Microplastics Biodegradation by Estuarine and Landfill Microbiomes.

Plastic pollution poses a worldwide environmental challenge, affecting wildlife and human health. Assessing the biodegradation capabilities of natural microbiomes in environments contaminated with microplastics is crucial for mitigating the effects of plastic pollution. In this work, we evaluated the potential of landfill leachate (LL) and estuarine sediments (ES) to biodegrade polyethylene (PE), polyethylene terephthalate (PET), and polycaprolactone (PCL), under aerobic, anaerobic, thermophilic, and mesophilic conditions. PCL underwent extensive aerobic biodegradation with LL (99 ± 7%) and ES (78 ± 3%) within 50-60 days. Under anaerobic conditions, LL degraded 87 ± 19% of PCL in 60 days, whereas ES showed minimal biodegradation (3 ± 0.3%). PE and PET showed no notable degradation. Metataxonomics results (16S rRNA sequencing) revealed the presence of highly abundant thermophilic microorganisms assigned to Coprothermobacter sp. (6.8% and 28% relative abundance in anaerobic and aerobic incubations, respectively). Coprothermobacter spp. contain genes encoding two enzymes, an esterase and a thermostable monoacylglycerol lipase, that can potentially catalyze PCL hydrolysis. These results suggest that Coprothermobacter sp. may be pivotal in landfill leachate microbiomes for thermophilic PCL biodegradation across varying conditions. The anaerobic microbial community was dominated by hydrogenotrophic methanogens assigned to Methanothermobacter sp. (21%), pointing at possible syntrophic interactions with Coprothermobacter sp. (a H2-producer) during PCL biodegradation. In the aerobic experiments, fungi dominated the eukaryotic microbial community (e.g., Exophiala (41%), Penicillium (17%), and Mucor (18%)), suggesting that aerobic PCL biodegradation by LL involves collaboration between fungi and bacteria. Our findings bring insights on the microbial communities and microbial interactions mediating plastic biodegradation, offering valuable perspectives for plastic pollution mitigation.

Principles, Advances, and Perspectives of Anaerobic Digestion of Lipids.

Several problems associated with the presence of lipids in wastewater treatment plants are usually overcome by removing them ahead of the biological treatment. However, because of their high energy content, waste lipids are interesting yet challenging pollutants in anaerobic wastewater treatment and codigestion processes. The maximal amount of waste lipids that can be sustainably accommodated, and effectively converted to methane in anaerobic reactors, is limited by several problems including adsorption, sludge flotation, washout, and inhibition. These difficulties can be circumvented by appropriate feeding, mixing, and solids separation strategies, provided by suitable reactor technology and operation. In recent years, membrane bioreactors and flotation-based bioreactors have been developed to treat lipid-rich wastewater. In parallel, the increasing knowledge on the diversity of complex microbial communities in anaerobic sludge, and on interspecies microbial interactions, contributed to extend the knowledge and to understand more precisely the limits and constraints influencing the anaerobic biodegradation of lipids in anaerobic reactors. This critical review discusses the most important principles underpinning the degradation process and recent key discoveries and outlines the current knowledge coupling fundamental and applied aspects. A critical assessment of knowledge gaps in the field is also presented by integrating sectorial perspectives of academic researchers and of prominent developers of anaerobic technology.

Detoxification of Ciprofloxacin in an Anaerobic Bioprocess Supplemented with Magnetic Carbon Nanotubes: Contribution of Adsorption and Biodegradation Mechanisms.

In anaerobic bioreactors, the electrons produced during the oxidation of organic matter can potentially be used for the biological reduction of pharmaceuticals in wastewaters. Common electron transfer limitations benefit from the acceleration of reactions through utilization of redox mediators (RM). This work explores the potential of carbon nanomaterials (CNM) as RM on the anaerobic removal of ciprofloxacin (CIP). Pristine and tailored carbon nanotubes (CNT) were first tested for chemical reduction of CIP, and pristine CNT was found as the best material, so it was further utilized in biological anaerobic assays with anaerobic granular sludge (GS). In addition, magnetic CNT were prepared and also tested in biological assays, as they are easier to be recovered and reused. In biological tests with CNM, approximately 99% CIP removal was achieved, and the reaction rates increased ≈1.5-fold relatively to the control without CNM. In these experiments, CIP adsorption onto GS and CNM was above 90%. Despite, after applying three successive cycles of CIP addition, the catalytic properties of magnetic CNT were maintained while adsorption decreased to 29 ± 3.2%, as the result of CNM overload by CIP. The results suggest the combined occurrence of different mechanisms for CIP removal: adsorption on GS and/or CNM, and biological reduction or oxidation, which can be accelerated by the presence of CNM. After biological treatment with CNM, toxicity towards Vibrio fischeri was evaluated, resulting in ≈ 46% detoxification of CIP solution, showing the advantages of combining biological treatment with CNM for CIP removal.

