Active microbial communities during biodegradation of biodegradable plastics by mesophilic and thermophilic anaerobic digestion.

Biodegradable plastics, if they are not properly managed at their end-of-life, can have the same hazardous environmental consequences as conventional plastics. This study investigates the treatment of the main biodegradable plastics under mesophilic and thermophilic anaerobic digestion using biochemical methane potential test and the microorganisms involved in the process using amplicon sequencing of the 16 S rRNA. Here we showed that, only PHB and TPS undergone important and rapid biodegradation under mesophilic condition (38 °C). By contrast, PCL and PLA exhibited very low biodegradation rate as 500 days were required to reach the ultimate methane yield. Little or no degradation occurred for PBAT and PBS at 38 °C. Under thermophilic conditions (58 °C), TPS, PHB, and PLA reached high levels of biodegradation in a relatively short period (< 100 d). While PBS, PBAT, and PCL could not be converted into methane at 58 °C. PHB degraders (Enterobacter and Cupriavidus) and lactate-utilizing bacteria (Moorella and Tepidimicrobium) appeared to play an important role in the PHB and PLA degradation, respectively. This work not only provides crucial data on the anaerobic digestion of the main biodegradable plastics but also enriches the understanding of the microorganisms involved in this process, which are of great importance for future development of the treatment of biodegradable plastics in anaerobic digestion systems.

Simulation of biowastes and biodegradable plastics co-digestion in semi-continuous reactors: Performances and agronomic evaluation.

The development of selective biowaste collection in most European countries provides new opportunities for the anaerobic digestion sector. In parallel, extensive development of biodegradable plastics like polylactic-acid (PLA) and polyhydroxybutyrate (PHB), which facilitates the replacement of conventional plastics, has taken place in the past decade. This study investigated anaerobic co-digestion in semi-continuous reactors of biowastes (75 % Volatil Solids) and biodegradable plastics (25 % Volatil Solids, PLA and PHB). PHB was estimated to be fully biodegraded in the reactors. By contrast, PLA accumulated in the reactor, and an average biodegradation of 47.6 ± 17.9 % was estimated during the third hydraulic retention time. Pretreatment of PLA, by thermo-alkaline hydrolysis at 70 °C, with 2.5 w/v of Ca(OH)2 for 48 h, improved the biodegradation yield of PLA to 77.5 ± 9.3 %. Finally, it was highlighted that PLA or PHB addition to the feed did not further affect the agronomic properties of the digestate.

Biochemical methane potential and active microbial communities during anaerobic digestion of biodegradable plastics at different inoculum-substrate ratios.

The influence of the inoculum-substrate ratio (ISR) on the mesophilic and thermophilic biochemical methane potential test of two biodegradable plastics was evaluated. Poly(lactic acid) (PLA) and polyhydroxybutyrate (PHB) were selected for this study, the first for being recalcitrant to mesophilic anaerobic digestion (AD) and the second, by contrast, for being readily biodegradable. Several ISRs, calculated on the basis of volatile solids (VS), were tested: 1, 2, 2.85, 4, and 10 g(VS of inoculum).g(VS of substrate)-1. A high ISR was associated with an enhanced methane production rate (i.e., biodegradation kinetics). However, the ultimate methane production did not change, except when inhibition was observed. Indeed, applying the lowest ISR to readily biodegradable plastics such as PHB resulted in inhibition of methane production. Based on these experiments, in order to have reproducible degradation kinetics and optimal methane production, an ISR between 2.85 and 4 is recommended for biodegradable plastics. The active microbial communities were analyzed, and the active bacteria differed depending on the plastic digested (PLA versus PHB) and the temperature of the process (mesophilic versus thermophilic). Previously identified PHB degraders (Ilyobacter delafieldii and Enterobacter) were detected in PHB-fed reactors. Thermogutta and Tepidanaerobacter were detected during the thermophilic AD of PLA, and they are probably involved in PLA hydrolysis and lactate conversion, respectively.

Can anaerobic digestion be a suitable end-of-life scenario for biodegradable plastics? A critical review of the current situation, hurdles, and challenges.

