WO2018124988A1

WO2018124988A1 - A method for improvement of methane production from microalgae

Info

Publication number: WO2018124988A1
Application number: PCT/TR2016/050558
Authority: WO
Inventors: Orhan INCE; Bahar INCE; Sevcan AYDIN; Elif YILDIRIM
Original assignee: Istanbul Teknik Universitesi
Priority date: 2016-12-28
Filing date: 2016-12-28
Publication date: 2018-07-05
Also published as: ES2743548A1; ES2743548B1

Abstract

The invention is particularly related with a method, which use bioaugmentation with anaerobic rumen fungi at varied ratios of inoculums on the performance of anaerobic digesters of microalgae biomass for increasing methane production. In that proposed method the composite obtain that 4 isolated species of anaerobic rumen fungi of Orpinomyces sp., Piromyces sp. And Anaeromyces sp., Neocallimastix frontalis were selected and these species were mixed. After that, mixture of rumen fungi containing 4 species was added into the anaerobic digesters fed with microalgae Haematoccus pluvialis at different inoculums ratios: %1 (F1), 5% (F2), 10% (F3), 15% (F4) and 20% (F5) (v/v).

Description

A METHOD FOR IMPROVEMENT OF METHANE PRODUCTION

FROM MICROALGAE

Technical Field

The invention is related with improvement of methane production with using anaerobic digesters of microalgae biomass

The invention is particularly related with a method, which use bioaugmentation with anaerobic rumen fungi at varied ratios of inoculums on the performance of anaerobic digesters of microalgae biomass for increasing methane production. Prior Art

As microalgae have high photo synthetic performance, advanced growth rates and the characteristic of not requiring an external organic carbon source, it is thought that microalgae are potential sources for bioenergy and biofuel production. Production of biogas comprising hydrogen or methane from anaerobic digestion of algae is currently rather conspicuous technology because they have the capacity for energy conservation and environmentally friendly features. In addition to environmental conditions promoting microbial activity, degradation of substrates is quite an important parameter through anaerobic process.

There are some developments present in the known state of the art that have been provide to bioenergy from the biomass.

For example in the Russian Patent document numbered RU2419594C1 within the known state of the art, the invention relates to agricultural production, particularly, to complete treatment and reclamation of animal framing wastes to produce electric and thermal power, circulation water and fertilisers. Liquid phase of over fermented dropping is evaporated to dry concentrated fertiliser. Note here that steam is converted to water to be used for process needs. Portion of homogeneous mass is combusted to clean obtained biogas by passing its through water to produce biomethane to be fed to consumer. Water is saturated with organic substances to be used as liquid fertiliser. Air from production premises is collected to facilitate combustion of said homogeneous mass with increased heat emission. Residue of combustion is used as a mineral fertiliser. Off-gases are cleaned from solid volatile admixtures by passing them through water and saturating with mineral substances for use as mineral fertilisers. Purified off-gas is used to generate electric power to be fed to green houses.

In the Global Patent document numbered WO2015044721A1 within the known state of the art, the invention relates to downstream processing of microalgal biomass to produce different products in a biorefinery process. The invention establishes interconnected stages from harvesting of microalgal biomass, following several productive processes, including alternatives to use the remaining biomass. The invention allows improving the overall downstream process by adapting each stage of the production process for a complete microalgal biomass use producing various added-value products of commercial interest: proteins, biodiesel, and biogas or biomethane. In addition, the invention includes wastewater reutilization alternatives to be reused in the same processes. The invention has application in processing biomass such as microalgae and other biomass types, for the production of biofuels and co-products.

In the Global Patent document numbered W09325671A1 within the known state of the art, a method of cloning of xylanase clones from an anaerobic rumen fungus including the steps of: (I) cultivation of an anaerobic rumen fungus; (II) isolating total RNA from the culture in step (III); (III) isolating poly A<+> mRNA from the total RNA referred to in step (II); (IV) constructing a cDNA expression library; (V) ligating cDNA to a bacteriophage expression vector selected from lambda ZAP, lambda ZAPII or vectors of similar properties; (VI) screening of xylanase positive recombinant clones in a culture medium incorporating xylan by detection of xylan hydrolysis; and (VII) purifying xylanase positive recombinant clones. There is also provided xylanase positive recombinant clones produced by the above-mentioned method as well as xylanase positive recombinant clones having the following properties: (I) production of xylan clearing zones in a culture containing xylanase cDNA derived from N. patriciarum; (II) having activity in hydrolysis of xylan but having no activity in relation to hydrolysis of CMC or crystalline cellulose. There is also provided various cDNA molecules which may be utilised in the above-mentioned method.