Multiple and flexible roles of facultative anaerobic bacteria in microaerophilic oleate degradation.

Anaerobic degradation of long-chain fatty acids (LCFA) involves syntrophic bacteria and methanogens, but facultative anaerobic bacteria (FAB) might have a relevant role as well. Here we investigated oleate degradation by a syntrophic synthetic co-culture of Syntrophomonas zehnderi (Sz) and Methanobacterium formicicum (Mf) and FAB (two oleate-degrading Pseudomonas spp. I1 + I2). Sz + Mf were first cultivated in a continuous bioreactor under strict anaerobic conditions. Thereafter, I1 + I2 were inoculated and microaerophilic conditions were provided. Methane and acetate were the main degradation products by Sz + Mf in anaerobiosis and by Sz + Mf + I1 + I2 in microaerophilic conditions. However, acetate production from oleate was higher in microaerophilic conditions (5% O2 ) with the four microorganisms together (0.41 ± 0.07 mmol day-1 ) than in anaerobiosis with Sz + Mf (0.23 ± 0.05 mmol day-1 ). Oleate degradation in batch assays was faster by Sz + Mf + I1 + I2 (under microaerophilic conditions) than by Sz + Mf alone (under strict anaerobic conditions). I1 + I2 were able to grow with oleate and with intermediates of oleate degradation (hydrogen, acetate and formate). This work highlights the importance of FAB, particularly Pseudomonas sp., in anaerobic reactors treating oleate-based wastewater, because they accelerate oleate conversion to methane, by protecting strict anaerobes from oxygen toxicity and also by acting as alternative hydrogen/formate and acetate scavengers for LCFA-degrading anaerobes.

Methane Production and Conductive Materials: A Critical Review.

Conductive materials (CM) have been extensively reported to enhance methane production in anaerobic digestion processes. The occurrence of direct interspecies electron transfer (DIET) in microbial communities, as an alternative or complementary to indirect electron transfer (via hydrogen or formate), is the main explanation given to justify the improvement of methane production. Not disregarding that DIET can be promoted in the presence of certain CM, it surely does not explain all the reported observations. In fact, in methanogenic environments DIET was only unequivocally demonstrated in cocultures of Geobacter metallireducens with Methanosaeta harundinacea or Methanosarcina barkeri and frequently Geobacter sp. are not detected in improved methane production driven systems. Furthermore, conductive carbon nanotubes were shown to accelerate the activity of methanogens growing in pure cultures, where DIET is not expected to occur, and hydrogenotrophic activity is ubiquitous in full-scale anaerobic digesters treating for example brewery wastewaters, indicating that interspecies hydrogen transfer is an important electron transfer mechanism in those systems. This paper presents an overview of the effect of several iron-based and carbon-based CM in bioengineered systems, focusing on the improvement in methane production and in microbial communities' changes. Control assays, as fundamental elements to support major conclusions in reported experiments, are critically revised and discussed.

Insight into the Role of Facultative Bacteria Stimulated by Microaeration in Continuous Bioreactors Converting LCFA to Methane.

Conversion of unsaturated long chain fatty acids (LCFA) to methane in continuous bioreactors is not fully understood. Palmitate (C16:0) often accumulates during oleate (C18:1) biodegradation in methanogenic bioreactors, and the reason why this happens and which microorganisms catalyze this reaction remains unknown. Facultative anaerobic bacteria are frequently found in continuous reactors operated at high LCFA loads, but their function is unclear. To get more insight on the role of these bacteria, LCFA conversion was studied under microaerophilic conditions. For that, we compared bioreactors treating oleate-based wastewater (organic loading rates of 1 and 3 kg COD m-3 d-1), operated under different redox conditions (strictly anaerobic-AnR, -350 mV; microaerophilic-MaR, -250 mV). At the higher load, palmitate accumulated 7 times more in the MaR, where facultative anaerobes were more abundant, and only the biomass from this reactor could recover the methanogenic activity after a transient inhibition. In a second experiment, the abundance of facultative anaerobic bacteria, particularly Pseudomonas spp. (from which two strains were isolated), was strongly correlated ( p < 0.05) with palmitate-to-total LCFA percentage in the biofilm formed in a continuous plug flow reactor fed with very high loads of oleate. This work strongly suggests that microaeration stimulates the development of facultative bacteria that are critical for achieving LCFA conversion to methane in continuous bioreactors. Microbial networks and interactions of facultative and strict anaerobes in microbial communities should be considered in future studies.

