From grass to gas and beyond: Anaerobic digestion as a key enabling technology for a residual grass biorefinery.

Roadside grass clippings hold potential as a sustainable source of bioenergy as they do not compete with crops for land use, and are only partially utilized for low-value applications. In this study, we proposed using roadside grass as a sole feedstock for anaerobic digestion (AD) in three different settings, and assessed the potential of producing biomaterials and fertilizers from grass-based digestate. Wet continuous digestion at pilot scale and dry batch digestion at pilot and large scales resulted in biogas yields up to 700 Nm3.t-1 DOM with a methane content of 49-55 %. Despite promising results, wet AD had operational problems such as clogging and poor mixing; once upscaled, the dry digestion initially also presented an operational problem with acidification, which was overcome by the second trial. Digested grass fibers from the pilot dry AD were processed into biomaterials and performed similarly or better than the undigested fibers, while around 20 % performance reduction was observed when compared to reference wood fibers. A mass balance indicated reduced fiber recovery when higher biogas production was obtained. The liquid fraction from the pilot dry AD was characterized for its nutrient content and used as a biofertilizer in another study. In contrast, the leachate collected from the large-scale dry AD had a low nitrogen content and high chloride content that could hinder its further use. Finally, a regional market analysis was conducted showing that the biocomposites produced with the available grass fibers could substitute at least half of the current European market based on our results.

Industrial hemp (Cannabis sativa L.) field cultivation in a phytoattenuation strategy and valorization potential of the fibers for textile production.

This paper evaluates the valorization potential of industrial hemp (Cannabis sativa L.) fibers produced on HM-contaminated soil as a safe feedstock for the textile industry. The chosen strategy was phytoattenuation, which combines the progressive soil quality improvement of contaminated land using phytoremediation techniques with the production of safe non-food biomass. A field experiment was set up with two hemp cultivars on a site contaminated with Cd, Pb, and Zn and on a nearby site containing clean soil as a control. Stem height and diameter were analyzed, as well as stem and fiber yield and the HM concentrations in the fibers, which were compared to legal safety standards and toxicity thresholds used in the textile industry. The hemp cultivar Carmagnola Selected (CS) had a significantly higher stem and bigger stem diameter compared to cultivar USO 31 on both sites. Stem yields showed a decrease of 30% and 50%, respectively, for both hemp cultivars grown on the contaminated site. However, the stem yield of CS growing on the contaminated site was similar to the stem yield of USO 31 growing on the control site, indicating that hemp cultivation on contaminated soil can be economically viable. Total and extractable Cd, Pb, and Zn fiber concentrations were far below the toxicity standards for textile production purposes. These results are promising in terms of the potential valorization of contaminated land with hemp cultivation and the development of a non-food value chain within a phytoattenuation strategy.

Maximizing nutrient recycling from digestate for production of protein-rich microalgae for animal feed application.

The integration of phototrophic microalgal production and anaerobic digestion can recycle excess nutrients across European surplus hotspots to produce protein-rich biomass for nutritional applications. However, the challenging physico-chemical properties of raw digestate constrain microalgal growth and limit digestate valorization potential. This study focused on the pre-treatment of food waste-based digestate using paper-filtration to improve its properties for cultivating Desmodesmus sp. and Chlorella vulgaris. The microalgal growth performance in paper-filtered digestate (PFD, 10 µm-pore size) was then compared to growth in membrane-filtered digestate (MFD, 0.2 µm-pore size). A microplate-based screening coupled with Cytation device assessment of PFD and MFD samples after dilution and with/without phosphorus supplementation showed that PFD was the best substrate. Moreover, phosphorus supplementation resulted in improved growth at higher digestate concentrations (5-10% v/v PFD), indicating the importance of using a balanced growth medium to increase the volumetric usage of digestate. Results were validated in a 3-L bioreactor at 10% PFD with phosphorus supplementation, reaching a biomass concentration of 2.4 g L-1 with a protein and carbohydrate content of 67% and 13% w/w respectively. This trial indicates that paper-filtration is a promising pre-treatment technique to maximize digestate recycling and deliver a sustainable animal feed-grade protein alternative.