Growing concern regarding non-biodegradable plastics and the impact of these materials on the environment has promoted interest in biodegradable plastics. The intensification of separate biowastes collection in most European countries has also contributed to the development of biodegradable plastics, and the subject of their end-of-life is becoming a key issue. To date, there has been relatively little research to evaluate the biodegradability of biodegradable plastics by anaerobic digestion (AD) compared to industrial and home composting. However, anaerobic digestion is a particularly promising strategy for treating biodegradable organic wastes in the context of circular waste management. This critical review aims to provide an in-depth update of anaerobic digestion of biodegradable plastics by providing a summary of the literature regarding process performance, parameters affecting biodegradability, the microorganisms involved, and some of the strategies (e.g., pretreatment, additives, and inoculum acclimation) used to enhance the degradation rate of biodegradable plastics. In addition, a critical section is dedicated to suggestions and recommendations for the development of biodegradable plastics sector and their treatment in anaerobic digestion.

Impact of mechanical and thermo-chemical pretreatments to enhance anaerobic digestion of poly(lactic acid).

To date, the introduction of biodegradable plastics such as PLA in anaerobic digestion systems has been limited by a very low rate of biodegradation. To overcome these limitations, pretreatment technologies can be applied. In this study, the impact of pretreatments (mechanical, thermal, thermo-acid, and thermo-alkaline) was investigated. Mechanical pretreatment of PLA improved its biodegradation rate but did not affect the ultimate methane potential (430-461 NL CH4 kg-1 VS). In parallel, thermal and thermo-acid pretreatments exhibited a similar trend for PLA solubilization. Both of these pretreatments only achieved substantial solubilization (>60%) at higher temperatures (120 and 150 °C). At lower temperatures (70 and 90 °C), negligible solubilization (between 1 and 6%) occurred after 48 h. By contrast, coupling of thermal and alkaline pretreatment significantly increased solubilization at the lower temperatures (70 and 90 °C). In terms of biodegradation, thermo-alkaline pretreatment (with 5% w/v Ca(OH)2) of PLA resulted in a similar methane potential (from 325 to 390 NL CH4 kg-1 VS) for 1 h at 150 °C, 6 h at 120 °C, 24 h at 90 °C, and 48 h at 70 °C. Reduction of the Ca(OH)2 concentration (from 5% to 0.5% w/v) highlighted that a concentration of 2.5% w/v was sufficient to achieve a substantial level of biodegradation. Pretreatment at 70 and 90 °C using 2.5% w/v Ca(OH)2 for 48 h resulted in biodegradation yields of 73% and 68%, respectively. Finally, a good correlation (R2 = 0.90) was found between the PLA solubilization and its biodegradation.

Methane production and active microbial communities during anaerobic digestion of three commercial biodegradable coffee capsules under mesophilic and thermophilic conditions.

Biodegradable plastics market is increasing these last decades, including for coffee capsules. Anaerobic digestion, as a potential end-of-life scenario for plastic waste, has to be investigated. For this purpose, mesophilic (38 °C) and thermophilic (58 °C) anaerobic digestion tests on three coffee capsules made up with biodegradable plastic (Beanarella®, Launay® or Tintoretto®) and spent coffee (control) were compared by their methane production and the microbial communities active during the process. Mesophilic biodegradation of the capsules was slow and did not reach completion after 100 days, methane production ranged between 67 and 127 NL (CH4) kg-1 (VS). Thermophilic anaerobic digestion resulted in a better biodegradation and reached completion around 100 days, methane productions were between 257 and 294 NL (CH4) kg-1 (VS). The microbial populations from the reactors fed with plastics versus spent coffee grounds were significantly different, under both the mesophilic and the thermophilic conditions. However, the different biodegradable plastics only had a small impact on the main microbial community composition at a similar operational temperature and sampling time. Interestingly, the genus Tepidimicrobium was identified as a potential key microorganisms involved in the thermophilic conversion of biodegradable plastic in methane.

Prospecting bacterial consortia from a geothermal site for metals biotransformation.