Anaerobic digestion can take place directly on algae following new collection or microalgal wastes after lipid extraction. With regard to the former, the resistance of the microalgal cell wall may be a significant restrictive factor for cell digestibility. Certain microalgal species, like Haematococcus sp. and Chlorella sp., feature recalcitrant cellulose in the cell wall, and it protects microalgae against invasion of enzymes and also limits algal biodegradability. Bioagumentation with a combination of rumen fungi in anaerobic process can represent an appropriate alternative to the use of chemical pre-treatments of microalgae biomass. Thus, anaerobic rumen fungi have promise for enhancing biogas production from different microalgae and macroalgae species and also various lignocellulosic substrates.

Brief Description of the Invention and its Aims

The aim of this invention is improving of bioenergy potential of anaerobic digesters of microalgae with using of rumen fungi.

Another aim of this invention is to improve the degradation of algal biomass by bioaugmentation using rumen anaerobic fungi. Another aim of this invention is to gain improvement of biomethane production from biomass of microalgae according to bioaugmentation with anaerobic rumen fungi at varied ratios of inoculums on the performance of anaerobic digesters. In this method, Anaerobic rumen fungi, Anaeromyces, Neocallimastix, Orpinomyces and Piromyces, were used and have groups of genes that originate from bacteria by the way of horizontal gene transfer. The results imply that rumen fungi improved the fermentation and degradation of microalgae biomass because they fostered cell wall degradation while methane production increased of 41% because of bioaugmentation with rumen fungi during anaerobic processes. Overall, the findings here indicate that bioaugmentation with a combination of rumen fungi in anaerobic process can represent an appropriate alternative to the use of chemical pre-treatments of microalgae biomass. Thus, anaerobic rumen fungi have promise for enhancing biogas production from different microalgae and macroalgae species and also various lignocellulosic substrates.

Detailed Description of the Invention

In this method of the invention firstly, rumen fluid was taken via rumen fistulae from a cow. The fluid was analysed through metagenomic analysis in order to determine the specific anaerobic rumen fungi within it. Isolated and cultivated rumen fungi were evaluated with strain identification and phylogenetic analysis techniques so as to characterize the species of anaerobic rumen fungi. Four species were isolated that had high lignocellulose-degrading enzyme expression and were selected and mixed. Afterwards, this mixture was added in the anaerobic digesters fed with microalgae H. pluvialis at different inoculums ratios: %1 (Fl), 5% (F2), 10% (F3), 15% (F4) and 20% (F5) (v/v). In order to understand the effect of anaerobic rumen fungi on biogas production, one digester was not biougmentated with anaerobic rumen fungi as a control digester: 0% (F0). Anaerobic digesters fed with microalgae were set up semi-continuously with 2000 ml volumes over 40 days at 41 °C. Reactors were operated in duplicate under the same conditions. Performance of anaerobic digesters was evaluated via biogas and biomethane production. The inhibitory effect of the digesters was controlled with measurement of volatile fatty acids (VFAs). Finally, microbial community dynamics during the anaerobic digestion process were distinguished according to Illumina Miseq and qPCR analyses. Samples for all rumen content comprising fluid and solids were taken via rumen fistulae from a cow (live weight: 400-450 kg) with confidential techniques by veterinarians. Cows were older than two years and fed with alfalfa hay, barely grass, legumes, silage and soybean meal during the summer and winter periods. All samples of ruminal fluid were flushed with nitrogen gas (N2) so as to provide anaerobic conditions after loading and sealing. A number of the samples of rumen fluid were stored at -20 °C in order to extract DNA for subsequent metagenomic survey of rumen fluid.

Metagenomics, also known as environmental genomics, was employed for determination of the abundance and identity of rumen fungi in a sample. The metagenomic survey, based on total purified DNA, was comprehensively investigated in species of rumen fungi classified by gene function and pathway analysis at the DNA level. Initially, qualified DNA samples were sheared into smaller fragments by nebulization. Next, T4 DNA polymerase, Klenow fragment and T4 polynucleotide kinase converted the results of the fragmentation into blunt ends. After addition of an Adenine (A) base to the 3' end of the blunt phosphorylated DNA fragments, the ends of the DNA fragments were linked with adapters. At that point, the short fragments were removed with Ampure beads. The sample libraries were qualified and quantified with an Agilent 2100 Bioanaylzer and an ABI StepOnePlus Real-Time PCR System.