Impact of time and phosphorus application rate on phosphorus bioavailability and efficiency of secondary fertilizers recovered from municipal wastewater.

Demand for phosphorus (P) resources other than non-renewable P rock has driven the development of several P recovery technologies from municipal wastewater treatment and directed recovery of P into valuable fertilizers (struvite, ash, iron phosphate, etc.). Although the bioavailability of novel secondary P fertilizers has been examined in previous studies, insufficient attention has been paid to defining optimal plant growth duration and monitoring conditions to assess the dynamic changes in P. Accordingly, five fertilizers recovered from municipal wastewater: two struvites (STRSL and STRLQ), two ashes (ASH1 and ASH2), and iron-phosphate pelletized sludge (FeP) using triple superphosphate (TSP) as a positive control and blank (zero P) as a negative control, were applied to P poor-sand at three P doses (equivalent to 30, 60, and 90 kg P2O5 ha-1). Fertilizer impact on perennial ryegrass (Lolium perenne) dry matter (DM) and P concentration were evaluated on a monthly basis for seven months. DM and relative agronomical efficiency (RAE) have shown the same trend between the fertilizers, but only at the lowest P dose (corresponding to 30 kg P2O5 ha-1). At higher P doses (60, and 90 kg P2O5 ha-1) the differences in DM and RAE among the fertilizers diminished. STRLQ, STRSL, ASH1 and FeP expressed a rather steady P release pattern, while ASH2 had a delay of four cuts and increase afterward. Monitoring the P uptake during four months of perennial ryegrass growth turned out to be the minimum, and seven months the optimum period for reaching the full capacity of the slow-release P fertilizers.

Looking for phosphate-accumulating bacteria in activated sludge processes: a multidisciplinary approach.

Over the past decades, an increasing need in renewable resources has progressively appeared. This trend concerns not only fossil fuels but also mineral resources. Wastewater and sewage sludge contain significant concentrations in phosphate and can be considered as a fertilizer source of the utmost importance. In wastewater treatment plants, the biological uptake of phosphate is performed by a specific microbiota: the phosphate-accumulating organisms. These microorganisms are recovered in sewage sludge. Here, we aimed to investigate the occurrence of phosphate accumulators in four wastewater treatment plants. A 16S metagenetic analysis identified the main bacterial phyla extracted from the aerobic treatment: α-Proteobacteria, ß-Proteobacteria, and Sphingobacteria. An enrichment stage was performed to stimulate the specific growth of phosphate-accumulating bacteria in an acetate medium. An analysis of metabolic activities of sulfur and phosphorus highlighted strong modifications related to phosphorus and much less distinguishable effects with sulfur. A solid acetate medium containing 5-Br-4-Cl-3-indolyl phosphate was used to select potential phosphate-accumulating bacteria from the enriched consortia. The positive strains have been found to belong in the genera Acinetobacter, Corynebacterium, and Pseudomonas. Finally, electron microscopy was applied to the strains and allowed to confirm the presence of polyphosphate granules. Some of these bacteria contained granules the size of which exceeded 100 nm.

Characterisation of Phosphate Accumulating Organisms and Techniques for Polyphosphate Detection: A Review.

Phosphate minerals have long been used for the production of phosphorus-based chemicals used in many economic sectors. However, these resources are not renewable and the natural phosphate stocks are decreasing. In this context, the research of new phosphate sources has become necessary. Many types of wastes contain non-negligible phosphate concentrations, such as wastewater. In wastewater treatment plants, phosphorus is eliminated by physicochemical and/or biological techniques. In this latter case, a specific microbiota, phosphate accumulating organisms (PAOs), accumulates phosphate as polyphosphate. This molecule can be considered as an alternative phosphate source, and is directly extracted from wastewater generated by human activities. This review focuses on the techniques which can be applied to enrich and try to isolate these PAOs, and to detect the presence of polyphosphate in microbial cells.

New perspectives for the design of sustainable bioprocesses for phosphorus recovery from waste.