Biomats that flourished in a fumarole located on the geothermal site Los Azufres (Mexico) were used as inocula to select aerobic and sulfate-reducing bacteria consortia for studying their capacity to reduce hexavalent chromium [Cr(VI)], aiming to use these consortia in biotransformation technologies. The sample site is characterized by slightly warm (nearly 27 [Formula: see text]C), acid (pH 3) and about hypoxic (1.8 mg L[Formula: see text] of dissolved oxygen) conditions. Four culture systems (2 aerobic and 2 anaerobic) were investigated, including their enzymatic activity, capacity to produce biofilms, and an analysis of the total bacterial populations. For the anaerobic condition (using sulfate and sulfur as electron acceptors), four pH values (from 2 to 8) and four carbon sources (pyruvate, glycerol, Na-lactate and Na-acetate) were probed. Significant biological Cr(VI) removal was observed for all the pH values probed, particularly during the first 12 h, being more effective at the most acid conditions. At a pH value of 4 and using pyruvate as carbon source, 100 mg L[Formula: see text] of Cr(VI) were completely depleted in less than 12 h, while the use of Na-lactate was less effective but still reasonable. These results indicate that sulfate-reducing bacteria consortia from geothermal sites like the one studied here are capable of biotransforming Cr(VI) and have the potential to provide metal bioremediation technologies.

Diurnal variability and biogeochemical reactivity of mercury species in an extreme high-altitude lake ecosystem of the Bolivian Altiplano.

Methylation and demethylation represent major transformation pathways regulating the net production of methylmercury (MMHg). Very few studies have documented Hg reactivity and transformation in extreme high-altitude lake ecosystems. Mercury (Hg) species concentrations (IHg, MMHg, Hg°, and DMHg) and in situ Hg methylation (M) and MMHg demethylation (D) potentials were determined in water, sediment, floating organic aggregates, and periphyton compartments of a shallow productive Lake of the Bolivian Altiplano (Uru Uru Lake, 3686 m). Samples were collected during late dry season (October 2010) and late wet season (May 2011) at a north (NS) and a south (SS) site of the lake, respectively. Mercury species concentrations exhibited significant diurnal variability as influenced by the strong diurnal biogeochemical gradients. Particularly high methylated mercury concentrations (0.2 to 4.5 ng L(-1) for MMHgT) were determined in the water column evidencing important Hg methylation in this ecosystem. Methylation and D potentials range were, respectively, <0.1-16.5 and <0.2-68.3 % day(-1) and were highly variable among compartments of the lake, but always higher during the dry season. Net Hg M indicates that the influence of urban and mining effluent (NS) promotes MMHg production in both water (up to 0.45 ng MMHg L(-1) day(-1)) and sediment compartments (2.0 to 19.7 ng MMHg g(-1) day(-1)). While the sediment compartment appears to represent a major source of MMHg in this shallow ecosystem, floating organic aggregates (dry season, SS) and Totora's periphyton (wet season, NS) were found to act as a significant source (5.8 ng MMHg g(-1) day(-1)) and a sink (-2.1 ng MMHg g(-1) day(-1)) of MMHg, respectively. This work demonstrates that high-altitude productive lake ecosystems can promote MMHg formation in various compartments supporting recent observations of high Hg contents in fish and water birds.

Investigations into the differential reactivity of endogenous and exogenous mercury species in coastal sediments.

Stable isotopic tracer methodologies now allow the evaluation of the reactivity of the endogenous (ambient) and exogenous (added) Hg to further predict the potential effect of Hg inputs in ecosystems. The differential reactivity of endogenous and exogenous Hg was compared in superficial sediments collected in a coastal lagoon (Arcachon Bay) and in an estuary (Adour River) from the Bay of Biscay (SW France). All Hg species (gaseous, aqueous, and solid fraction) and ancillary data were measured during time course slurry experiments under variable redox conditions. The average endogenous methylation yield was higher in the estuarine (1.2 %) than in the lagoonal sediment (0.5 %), although both methylation and demethylation rates were higher in the lagoonal sediment in relation with a higher sulfate-reducing activity. Demethylation was overall more consistent than methylation in both sediments. The endogenous and exogenous Hg behaviors were always correlated but the exogenous inorganic Hg (IHg) partitioning into water was 2.0-4.3 times higher than the endogenous one. Its methylation was just slightly higher (1.4) in the estuarine sediment while the difference in the lagoonal sediment was much larger (3.6). The relative endogenous and exogenous methylation yields were not correlated to IHg partitioning, demonstrating that the bioavailable species distributions were different for the two IHg pools. In both sediments, the exogenous IHg partitioning equaled the endogenous one within a week, while its higher methylation lasted for months. Such results provide an original assessment approach to compare coastal sediment response to Hg inputs.