Carbon nanotubes accelerate methane production in pure cultures of methanogens and in a syntrophic coculture.

Carbon materials have been reported to facilitate direct interspecies electron transfer (DIET) between bacteria and methanogens improving methane production in anaerobic processes. In this work, the effect of increasing concentrations of carbon nanotubes (CNT) on the activity of pure cultures of methanogens and on typical fatty acid-degrading syntrophic methanogenic coculture was evaluated. CNT affected methane production by methanogenic cultures, although acceleration was higher for hydrogenotrophic methanogens than for acetoclastic methanogens or syntrophic coculture. Interestingly, the initial methane production rate (IMPR) by Methanobacterium formicicum cultures increased 17 times with 5 g·L-1 CNT. Butyrate conversion to methane by Syntrophomonas wolfei and Methanospirillum hungatei was enhanced (∼1.5 times) in the presence of CNT (5 g·L-1 ), but indications of DIET were not obtained. Increasing CNT concentrations resulted in more negative redox potentials in the anaerobic microcosms. Remarkably, without a reducing agent but in the presence of CNT, the IMPR was higher than in incubations with reducing agent. No growth was observed without reducing agent and without CNT. This finding is important to re-frame discussions and re-interpret data on the role of conductive materials as mediators of DIET in anaerobic communities. It also opens new challenges to improve methane production in engineered methanogenic processes.

Biodegradation of PHB/PBAT films and isolation of novel PBAT biodegraders from soil microbiomes.

Polyhydroxyalkanoates (PHAs) are important candidates for replacing petroleum-based plastics. This transition is urgent for the development of a biobased economy and to protect human health and natural ecosystems. PHAs are biobased and biodegradable polyesters that when blended with other polymers, such as poly(butylene adipate-co-terephthalate) (PBAT), acquire remarkable improvements in their properties, which allow them to comply with the requirements of packaging applications. However, the biodegradation of such blends should be tested to evaluate the impact of those polymers in the environment. For instance, PBAT is a compostable aliphatic-aromatic copolyester, and its biodegradation in natural environments, such as soil, is poorly studied. In this work, we evaluated the biodegradation of a bilayer film composed of PHB and PBAT, by a soil microbiome. The bilayer film reached 47 ± 1 % mineralization in 180 days and PHB was no longer detected after this period. The increased crystallinity of the PBAT residue was a clear sign of biodegradation, indicating that the amorphous regions were preferentially biodegraded. Seven microorganisms were isolated, from which 4 were closely related to microorganisms already known as PHB degraders, but the other 3 species, closely related to Streptomyces coelicoflavus, Clonostachys rosea and Aspergillus insuetus, were found for the first time as PHB degraders. Most remarkably, two fungi closely related to Purpureocillium lilacinum and Aspergillus pseudodeflectus (99.83 % and 100 % identity by ITS sequencing) were isolated and identified as PBAT degraders. This is very interesting due to the rarity of isolating PBAT-degrading microorganisms. These results show that the bilayer film can be biodegraded in soil, at mesophilic temperatures, showing its potential to replace synthetic plastics in food packaging.

Activity and viability of methanogens in anaerobic digestion of unsaturated and saturated long-chain fatty acids.

