all metals tested, an inhibition of esterase activity, in a dose-dependent manner, was observed. Reduction of chlorophyll a content, decrease of maximum quantum yield of photosystem II and modification of mitochondrial membrane potential was also verified. In conclusion, the exposure of P. subcapitata to metals resulted in a perturbation of the cell physiological status. Principal component analysis revealed that the impairment of esterase activity combined with the reduction of chlorophyll a content were related with the inhibition of growth caused by a prolonged exposure to the heavy metals.

Bacillus purgationiresistans sp. nov., isolated from a drinking-water treatment plant

A Gram-positive, aerobic, non-motile, endospore-forming rod, designated DS22(T), was isolated from a drinking-water treatment plant. Cells were catalase- and oxidase-positive. Growth occurred at 15-37 °C, at pH 7-10 and with <8% (w/v) NaCl (optimum growth: 30 °C, pH 7-8 and 1-3% NaCl). The major respiratory quinone was menaquinone 7, the G+C content of the genomic DNA was 36.5 mol% and the cell wall contained meso-diaminopimelic acid. On the basis of 16S rRNA gene sequence analysis, strain DS22(T) was a member of the genus Bacillus. Its closest phylogenetic neighbours were Bacillus horneckiae NRRL B-59162(T) (98.5% 16S rRNA gene sequence similarity), Bacillus oceanisediminis H2(T) (97.9%), Bacillus infantis SMC 4352-1(T) (97.4%), Bacillus firmus IAM 12464(T) (96.8%) and Bacillus muralis LMG 20238(T) (96.8%). DNA-DNA hybridization, and biochemical and physiological characterization allowed the differentiation of strain DS22(T) from its closest phylogenetic neighbours. The data supports the proposal of a novel species, Bacillus purgationiresistans sp. nov.; the type strain is DS22(T) (=DSM 23494(T)=NRRL B-59432(T)=LMG 25783(T)).

Biological treatment of propanil and 3,4-dichloroaniline: Kinetic and microbiological characterisation

Propanil (3,4-dichloropropionanilide) is a widely used herbicide, applied worldwide in rice paddies. Propanil is primarily transformed in nature to 3,4-dichloroaniline (DCA), which is more slowly biodegradable. Both compounds have adverse health and ecotoxicity effects. This work investigated the microbial ecology and kinetics of propanil-degrading enrichments obtained from soil in a sequencing batch reactor (SBR) operated with different feeding strategies, aiming at the enhanced biological removal of propanil and DCA from contaminated waters. During SBR operation with a dump feeding strategy, a high propanil concentration led to DCA accumulation, which was only fully degraded after 5 days, likely due to DCA inhibition. For this reason, the operational mode was changed to fed-batch operation with lower initial propanil concentrations, which resulted in faster propanil and DCA biodegradation. Thus a fed-batch operation seems more appropriate for the acclimatisation of an effective propanil- and DCA-degrading population. The changes in performance were accompanied by a shift in the microbial population structure, as determined by DGGE of the 16S rRNA gene, particularly after a feed of DCA as the sole carbon source. Isolates obtained from the acclimatised population included members of the genera Enterococcus and Rhodococcus, as well as Brevundimonas, which displayed >90% propanil biodegradation efficiency.

Paenibacillus residui sp. nov., isolated from urban waste compost.

Two bacterial strains, MC-246(T) and MC-247, were isolated from municipal urban waste compost and characterized by a polyphasic approach. Both isolates were Gram-stain-variable, endospore-forming rods that were catalase-, oxidase- and β-galactosidase-positive, and able to grow at 25-50°C and pH 7.0-9.0, with optimum growth at 37°C and pH 7. The predominant cellular fatty acids were anteiso-C₁₅:₀, iso-C₁₅:₀, iso-C₁₆: ₀, anteiso-C₁₇:₀ and iso-C₁₇:₀; the major respiratory quinone was menaquinone MK-7; the cell wall peptidoglycan was of type A1γ; and the DNA G+C content was 49 mol%. These characteristics, as well as data from 16S RNA gene sequence analysis, showed that these strains were affiliated with the genus Paenibacillus; the type strains of Paenibacillus ginsengarvi and Paenibacillus hodogayensis were among their closest neighbours (< 94.2 % sequence similarity). Nevertheless, the hypothesis that strains MC-246(T) and MC-247 could represent a novel species was supported by the low 16S rRNA gene sequence similarity values shared with other members of the genus Paenibacillus and by the observation of distinct biochemical and physiological traits. Strains MC-246(T) and MC-247 shared 99.6 % 16S rRNA gene sequence similarity and showed almost identical MALDI-TOF mass spectra, but could be distinguished at the phenotypic and genotypic level. However, DNA-DNA hybridization between strains MC-246(T) and MC-247 resulted in values above 70 % indicating that both organisms represent a single species, for which the name Paenibacillus residui sp. nov. is proposed; the type strain is MC-246(T) (=DSM 22072(T) =CCUG 57263(T)).