Phosphate rock has long been used for the production of phosphorus based chemicals. However, considering the depletion of the reservoirs and the decrease of the quality of phosphate rocks, a potential market is now emerging for the recovery of phosphate from waste and its reuse for different applications. Notably, phosphate recovery from wastewater could be included in a circular economy approach. This review focuses on the use of microbial systems for phosphorus accumulation and recovery, by considering the actual range of analytical techniques available for the monitoring of phosphorus accumulating organisms, as well as the actual biochemical and metabolic engineering toolbox available for the optimization of bioprocesses. In this context, knowledge gathered from process, system and synthetic biology could potentially lead to innovative process design.

Environmental assessment of digestate treatment technologies using LCA methodology.

The production of biogas from energy crops, organic waste and manure has augmented considerably the amounts of digestate available in Flanders. This has pushed authorities to steadily introduce legislative changes to promote its use as a fertilising agent. There is limited arable land in Flanders, which entails that digestate has to compete with animal manure to be spread. This forces many anaerobic digestion plants to further treat digestate in such a way that it can either be exported or the nitrogen be removed. Nevertheless, the environmental impact of these treatment options is still widely unknown, as well as the influence of these impacts on the sustainability of Flemish anaerobic digestion plants in comparison to other regions where spreading of raw digestate is allowed. Despite important economic aspects that must be considered, the use of Life Cycle Assessment (LCA) is suggested in this study to identify the environmental impacts of spreading digestate directly as compared to four different treatment technologies. Results suggest relevant environmental gains when the digestate mix is treated using the examined conversion technologies prior to spreading, although important trade-offs between impact categories were observed and discussed. The promising results of digestate conversion technologies suggest that further LCA analyses should be performed to delve into, for instance, the appropriateness to shift to nutrient recovery technologies rather than digestate conversion treatments.

Chromosome 3p microsatellite allelotyping in neuroblastoma: a report on the technical hurdles.

Pinpointing critical regions of recurrent loss may help localize tumor suppressor genes. To determine the regions of loss on chromosome 3p in neuroblastoma, we performed loss of heterozygosity analysis using 16 microsatellite markers in a series of 65 primary tumors and 29 neuroblastoma cell lines. In this study, we report the results and discuss the technical hurdles that we encountered during data generation and interpretation that are of relevance for current studies or tests employing microsatellites. To provide functional support for the implication of 3p tumor suppressor genes in this childhood malignancy, we performed a microcell-mediated chromosome 3 transfer in neuroblastoma cells.

Overall genomic pattern is a predictor of outcome in neuroblastoma.

PURPOSE: For a comprehensive overview of the genetic alterations of neuroblastoma, their association and clinical significance, we conducted a whole-genome DNA copy number analysis. PATIENTS AND METHODS: A series of 493 neuroblastoma (NB) samples was investigated by array-based comparative genomic hybridization in two consecutive steps (224, then 269 patients). RESULTS: Genomic analysis identified several types of profiles. Tumors presenting exclusively whole-chromosome copy number variations were associated with excellent survival. No disease-related death was observed in this group. In contrast, tumors with any type of segmental chromosome alterations characterized patients with a high risk of relapse. Patients with both numerical and segmental abnormalities clearly shared the higher risk of relapse of segmental-only patients. In a multivariate analysis, taking into account the genomic profile, but also previously described individual genetic and clinical markers with prognostic significance, the presence of segmental alterations with (HR, 7.3; 95% CI, 3.7 to 14.5; P < .001) or without MYCN amplification (HR, 4.5; 95% CI, 2.4 to 8.4; P < .001) was the strongest predictor of relapse; the other significant variables were age older than 18 months (HR, 1.8; 95% CI, 1.2 to 2.8; P = .004) and stage 4 (HR, 1.8; 95% CI, 1.2 to 2.7; P = .005). Finally, within tumors showing segmental alterations, stage 4, age, MYCN amplification, 1p and 11q deletions, and 1q gain were independent predictors of decreased overall survival. CONCLUSION: The analysis of the overall genomic pattern, which probably unravels particular genomic instability mechanisms rather than the analysis of individual markers, is essential to predict relapse in NB patients. It adds critical prognostic information to conventional markers and should be included in future treatment stratification.

Aberrant methylation of candidate tumor suppressor genes in neuroblastoma.