Lipids can be anaerobically digested to methane, but methanogens are often considered to be highly sensitive to the long-chain fatty acids (LCFA) deriving from lipids hydrolysis. In this study, the effect of unsaturated (oleate [C18:1]) and saturated (stearate [C18:0] and palmitate [C16:0]) LCFA toward methanogenic archaea was studied in batch enrichments and in pure cultures. Overall, oleate had a more stringent effect on methanogens than saturated LCFA, and the degree of tolerance to LCFA was different among distinct species of methanogens. Methanobacterium formicicum was able to grow in both oleate- and palmitate-degrading enrichments (OM and PM cultures, respectively), whereas Methanospirillum hungatei only survived in a PM culture. The two acetoclastic methanogens tested, Methanosarcina mazei and Methanosaeta concilii, could be detected in both enrichment cultures, with better survival in PM cultures than in OM cultures. Viability tests using live/dead staining further confirmed that exponential growth-phase cultures of M. hungatei are more sensitive to oleate than are M. formicicum cultures; exposure to 0.5 mM oleate damaged 99% ± 1% of the cell membranes of M. hungatei and 53% ± 10% of the cell membranes of M. formicicum. In terms of methanogenic activity, M. hungatei was inhibited for 50% by 0.3, 0.4, and 1 mM oleate, stearate, and palmitate, respectively. M. formicicum was more resilient, since 1 mM oleate and >4 mM stearate or palmitate was needed to cause 50% inhibition on methanogenic activity.

Endurance of methanogenic archaea in anaerobic bioreactors treating oleate-based wastewater.

Methanogenic archaea are reported as very sensitive to lipids and long chain fatty acids (LCFA). Therefore, in conventional anaerobic processes, methane recovery during LCFA-rich wastewater treatment is usually low. By applying a start-up strategy, based on a sequence of step feeding and reaction cycles, an oleate-rich wastewater was efficiently treated at an organic loading rate of 21 kg COD m(-3) day(-1) (50 % as oleate), showing a methane recovery of 72 %. In the present work, the archaeal community developed in that reactor is investigated using a 16S rRNA gene approach. This is the first time that methanogens present in a bioreactor converting efficiently high loads of LCFA to methane are monitored. Denaturing gradient gel electrophoresis profiling showed that major changes on the archaeal community took place during the bioreactor start-up, where phases of continuous feeding were alternated with batch phases. After the start-up, a stable archaeal community (similarity higher than 84 %) was observed and maintained throughout the continuous operation. This community exhibited high LCFA tolerance and high acetoclastic and hydrogenotrophic activity. Cloning and sequencing results showed that Methanobacterium- and Methanosaeta-like microorganisms prevailed in the system and were able to tolerate and endure during prolonged exposure to high LCFA loads, despite the previously reported LCFA sensitivity of methanogens.

UPIMAPI, reCOGnizer and KEGGCharter: Bioinformatics tools for functional annotation and visualization of (meta)-omics datasets.

Omics and meta-omics technologies are powerful approaches to explore microorganisms' functions, but the sheer size and complexity of omics datasets often turn the analysis into a challenging task. Software developed for omics and meta-omics analyses, together with knowledgebases encompassing information on genes, proteins, taxonomic and functional annotation, among other types of information, are valuable resources for analyzing omics data. Although several bioinformatics resources are available for meta-omics analyses, many require significant computational expertise. Web interfaces are more user-friendly, but often struggle to handle large data files, such as those obtained in metagenomics, metatranscriptomics, or metaproteomics experiments. In this work, we present three novel bioinformatics tools, which are available through user-friendly command-line interfaces, can be run sequentially or stand-alone, and combine popular resources for functional annotation. UPIMAPI performs sequence homology-based annotation and obtains data from UniProtKB (e.g., protein names, EC numbers, Gene Ontology, Taxonomy, cross-references to external databases). reCOGnizer performs multithreaded domain homology-based annotation of protein sequences with several functional databases (i.e., CDD, NCBIfam, Pfam, Protein Clusters, SMART, TIGRFAM, COG and KOG) and in addition, obtains information on domain names and descriptions and EC numbers. KEGGCharter represents omics results, including differential gene expression, in KEGG metabolic pathways. In addition, it shows the taxonomic assignment of the enzymes represented, which is particularly useful in metagenomics studies in which several microorganisms are present. reCOGnizer, UPIMAPI and KEGGCharter together provide a comprehensive and complete functional characterization of large datasets, facilitating the interpretation of microbial activities in nature and in biotechnological processes.

Iron Compounds in Anaerobic Degradation of Petroleum Hydrocarbons: A Review.