The qualified libraries were sequenced via the Illumina HiSeqTM platform. The ABI StepOnePlus Real-Time PCR System was utilised to qualify and quantify the sample libraries. These libraries were then sequenced via the Illumina HiSeqTM platform. Qualified sequencing reads produced by the Illumina platform were preprocessed and then assembled de novo with SOAPdenovo2 and Rabbit. MetaGeneMark was then employed to predict genes from assembled contigs, building a project-specific gene catalogue. Pre-processed reads were also mapped to the IGC database and mapped genes were retrieved and integrated into the gene catalogue. CD-Hit was used to eliminate redundancy. The gene catalogue was blasted against public databases, including nr, Swiss-Prot, COG, KEGG, GO, CAZy, eggNOG and ARDB to obtain functional and taxonomic annotations. Before isolation of anaerobic rumen fungi, in order to culture them, complex media was prepared using previously described protocols. Two different salt solutions were prepared to use in the media. While salt solution I involved (g/L) KH2P04 - 3.0, ( H)2SO - 3.0, NaCl - 6.0, MgSO - 0.6 and CaCl - 0.6, salt solution II included K2HP04 (3 g/L). Salt solution I featured 150 ml of salt solution II, 150 ml of centrifuged rumen fluid, 200 ml of Bactocasitone (Difco), 10 g of yeast extract (Oxoid), 2.5 g of NaHCO, 6 g of L-cysteine. lg of HCl, 2g of fructose, 2 g of xylose, 2 g of cellobiose, 8 g of trace elements solution, 10 ml of haemin solution, 10 ml of resazurin solution (0.1 %, w/v) and 1 ml of deionized water to 900 ml were added. The media was then autoclaved for 20 min at 115 °C. After autoclaving, 0.1 % (v/v) of two different vitamin solutions were added to the media. Vitamin solution I contained (g/L): thiamin HCl, 0.10; riboflavin, 0.20; D-calcium pantothenate, 0-60; nicotinic acid, 1.00; nicotinamide, 1.00; folic acid, 0.05; cyanocobalamin, 0.20; biotin, 0.20; pyridoxine.HCl, 0.10; and paminobenzoic acid, 0.01. Vitamin solution II featured (mg/L): thiamin HCl, 5; riboflavin, 5; D-calcium pantothenate, 5; nicotinic acid, 5; folic acid, 2; cyanocobalamin, 1 ; biotin, 1 ; pyridoxine HCl, 10; paminobenzoic acid, 5. Antibiotics solution 0.1 % (v/v) containing penicillin (5 g/L), streptomycin (5 g/L), neomycin (5 g/L) and chloramphenicol (5 g/L) was added to the isolation media to suppress bacterial growth. After media preparation, all cultures were incubated under C02 at 39 °C over the course of a week to reproduce rumen fungi. Fungal DNA was sequenced with strain identification and phylogenetic analysis to identify the species isolated from ruminal fluid and cow manure. This analysis was carried with the complete internally transcribed spacer (ITS; partial 18S, complete ITS 1, 5.8S, ITS 2, and partial 28S) and D1/D2 domain at the 5' end of the large-subunit (LSU) ribosomal DNA being amplified using the primer pairs, ITS1 (5'- TCC GTA GGT GAA CCT GCG G-3 ')/ITS4 (5'- TCC TCC GCT TAT TGA TAT GC-3 ') and L1 5'-GCA TAT CAA TAA GCG GAG GAA AAG- 3')/NL4 (5'-GGT CCG TGT TTC AAG ACG G-3'). The consensus sequences, CATTA/CAACTTCAG (end of 18S/start of 5.8S) and GAGTGTCATTA/TTGACCTCAAT (end of 5.8S/start of 28S) were employed to limit the different regions of the rRNA locus in a consistent manner as suggested by Hibbett (2016). The Geneious v6 Bioinformatics package using MAFFT for sequence alignment and Mr Bayes for phylogenetic analysis were applied to conduct the phylogenetic reconstruction.

2% C02-enriched air was photoautotrophically utilised to cultivate the H. pluvialis strain SCCAP 34/7. Bold basal medium with three-fold N2 and vitamins (3N-BBM+V; CCAP 2015) at 25 ⁰ C was used to grow microalgae cells in a 2 L photo bioreactor system. A 9" x 9" x 9" chamber with 8000-10000 lux LED lights was the source of light. After incubation, the micro algal biomass was obtained, which was concentrated by centrifugation at 3600 x g for 15 min. The concentrated algal biomass featured 11% (on wet-weight basis) of total solid (TS), 91.6% (on dry weight basis) of volatile solid (VS) and 44, 16 and 26% (on dry weight basis) of proteins, lipids, and carbohydrates, respectively.

Granular sludge that was cultivated in a laboratory-scale (1.5 L) anaerobic sequenced batch reactor (ASBR) was employed for the methanogenic inoculums. The ASBR had a temperature of 41°C, and glucose and acetate (80%:20%, calculated as COD) were used as the feedstock at an organic loading rate of 1 g COD/(L-day). Different initial concentrations of H. pluvialis (2 g VS/L of the algal biomass) and 3 g VS/L of methanogenic sludge were employed for the batch experiments performed at 41 °C. The culture medium consisting of anaerobic fungi were used at different inoculum ratios: 0% (F0), %1 (Fl), 5% (F2), 10% (F3), 15% (F4) and 20% (F5) (v/v). The buffer contained (per L): 1.0 g of NH4C1, 0.4 g of K2HP04.3H20, 0.2 g of MgC12.6H20, 0.08 g of CaC12.2H20, 10 ml of trace element solution and 10 ml of stock vitamin solution. Next, a stock trace element and vitamin solution were prepared and then adjusted in accordance with the procedure described by our previous study.