Hexavalent chromium reduction by bacterial consortia and pure strains from an alkaline industrial effluent.

AIMS: To characterize the bacterial consortia and isolates selected for their role in hexavalent chromium removal by adsorption and reduction. METHODS AND RESULTS: Bacterial consortia from industrial wastes revealed significant Cr(VI) removal after 15 days when incubated in medium M9 at pH 6·5 and 8·0. The results suggested chromium reduction. The bacterial consortia diversity (T-RFLP based on 16S rRNA gene) indicated a highest number of operational taxonomic units in an alkaline carbonate medium mimicking in situ conditions. However, incubations under such conditions revealed low Cr(VI) removal. Genomic libraries were obtained for the consortia exhibiting optimal Cr(VI) removal (M9 medium at pH 6·5 and 8·0). They revealed the dominance of 16S rRNA gene sequences related to the genera Pseudomonas/Stenotrophomonas or Enterobacter/Halomonas, respectively. Isolates related to Pseudomonas fluorescens and Enterobacter aerogenes were efficient in Cr(VI) reduction and adsorption to the biomass. CONCLUSIONS: Cr(VI) reduction was better at neutral pH rather than under in situ conditions (alkaline pH with carbonate). Isolated strains exhibited significant capacity for Cr(VI) reduction and adsorption. SIGNIFICANCE AND IMPACT OF STUDY: Bacterial communities from chromium-contaminated industrial wastes as well as isolates were able to remove Cr(VI). The results suggest a good potential for bioremediation of industrial wastes when optimal conditions are applied.

Mercury methylation by a microbial community from sediments of the Adour Estuary (Bay of Biscay, France).

In order to study the influence of microorganisms on the mercury biogeochemistry, the metal content and the structure of microbial communities were determined in sediments from stations along the Adour Estuary. The comparison of the bacterial communities and their distribution in function of the environmental parameters by Canonical Correspondence Analysis (CCA) revealed the influence of metals on the bacterial communities structure. Sediments where the bacterial communities are mostly influenced by methylmercury were incubated in slurries with or without mercury, under oxic and anoxic conditions. Methylmercury production was detected in the anoxic biotic slurries with a net methylation yield of 0.3% after 24 h. CCA based on T-RFLP profiles revealed the impact of mercury addition on the bacterial communities structure. In addition, 17 bacterial strains, mainly sulphate-reducing bacteria involved in mercury methylation, were isolated and identified.

Characterization of Desulfomicrobium salsuginis sp. nov. and Desulfomicrobium aestuarii sp. nov., two new sulfate-reducing bacteria isolated from the Adour estuary (French Atlantic coast) with specific mercury methylation potentials.

Three strains of sulfate-reducing bacteria (ADR21, ADR26 and ADR28) were isolated from Adour estuary sediments (French South Atlantic coast). Cells of these isolates were rod-shaped, motile and stained Gram-negative. The 16S rRNA and dsrAB genes sequence analyses indicated that these three strains belonged to the genus Desulfomicrobium within the delta Proteobacteria, with Desulfomicrobium escambiense strain DSM10707T as their closest relative. According to phenotypic characteristics, strains ADR21 and ADR28 could be considered as members of the same species. The relatedness values, based on DNA-DNA hybridization studies, between strains ADR21/DSM10707T, ADR26/DSM10707T and ADR21/ADR26 ranged between 30.6-40.8%, 45.2-43.0% and 19.0-26.4%, respectively. Strains ADR21 and ADR28 grew well on lactate, fumarate, malate, formate, ethanol and H2/acetate in the presence of sulfate as an electron acceptor. Thiosulfate, nitrate, fumarate and DMSO were alternative electron acceptors. Malate was well fermented but pyruvate and fumarate only poorly. Strain ADR26 could not grow on ethanol or fumarate and was unable to use DMSO or fumarate as electron acceptors. The three new strains exhibited differences compared to the type strain of D. escambiense, such as temperature optima, substrate utilization and mercury methylation capacities. On the basis of both genetic and phenotypic evidences, strain ADR21 is proposed as the type strain of the species Desulfomicrobium salsuginis sp. nov., and strain ADR26 as the type strain of the species Desulfomicrobium aestuarii sp. nov.