Waste and wastewater containing hydrocarbons are produced worldwide by various oil-based industries, whose activities also contribute to the occurrence of oil spills throughout the globe, causing severe environmental contamination. Anaerobic microorganisms with the ability to biodegrade petroleum hydrocarbons are important in the treatment of contaminated matrices, both in situ in deep subsurfaces, or ex situ in bioreactors. In the latter, part of the energetic value of these compounds can be recovered in the form of biogas. Anaerobic degradation of petroleum hydrocarbons can be improved by various iron compounds, but different iron species exert distinct effects. For example, Fe(III) can be used as an electron acceptor in microbial hydrocarbon degradation, zero-valent iron can donate electrons for enhanced methanogenesis, and conductive iron oxides may facilitate electron transfers in methanogenic processes. Iron compounds can also act as hydrocarbon adsorbents, or be involved in secondary abiotic reactions, overall promoting hydrocarbon biodegradation. These multiple roles of iron are comprehensively reviewed in this paper and linked to key functional microorganisms involved in these processes, to the underlying mechanisms, and to the main influential factors. Recent research progress, future perspectives, and remaining challenges on the application of iron-assisted anaerobic hydrocarbon degradation are highlighted.

Corksorb Enhances Alkane Degradation by Hydrocarbonoclastic Bacteria.

Biosorbent materials are effective in the removal of spilled oil from water, but their effect on hydrocarbonoclastic bacteria is not known. Here, we show that corksorb, a cork-based biosorbent, enhances growth and alkane degradation by Rhodococcus opacus B4 (Ro) and Alcanivorax borkumensis SK2 (Ab). Ro and Ab degraded 96 ± 1% and 72 ± 2%, respectively, of a mixture of n-alkanes (2 g L-1) in the presence of corksorb. These values represent an increase of 6 and 24%, respectively, relative to the assays without corksorb. The biosorbent also increased the growth of Ab by 51%. However, no significant changes were detected in the expression of genes involved in alkane uptake and degradation in the presence of corksorb relative to the control without the biosorbent. Nevertheless, transcriptomics analysis revealed an increased expression of rRNA and tRNA coding genes, which confirms the higher metabolic activity of Ab in the presence of corksorb. The effect of corksorb is not related to the release of soluble stimulating compounds, but rather to the presence of the biosorbent, which was shown to be essential. Indeed, scanning electron microscopy images and downregulation of pili formation coding genes, which are involved in cell mobility, suggest that cell attachment on corksorb is a determinant for the improved activity. Furthermore, the existence of native alkane-degrading bacteria in corksorb was revealed, which may assist in situ bioremediation. Hence, the use of corksorb in marine oil spills may induce a combined effect of sorption and stimulated biodegradation, with high potential for enhancing in situ bioremediation processes.

Effect of Sub-Stoichiometric Fe(III) Amounts on LCFA Degradation by Methanogenic Communities.

Long-chain fatty acids (LCFA) are common contaminants in municipal and industrial wastewater that can be converted anaerobically to methane. A low hydrogen partial pressure is required for LCFA degradation by anaerobic bacteria, requiring the establishment of syntrophic relationships with hydrogenotrophic methanogens. However, high LCFA loads can inhibit methanogens, hindering biodegradation. Because it has been suggested that anaerobic degradation of these compounds may be enhanced by the presence of alternative electron acceptors, such as iron, we investigated the effect of sub-stoichiometric amounts of Fe(III) on oleate (C18:1 LCFA) degradation by suspended and granular methanogenic sludge. Fe(III) accelerated oleate biodegradation and hydrogenotrophic methanogenesis in the assays with suspended sludge, with H2-consuming methanogens coexisting with iron-reducing bacteria. On the other hand, acetoclastic methanogenesis was delayed by Fe(III). These effects were less evident with granular sludge, possibly due to its higher initial methanogenic activity relative to suspended sludge. Enrichments with close-to-stoichiometric amounts of Fe(III) resulted in a microbial community mainly composed of Geobacter, Syntrophomonas, and Methanobacterium genera, with relative abundances of 83-89%, 3-6%, and 0.2-10%, respectively. In these enrichments, oleate was biodegraded to acetate and coupled to iron-reduction and methane production, revealing novel microbial interactions between syntrophic LCFA-degrading bacteria, iron-reducing bacteria, and methanogens.

Long-Chain Fatty Acids Degradation by Desulfomonile Species and Proposal of "Candidatus Desulfomonile Palmitatoxidans".