CpG island hypermethylation has been recognized as an alternative mechanism for tumor suppressor gene inactivation. In this study, we performed methylation-specific PCR (MSP) to investigate the methylation status of 10 selected tumor suppressor genes in neuroblastoma. Seven of the investigated genes (CD44, RASSF1A, CASP8, PTEN, ZMYND10, CDH1, PRDM2) showed high frequencies (> or =30%) of methylation in 33 neuroblastoma cell lines. In 42 primary neuroblastoma tumors, the frequencies of methylation were 69%, CD44; 71%, RASSF1A; 56%, CASP8; 25%, PTEN; 15%, ZMYND10; 8%, CDH1; and 0%, PRDM2. Furthermore, CASP8 and CDH1 hypermethylation was significantly associated with poor event-free survival. Meta-analysis of 115 neuroblastoma tumors demonstrated a significant correlation between CASP8 methylation and MYCN amplification. In addition, there was a correlation between ZMYND10 methylation and MYCN amplification. The MSP data, together with optimized mRNA re-expression experiments (in terms of concentration and time of treatment and use of proper reference genes) further strengthen the notion that epigenetic alterations could play a significant role in NB oncogenesis. This study thus warrants the need for a global profiling of gene promoter hypermethylation to identify genome-wide aberrantly methylated genes in order to further understand neuroblastoma pathogenesis and to identify prognostic methylation markers.

CADM1 is a strong neuroblastoma candidate gene that maps within a 3.72 Mb critical region of loss on 11q23.

BACKGROUND: Recurrent loss of part of the long arm of chromosome 11 is a well established hallmark of a subtype of aggressive neuroblastomas. Despite intensive mapping efforts to localize the culprit 11q tumour suppressor gene, this search has been unsuccessful thus far as no sufficiently small critical region could be delineated for selection of candidate genes. METHODS: To refine the critical region of 11q loss, the chromosome 11 status of 100 primary neuroblastoma tumours and 29 cell lines was analyzed using a BAC array containing a chromosome 11 tiling path. For the genes mapping within our refined region of loss, meta-analysis on published neuroblastoma mRNA gene expression datasets was performed for candidate gene selection. The DNA methylation status of the resulting candidate gene was determined using re-expression experiments by treatment of neuroblastoma cells with the demethylating agent 5-aza-2'-deoxycytidine and bisulphite sequencing. RESULTS: Two small critical regions of loss within 11q23 at chromosomal band 11q23.1-q23.2 (1.79 Mb) and 11q23.2-q23.3 (3.72 Mb) were identified. In a first step towards further selection of candidate neuroblastoma tumour suppressor genes, we performed a meta-analysis on published expression profiles of 692 neuroblastoma tumours. Integration of the resulting candidate gene list with expression data of neuroblastoma progenitor cells pinpointed CADM1 as a compelling candidate gene. Meta-analysis indicated that CADM1 expression has prognostic significance and differential expression for the gene was noted in unfavourable neuroblastoma versus normal neuroblasts. Methylation analysis provided no evidence for a two-hit mechanism in 11q deleted cell lines. CONCLUSION: Our study puts CADM1 forward as a strong candidate neuroblastoma suppressor gene. Further functional studies are warranted to elucidate the role of CADM1 in neuroblastoma development and to investigate the possibility of CADM1 haploinsufficiency in neuroblastoma.

Low-cost dedicated mini-arrays for high-throughput analysis of DNA copy-number alterations in neuroblastoma.

ArrayCGH is commonly used for high-resolution detection of copy-number alterations in tumours, allowing identification of chromosomal aberrations with prognostic or diagnostic relevance. Currently available arrayCGH platforms are still very expensive for analysis of large sets of samples. For this purpose, we have constructed a dedicated mini-array that is enriched for BAC/PAC clones in the prognostic important regions for neuroblastoma and that only covers a small area on the slide, allowing down-scaling of the labelling and hybridisation reagents and hence reducing the price. The mini-arrays were validated on neuroblastoma samples and comparison with high-resolution whole-genome arrayCGH data yielded complete concordant results.

Identification of 2 putative critical segments of 17q gain in neuroblastoma through integrative genomics.