Triplicate samples were collected from the F0, Fl, F2, F3, F4 and F5 reactor sludges on the 5th, 10th, 20th and 30th and 40th days during the anaerobic digesters operation for total DNA isolation. The total DNAs were isolated from the 1 mL sludge samples with PureLink Genomic DNA extraction kits. A NanoDrop Spectrophotometer determined the concentration.

The V4-V5 hypervariable region of the 16S rRNA gene was reproduced with region-specific primers that were designed to contain an illumina adaptor and barcode sequences 518F-926R for bacteria and 518F-958R for archaea. A double round of PCR and dual indexing on a PTC-200 DNA Engine Peltier Thermal Cycler was employed to generate the sample amplicons. A picogreen assay and a Fluorometer (SpectraMax GeminiXPS 96-well plate reader) were utilized in order to adjust the concentrations of the amplicons. After determination of concentration, they were placed in the same amounts (-100 ng) into a single tube. The following technique to move away short undesirable fragments and clean the amplicon pool was applied. Firstly, the size of the pool was determined using AMPure beads (Beckman Coulter), and the product was characterized on a 1 % gel that was cut and purified with a Qiagen MiniElute PCR purification kit, after which the pool size was determined again with AMPure beads. PCR containing illumina adaptor-specific primers was then used to adjust the quality of the amplicon pool. Thereafter, the PCR products were run on a DNA 1000 chip for the Agilent 2100 Bioanalyzer. The last amplicon pool was accepted on the condition that long fragments would not be identified after PCR and if there were short fragments, the procedure was repeated again. The KAPA 454 library quantification kit (KAPA Biosciences) and the Applied Biosystems StepOnePlus Real-Time PCR system quantified the amplicon pool, which was determined to be clean. Finally, the illumina MiSeq paired-end 300 bp protocol was followed to procure the sequences. qPCR was applied for all anaerobic digesters to assess optimum fungi concentrations. The qPCR assay was performed in triplicate using an ABI 7500 SDS system with all digesters amplified using specific previous primer sets for anaerobic fungi (Forward primer: 5'-

GAGGAAGT AAAAGTCGT A AC AAGGTTTC-3 ' , Reverse primer: 5'- CAAATTC AC AAAGGGT AGGATGATTT-3 '). An optimum primer concentration of 350 nm and a final MgC12 concentration of 4mM were utilized for the qPCR under the following cycle conditions: denaturation at 94 ⁰ C for 4 min followed by 35 cycles of 96 ⁰ C for 45 s, 56 ⁰ C for 45 s, and 72 ⁰ C for 1 min with a subsequent final extension at 72 ⁰ C for 5 min. Detailed information on the qPCR analysis was already described.

The analysis for alkalinity, total solids (TS), and volatile solids (VS) were carried out appropriately with standard methods. Biogas production was measured with Milligas counters in both ASBRs. Gas chromatography with a flame ionization detector and Elite-FFAP column (30 m X 0.32 mm) measured the gas compositions and VFA concentrations. The set point of the oven was 100 °C and the maximum temperature of the inlet was 240 °C. In addition, helium gas was used as a carrier gas at a rate of 0.8 mL/min. Histogram, q-q plots and the Shapiro-Wilk' s test were performed to examine data normality. Variance homogeneity was also investigated by Levene's test. Oneway analysis of variance (ANOVA) or independent-samples t-test was utilised to check against the variations in biogas production and microbial community dynamics among various inoculum ratios of anaerobic fungi and F0. To facilitate multiple comparisons, Tukey's test was also applied. The values of tests were interpreted as mean and standard deviation. The applicability of microbial community and inoculum ratios was determined by Pearson's test in terms of correlation. Significant differences were detected at the p< 0.05 level.

Claims

CLAIMS A method for improvement of methane production with using anaerobic digesters of microalgae biomass characterized in that the said method comprises the following steps;

selecting of 4 isolated species of anaerobic rumen fungi with ratio of 30% Orpinomyces sp., 25% Piromyces sp. and 25 % Anaeromyces sp., 20% Neocallimastix frontalis to mixture adding these 4 species into the anaerobic digesters fed with microalgae Haematoccus pluvialis at different inoculums ratios: %1 (Fl), 5% (F2), 10% (F3), 15% (F4) and 20% (F5) (v/v).