Microbial communities with the ability to convert long-chain fatty acids (LCFA) coupled to sulfate reduction can be important in the removal of these compounds from wastewater. In this work, an enrichment culture, able to oxidize the long-chain fatty acid palmitate (C16 : 0) coupled to sulfate reduction, was obtained from anaerobic granular sludge. Microscopic analysis of this culture, designated HP culture, revealed that it was mainly composed of one morphotype with a typical collar-like cell wall invagination, a distinct morphological feature of the Desulfomonile genus. 16S rRNA gene amplicon and metagenome-assembled genome (MAG) indeed confirmed that the abundant phylotype in HP culture belong to Desulfomonile genus [ca. 92% 16S rRNA gene sequences closely related to Desulfomonile spp.; and ca. 82% whole genome shotgun (WGS)]. Based on similar cell morphology and average nucleotide identity (ANI) (77%) between the Desulfomonile sp. in HP culture and the type strain Desulfomonile tiedjei strain DCB-1T, we propose a novel species designated as "Candidatus Desulfomonile palmitatoxidans." This bacterium shares 94.3 and 93.6% 16S rRNA gene identity with Desulfomonile limimaris strain DCB-MT and D. tiedjei strain DCB-1T, respectively. Based on sequence abundance of Desulfomonile-morphotype in HP culture, its predominance in the microscopic observations, and presence of several genes coding for enzymes involved in LCFA degradation, the proposed species "Ca. Desulfomonile palmitatoxidans" most probably plays an important role in palmitate degradation in HP culture. Analysis of the growth of HP culture and D. tiedjei strain DCB-1T with short- (butyrate), medium- (caprylate) and long-chain fatty acids (palmitate, stearate, and oleate) showed that both cultures degraded all fatty acids coupled to sulfate reduction, except oleate that was only utilized by HP culture. In the absence of sulfate, neither HP culture, nor D. tiedjei strain DCB-1T degraded palmitate when incubated with Methanobacterium formicicum as a possible methanogenic syntrophic partner. Unlike D. tiedjei strain DCB-1T, "Ca. Desulfomonile palmitatoxidans" lacks reductive dehalogenase genes in its genome, and HP culture was not able to grow by organohalide respiration. An emended description of the genus Desulfomonile is proposed. Our study reveals an unrecognized LCFA degradation feature of the Desulfomonile genus.

Toxicity of long chain fatty acids towards acetate conversion by Methanosaeta concilii and Methanosarcina mazei.

Long-chain fatty acids (LCFA) can inhibit methane production by methanogenic archaea. The effect of oleate and palmitate on pure cultures of Methanosaeta concilii and Methanosarcina mazei was assessed by comparing methane production rates from acetate before and after LCFA addition. For both methanogens, a sharp decrease in methane production (> 50%) was observed at 0.5 mmol L(-1) oleate, and no methane was formed at concentrations higher than 2 mmol L(-1) oleate. Palmitate was less inhibitory than oleate, and M. concilii was more tolerant to palmitate than M. mazei, with 2 mmol L(-1) palmitate causing 11% and 64% methanogenic inhibition respectively. This study indicates that M. concilii and M. mazei tolerate LCFA concentrations similar to those previously described for hydrogenotrophic methanogens. In particular, the robustness of M. concilii might contribute to the observed prevalence of Methanosaeta species in anaerobic bioreactors used to treat LCFA-rich wastewater.

Continuous high rate anaerobic treatment of oleic acid based wastewater is possible after a step feeding start-up.

Mineralization of a synthetic effluent containing 50% COD as oleic acid was achieved in a continuous anaerobic reactor at organic loading rates up to 21 kg COD m(-3) day(-1), HRT of 9 h, attaining 99% of COD removal efficiency and a methane yield higher than 70%. A maximum specific methane production rate of 1170 +/- 170 mg COD-CH4 g VS(-1) day(-1) was measured during the reactor's operation. A start-up strategy combining feeding phases and batch degradation phases was applied to promote the development of an anaerobic community efficient for long chain fatty acids (LCFA) mineralization. Through the start-up period, the methane yield increased gradually from 67% to 91%, and LCFA accumulated onto the sludge only during the first 60 days of operation. For the first time, it is demonstrated that a step feeding start-up is required to produce a specialized and efficient anaerobic community for continuous high rate anaerobic treatment of LCFA-rich wastewater.