Partial gain of chromosome arm 17q is the most frequent genetic change in neuroblastoma (NB) and constitutes the strongest independent genetic factor for adverse prognosis. It is assumed that 1 or more genes on 17q contribute to NB pathogenesis by a gene dosage effect. In the present study, we applied chromosome 17 tiling path BAC arrays on a panel of 69 primary tumors and 28 NB cell lines in order to reduce the current smallest region of gain and facilitate identification of candidate dosage sensitive genes. In all tumors and cell lines with 17q gain, large distal segments were consistently present in extra copies and no interstitial gains were observed. In addition to these large regions of distal gain with breakpoints proximal to coordinate 44.3 Mb (17q21.32), smaller regions of gain (distal to coordinate 60 Mb at 17q24.1) were found superimposed on the larger region in a minority of cases. Positional gene enrichment analysis for 17q genes overexpressed in NB showed that dosage sensitive NB oncogenes are most likely located in the gained region immediately distal to the most distal breakpoint of the 2 breakpoint regions. Interestingly, comparison of gene expression profiles between primary tumors and normal fetal adrenal neuroblasts revealed 2 gene clusters on chromosome 17q that are overexpressed in NB, i.e. a region on 17q21.32 immediately distal to the most distal breakpoint (in cases with single regions of gain) and 17q24.1, a region coinciding with breakpoints leading to superimposed gain.

Detection of DNA copy number alterations in cancer by array comparative genomic hybridization.

Over the past few years, various reliable platforms for high-resolution detection of DNA copy number changes have become widely available. Together with optimized protocols for labeling and hybridization and algorithms for data analysis and representation, this has lead to a rapid increase in the application of this technology in the study of copy number variation in the human genome in normal cells and copy number imbalances in genetic diseases, including cancer. In this review, we briefly discuss specific technical issues relevant for array comparative genomic hybridization analysis in cancer tissues. We specifically focus on recent successes of array comparative genomic hybridization technology in the progress of our understanding of oncogenesis in a variety of cancer types. A third section highlights the potential of sensitive genome-wide detection of patterns of DNA imbalances or molecular portraits for class discovery and therapeutic stratification.

ArrayCGH-based classification of neuroblastoma into genomic subgroups.

High-resolution array comparative genomic hybridization (arrayCGH) profiling was performed on 75 primary tumors and 29 cell lines to gain further insight into the genetic heterogeneity of neuroblastoma and to refine genomic subclassification. Using a novel data-mining strategy, three major and two minor genomic subclasses were delineated. Eighty-three percent of tumors could be assigned to the three major genomic subclasses, corresponding to the three known clinically and biologically relevant subsets in neuroblastoma. The remaining subclasses represented (1) tumors with no/few copy number alterations or an atypical pattern of aberrations and (2) tumors with 11q13 amplification. Inspection of individual arrayCGH profiles showed that recurrent genomic imbalances were not exclusively associated with a specific subclass. Of particular notice were tumors with numerical imbalances typically observed in subtype 1 neuroblastoma, in association with genomic features of subtype 2A or 2B. A search for prognostically relevant genomic alterations disclosed 1q gain as a predictive marker for therapy failure within the group of subtype 2A and 2B tumors. In cell lines, a high incidence of 6q loss was observed, with a 3.87-5.32 Mb region of common loss within 6q25.1-6q25.2. Our study clearly illustrates the importance of genomic profiling in relation to tumor behavior in neuroblastoma. We propose that genome-wide assessment of copy number alterations should ideally be included in the genetic workup of neuroblastoma. Further multicentric studies on large tumor series are warranted in order to improve therapeutic stratification in conjunction with other features such as age at diagnosis, tumor stage, and gene expression signatures.

High resolution tiling-path BAC array deletion mapping suggests commonly involved 3p21-p22 tumor suppressor genes in neuroblastoma and more frequent tumors.