Light responses in the green sulfur bacterium Prosthecochloris aestuarii: changes in prosthecae length, ultrastructure, and antenna pigment composition.

The morphology (mainly prosthecae length), ultrastructure, and antenna pigment composition of the green sulfur bacterium Prosthecochloris aestuarii changed when grown under different light intensities. At light intensities of 0.5 and 5 micromol quanta m(-2) s(-1), the cells had a star-like morphology. Prosthecae, the characteristic appendages of the genus Prosthecochloris, were 232 nm and 194 nm long, respectively. In contrast, when grown at 100 micromol quanta m(-2) s(-1), these appendages were shorter (98 nm) and the cells appeared more rod-shaped. Transmission electron microscopy revealed a significant decrease in the cell perimeter to area ratio and in the number of chlorosomes per linear microm of membrane as light intensity increased. In addition to these morphological and ultrastructural responses, Prosthecochloris aestuarii exhibited changes in its pigment composition as a function of light regime. Lower specific pigment content and synthesis rates were found in cultures grown at light intensities above 5 micromol quanta m(-2) s(-1). A blue shift in the bacteriochlorophyll (BChl) c Q(y) absorption maximum of up to 17.5 nm was observed under saturating light conditions (100 micromol quanta m(-2) s(-1)). This displacement was accompanied by changes in the composition of BChl c homologs and by a very low carotenoid content. The morphological, ultrastructural and functional changes exhibited by Prosthecochloris aestuarii revealed the strong light-response capacity of this bacterium to both high and low photon-flux densities.

Effect of carotenoid biosynthesis inhibition on the chlorosome organization in Chlorobium phaeobacteroides strain CL1401.

We have studied the effect of the absence of carotenoids on the organization of bacteriochlorophylls (BChls) in chlorosomes of Chlorobium (Chl.) phaeobacteroides strain CL1401. Carotenoid-depleted chlorosomes were obtained by means of 2-hydroxybiphenyl-supplemented cultures. In the presence of the inhibitor, isorenieratene (Isr) and beta-Isr biosynthesis were inhibited to more than 95%, leading to an accumulation of the colorless precursor phytoene inside the chlorosomes. In addition, there was a 30-40% decrease in the baseplate BChl a content. The absorption spectrum of the carotenoid-depleted chlorosomes showed a 10 nm blue shift in the BChl e Qy absorption peak. Under reducing conditions, a decrease in the BChl a/BChl e fluorescence emission ratio was observed in carotenoid-depleted chlorosomes relative to that in control chlorosomes, caused mainly by the decrease in the BChl a content. The steady-state fluorescence emission anisotropy in the BChl e region dropped from approximately 0.24 for native chlorosomes to approximately 0.14 for carotenoid-depleted ones, indicating reorganization of BChl e. The circular dichroism (CD) signal of the carotenoid-depleted chlorosomes was increased two times in the BChl e Qy region. A simple model based on the structure proposed was used to explain the observed effects. Carotenoids might affect the angle between the direction of the BChl e Qy transition and the axis of the rod. The orientation of BChl a in the baseplate remains unchanged in carotenoid-depleted chlorosomes, although there is a partial loss of BChl a as a consequence of a decrease in the baseplate size. The carotenoids are most likely rather close to the BChls and appear to be important for the aggregate structure in Chl. phaeobacteroides.

Dynamics of anoxygenic photosynthesis in an experimental green sulphur bacteria biofilm.