Sphingobium vermicomposti sp. nov., isolated from vermicompost

Strain VC-230(T) was isolated from homemade vermicompost produced from kitchen waste. The isolate was a Gram-negative-staining, catalase- and oxidase-positive, motile rod-shaped bacterium able to grow at 15-37 degrees C and pH 6-8. On the basis of 16S rRNA gene sequence analysis, strain VC-230(T) was determined to belong to the family Sphingomonadaceae by its clustering with type strains of the genus Sphingobium, with Sphingobium chlorophenolicum ATCC 33790(T) (97.7 %) and Sphingobium herbicidovorans DSM 11019(T) (97.4 %) as its closest neighbours. The polar lipid pattern, the presence of spermidine and ubiquinone 10, the predominance of the cellular fatty acids C(18 : 1)omega7c/9t/12t, C(16 : 1)omega7c and C(16 : 0) and the G+C content of the genomic DNA supported the affiliation of this organism to the genus Sphingobium. The phylogenetic, chemotaxonomic, phenotypic and DNA-DNA hybridization analyses verify that strain VC-230(T) represents a novel species, for which the name Sphingobium vermicomposti sp. nov. is proposed. The type strain is VC-230(T) (=CCUG 55809(T) =DSM 21299(T)).

Microbial degradation of the herbicide molinate by defined cultures and in the environment

Molinate is a thiocarbamate herbicide used worldwide in rice crop protection. As with other pesticides, molinate is a recognized environmental pollutant, detected in soils, irrigation water, or rivers and bio-accumulated by some wildlife forms. For this reason, and in spite of its low toxicity to humans, environmental protection measures, which include reduction of use and/or remediation processes, are recommended. Due to its physic-chemical properties, molinate can easily disperse and react in the environment, originating diverse transformation products, some with increased toxicity. In spite of being a xenobiotic compound, molinate can also suffer microbial transformation by bacteria or fungi, sometimes serving as nutrient and energy source. In an attempt to isolate microorganisms to be used in the bioremediation of molinate-contaminated sites, a mixed culture, dominated by the actinobacterium Gulosibacter molinativorax ON4T, was recovered from the runoff of a molinate-producing plant. Beyond a promising tool to decontaminate molinate-polluted sites, this culture also brought interesting insights into the biology of the degradation of this herbicide. In this review, an overview of the distribution and properties of molinate as environmental contaminant, the capability of microorganisms to transform this herbicide, and some reflections about possible bioremediation approaches are made.

Candidimonas nitroreducens gen. nov., sp. nov. and Candidimonas humi sp. nov., isolated from sewage sludge compost.

Two bacterial strains (SC-089(T) and SC-092(T)) isolated from sewage sludge compost were characterized by using a polyphasic approach. The isolates were Gram-negative short rods, catalase- and oxidase-positive, and showed good growth at 30 °C, at pH 7 and with 1 % (w/v) NaCl. Ubiquinone 8 was the major respiratory quinone, and phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol were amongst the major polar lipids. On the basis of 16S rRNA gene sequence analysis, the strains were observed to be members of the family Alcaligenaceae, but could not be identified as members of any validly described genus. The low levels of 16S rRNA gene sequence similarity to other recognized taxa, together with comparative analysis of phenotypic traits and chemotaxonomic markers, supported the proposal of a new genus within the family Alcaligenaceae, for which the name Candidimonas gen. nov. is proposed. Strains SC-089(T) and SC-092(T), which shared 99.1 % 16S rRNA gene sequence similarity, could be differentiated at the phenotypic level, and DNA-DNA hybridization results supported their identification as representing distinct species. The names proposed for these novel species are Candidimonas nitroreducens sp. nov. (type strain, SC-089(T) = LMG 24812(T) = CCUG 55806(T)) and Candidimonas humi sp. nov. (type strain, SC-092(T) = LMG 24813(T) = CCUG 55807(T)).

Shinella fusca sp. nov., isolated from domestic waste compost.

A bacterium, designated strain DC-196(T), isolated from kitchen refuse compost was analysed by using a polyphasic approach. Strain DC-196(T) was characterized as a Gram-negative short rod that was catalase- and oxidase-positive, and able to grow at 10-40 degrees C, pH 6-9 and in NaCl concentrations as high as 3 %. Chemotaxonomically, C(18 : 1) was observed to be the predominant cellular fatty acid and ubiquinone 10 (Q10) was the predominant respiratory quinone. The G+C content of the genomic DNA was determined to be 66 mol%. On the basis of the genotypic, phenotypic and chemotaxonomic characteristics, strain DC-196(T) was assigned to the genus Shinella, although with distinctive features. At the time of writing, 16S rRNA gene sequence similarities of 97.6-96.8 % and the low DNA-DNA hybridization values of 38.2-32.2 % with the type strains of the three recognized Shinella species confirmed that strain DC-196(T) represents a novel species of the genus, for which the name Shinella fusca sp. nov. is proposed (type strain DC-196(T)=CCUG 55808(T)=LMG 24714(T)).