The recurrent loss of 3p segments in neuroblastoma suggests the implication of 1 or more tumor suppressor genes but thus far few efforts have been made to pinpoint their detailed chromosomal position. To achieve this goal, array-based comparative genomic hybridization was performed on a panel of 23 neuroblastoma cell lines and 75 primary tumors using a tiling-path bacterial artificial chromosome array for chromosome 3p. A total of 45 chromosome 3 losses were detected, including whole chromosome losses, large terminal deletions and interstitial deletions. The latter, observed in cell lines as well as a number of distal deletions detected in primary tumors, allowed us to demarcate 3 minimal regions of loss of 3.6 Mb [3p21.31-p21.2, shortest regions of overlap (SRO)1], 1.4 Mb (3p22.3-3p22.2, SRO2) and 3.8 Mb (3p25.3-p25.1, SRO3) in size. The present data significantly extend previous findings and now firmly establish critical regions on 3p implicated in neuroblastoma. Interestingly, the 2 proximal regions coincide with previously defined SROs on 3p21.3 in more frequent tumors including lung and breast cancer. As such, similar tumor suppressor genes may play a critical role in development or progression of a variety of neoplasms, including neuroblastoma.

methBLAST and methPrimerDB: web-tools for PCR based methylation analysis.

BACKGROUND: DNA methylation plays an important role in development and tumorigenesis by epigenetic modification and silencing of critical genes. The development of PCR-based methylation assays on bisulphite modified DNA heralded a breakthrough in speed and sensitivity for gene methylation analysis. Despite this technological advancement, these approaches require a cumbersome gene by gene primer design and experimental validation. Bisulphite DNA modification results in sequence alterations (all unmethylated cytosines are converted into uracils) and a general sequence complexity reduction as cytosines become underrepresented. Consequently, standard BLAST sequence homology searches cannot be applied to search for specific methylation primers. RESULTS: To address this problem we developed methBLAST, a sequence similarity search program, based on the original BLAST algorithm but querying in silico bisulphite modified genome sequences to evaluate oligonucleotide sequence similarities. Apart from the primer specificity analysis tool, we have also developed a public database termed methPrimerDB for the storage and retrieval of validated PCR based methylation assays. The web interface allows free public access to perform methBLAST searches or database queries and to submit user based information. Database records can be searched by gene symbol, nucleotide sequence, analytical method used, Entrez Gene or methPrimerDB identifier, and submitter's name. Each record contains a link to Entrez Gene and PubMed to retrieve additional information on the gene, its genomic context and the article in which the methylation assay was described. To assure and maintain data integrity and accuracy, the database is linked to other reference databases. Currently, the database contains primer records for the most popular PCR-based methylation analysis methods to study human, mouse and rat epigenetic modifications. methPrimerDB and methBLAST are available at http://medgen.ugent.be/methprimerdb and http://medgen.ugent.be/methblast. CONCLUSION: We have developed two integrated and freely available web-tools for PCR based methylation analysis. methBLAST allows in silico assessment of primer specificity in PCR based methylation assays that can be stored in the methPrimerDB database, which provides a search portal for validated methylation assays.

arrayCGHbase: an analysis platform for comparative genomic hybridization microarrays.

BACKGROUND: The availability of the human genome sequence as well as the large number of physically accessible oligonucleotides, cDNA, and BAC clones across the entire genome has triggered and accelerated the use of several platforms for analysis of DNA copy number changes, amongst others microarray comparative genomic hybridization (arrayCGH). One of the challenges inherent to this new technology is the management and analysis of large numbers of data points generated in each individual experiment. RESULTS: We have developed arrayCGHbase, a comprehensive analysis platform for arrayCGH experiments consisting of a MIAME (Minimal Information About a Microarray Experiment) supportive database using MySQL underlying a data mining web tool, to store, analyze, interpret, compare, and visualize arrayCGH results in a uniform and user-friendly format. Following its flexible design, arrayCGHbase is compatible with all existing and forthcoming arrayCGH platforms. Data can be exported in a multitude of formats, including BED files to map copy number information on the genome using the Ensembl or UCSC genome browser. CONCLUSION: ArrayCGHbase is a web based and platform independent arrayCGH data analysis tool, that allows users to access the analysis suite through the internet or a local intranet after installation on a private server. ArrayCGHbase is available at http://medgen.ugent.be/arrayCGHbase/.