The dynamics of sulphide oxidation in an experimental biofilm of the green sulphur bacterium, Prosthecochloris aestuarii, were studied using a newly developed light-dark cycling procedure. The biofilm was grown for 6 weeks in a benthic gradient chamber, in which gradients of light, sulphide and oxygen were imposed experimentally. The H2S concentrations and pH were measured with microsensors as a function of depth in the biofilm and of time after a change in illumination status. The sulphide oxidation rates were calculated as a function of time and depth in the biofilm using a numerical procedure to solve the non-stationary general diffusion equation. A close agreement was found between the areal rates of anoxygenic photosynthesis during the cycling procedure and the steady state before the cycling experiment. For the different layers of the biofilm, the maximum activity was observed after 10-12min of light exposure. After this maximum, sulphide oxidation decreased concomitantly with sulphide concentration, indicating sulphide limitation of anoxygenic photosynthesis. This lag time limits the application of the standard dark-light shift method with a brief light exposure of a few seconds and, therefore, the numerical procedure described in this study enables the depth distribution of anoxygenic photosynthesis rates in microbial mats to be determined more accurately.

Taxonomic rearrangements of the genera Thiocapsa and Amoebobacter on the basis of 16S rDNA sequence analyses, and description of Thiolamprovum gen. nov.

Complete nucleotide sequences of the 16S rDNAs were determined from Thiocapsa and Amoebobacter species, including all available type strains and some additional isolates. The distance-matrix analysis and the dendrogram for estimating the genetic relationships revealed that the investigated strains were found in two major clusters within the Chromatiaceae. One cluster comprises all Amoebobacter species, Thiocapsa roseopersicina and several isolates related to Thiocapsa roseopersicina. Representatives of the species Amoebobacter roseus, Amoebobacter pendens and Thiocapsa roseopersicina, the so called 'Thiocapsa roseopersicina group', are very closely related, justifying their inclusion into one genus, Thiocapsa, for which an emended description is presented. Amoebobacter purpureus and Amoebobacter pedioformis formed two separate lines of descent with less than 93% (89.6-92.9%) similarity to strains of the 'Thiocapsa roseopersicina group'. Therefore, they will be considered as two separate genera. As a consequence, an emended description is presented for the genus Amoebobacter, with Amoebobacter purpureus as the new type species and A. pedioformis is transferred to Thiolamprovum pedioforme gen. nov., comb. nov. Two species, Thiocapsa pfennigii and Thiocapsa halophila, which have been classified with the genus Thiocapsa because of their morphological properties, were found within another major cluster of the Chromatiaceae and are only distantly phylogenetically related to the first cluster with 88.4-90.6% and 90.4-92.2% sequence similarity, respectively.

Thiorhodococcus minus, gen. nov., sp. nov., A new purple sulfur bacterium isolated from coastal lagoon sediments.

A new marine phototrophic purple sulfur bacterium (strain CE2203) was isolated in pure culture from a man-made coastal lagoon located on the Atlantic coast (Arcachon Bay, France). Single cells were coccus-shaped, did not contain gas vesicles, and were highly motile. Intracellular photosynthetic membranes were of the vesicular type. Bacteriochlorophyll a and carotenoids of the normal spirilloxanthin series were present as photosynthetic pigments. Hydrogen sulfide, thiosulfate, elemental sulfur, and molecular hydrogen were used as electron donors during photolithotrophic growth under anoxic conditions, while carbon dioxide was utilized as carbon source. Acetate, propionate, lactate, glycolate, pyruvate, fumarate, succinate, fructose, sucrose, ethanol, and propanol were photoassimilated in the presence of hydrogen sulfide. During growth on sulfide, elemental sulfur globules were stored inside the cells. Chemotrophic growth under microoxic conditions in the dark was possible. The DNA base composition was 66.9 mol% G+C. Comparative sequence analysis of the 16S rRNA gene confirmed the membership of strain CE2203 in the family Chromatiaceae. Morphological characteristics of strain CE2203 indicated a close affiliation to the genera Thiocystis and Thiocapsa. However, the phylogenetic treeing revealed no closer relationship to Thiocystis spp. than to Thiocapsa roseopersicina or other known members of the Chromatiaceae. Consequently, strain CE2203 is proposed as the type strain of a new genus and species, Thiorhodococcus minus gen. nov., sp. nov.