Bio-Refining of Carbohydrate-Rich Food Waste for Biofuels

The global dependence on finite fossil fuel-derived energy is of serious concern given the predicted population increase. The potential of carbohydrate-rich food waste (CRFW) for biofuel and biogas production was therefore investigated using a novel integrated bio-refinery approach. In this approach, hydrolyzed CRFW from three different conditions was used for Rhodotorulla glutinis cultivation to produce biolipids, whilst residual solids after hydrolysis were characterized for methane recovery potential via anaerobic digestion. Initially, CRFW was hydrolysed using thermal- (Th), chemical- (Ch) and Th-Ch combined hydrolysis (TCh), with the CRFW-leachate serving as a control (Pcon). Excessive foaming led to the loss of TCh cultures, while day-7 biomass yields were similar (3.4–3.6 g dry weight (DW)/L) for the remaining treatments. Total fatty acid methyl ester (FAME) content of R. glutinis cultivated on CRFW hydrolysates were relatively low (~6.5%) but quality parameters (i.e., cetane number, density, viscosity and higher heating values) of biomass extracted biodiesel complied with ASTM standards. Despite low theoretical RS-derived methane potential, further research under optimised and scaled conditions will reveal the potential of this approach for the bio-refining of CRFW for energy recovery and value-added co-product production.

Materials and Methods

R. glutinis Culturing and Acclimatization

A pure culture of R. glutinis FRR-4522, an isolate from dairy produce was supplied by the Commonwealth Scientific and Industrial Research Organisation (CSIRO), Australia. R. glutinis was subcultured and maintained on 2% agar plates prepared with Yeast Malt (YM) medium (3 g/L yeast extract; 3 g/L malt extract; 5 g/L casein peptone; 10 g/L dextrose). Cultivation of R. glutinis for CRFW conversion consisted of 350 mL YM broth and 100 mL hydrolysed CRFW at a pH of 4 ± 0.01 in 1 L Erlenmeyer flasks. Seed cultures were maintained in YM liquid medium on a rotary shaker at 95 rpm for a minimum of 5 days (to a maximum of 7 days) at 28 ± 2 °C for biomass enrichment (≥5 × 108 cells/mL) prior to inoculations. The axenic state of seed cultures was confirmed by light microscopy at every stage of the cultivation process.

Bio-Refining of Carbohydrate-Rich Food Waste

CRFW was a mixture of bread (25.9%), oats (29.8%), cooked pasta (27.7%) and boiled rice (16.6%) which was homogenised (<1 cm³ particle size) by grinding. The homogenized CFRW was characterized for various parameters before and after pre-treatment.

Hydrolysis of CRFW

Feed slurry was prepared by adding 250 g CRFW to 1 L−1 of deionised (DI) water and refrigerated at 4 °C for 24 h to allow passive leaching of nutrients, whilst minimising microbial growth. The resultant mixtures represented the physical control (PCon) throughout the study (i.e., 4 °C 24 h-leachate). The slurry was hydrolyzed using three different approaches:

(i)                 Chemical hydrolysis (Ch)—acidic hydrolysis of the CRFW slurry at a pH of 3 ± 0.01 adjusted with 2 M HCl for 24 h at room temperature (25 °C);

(ii)               Thermal hydrolysis (Th)—autoclaving of the CRFW slurry using a standard moisture-heat procedure of 121 °C at 1013.25 hPa for 30 min;

(iii)             Thermochemical hydrolysis (TCh)—a combined double hydrolysing procedure, where the chemical hydrolysis of the slurry preceded the thermal hydrolysis.

Following hydrolysis, hydrolysates were centrifuged at 15,900 × g for 20 min with 10 min deceleration at 4 °C. The supernatants (hydrolysates) were decanted into sterilised 2 L Simax bottles for R. glutinis enrichment. Prior to inoculation with R. glutinis, total carbohydrate was determined for each hydrolysate and pH was adjusted to 4 with addition of either 1 M HCl or NaOH. pH probes were cleaned and sterilized with an ethanol wash before each measurement. Residual solids (RS) were characterized for biogas and corresponding bioenergy (methane) potential was estimated using Buswell equation.

Cultivation of R. Glutinis in Hydrolysates for Bio-Product Development

500 mL of undiluted hydrolysates of each treatment were inoculated with R. glutinis ~ 1.0 × 109 cells in sterile 1 L Simax reagent bottles with a modified polypropylene cap for aeration and ventilation. The culture bottles were maintained with a continuous airflow of 130 ± 0.7 L/h at 28 ± 2 °C for 7 days. Filtered air 0.45 µm syringe filter was supplied via a 2 mL glass pipette connected to a Precision Air Pump 7500 and venting occurred via pipette tips filled with cotton wool. Although photo periods and light intensities are not limiting factors for lipid accumulation in saprophytic microorganisms, R. glutinis experiments were conducted under a 12:12 light:dark cycle as cultivation was carried out in the algal culture room. Growth of R. glutinis was monitored daily by cell count and measuring dry weight (DW) gravimetrically at days 0, 1, 3, 5 and 7. Total carbohydrates were measured using the UV-sulphuric acid method at days 1, 4 and 7 and the system pH was measuered at days 0 and 7. Experiments were performed in triplicates and all sampling occurred in a sterile laminar flow cabinet to minimise contamination. Rhodotorula glutinis biomass was harvested from hydrolysates by centrifugation, was freeze-dried and extracted for transesterification into fatty acid methyl esters (FAME). In brief, ~ 30 ± 4 mg lyophilised R. glutinis biomass was measured into 8 mL Teflon capped glass vials. An equal volume (50 mg) of 0.5 mm zirconium oxide beads was added to the biomass, serving as abrasive particles for mechanical cell wall disruption. 2 mL of freshly prepared methylation mixture, HPLC-grade methanol and acetyl chloride (95:5 v/v), was added and supplemented with 300 µL internal standard solution. The methylated-biomass mixture was vortexed at 2,200 rpm for 30 s at 30 s intervals. Once homogenised, vials were placed into a block heater at 100 °C for 60 min to facilitate transesterification of fatty acids to methyl esters. Heated samples were allowed to cool to room temperature, before adding 1 mL non-polar organic solvent (0.01% BHT w/w HPLC-Hexane) and mixing by inversion. Sample vials were replaced into the warm block heater for 60 s, enabling the formation of a miscible mixture. Once cooled, addition of 1 mL UltraPure water separated the two phases. The upper FAME-hexane mixture was collected and filtered through a 0.2 µm PTFE filter (Agilent) prior to its injection into GC vials. Gas chromatography determination of FAME profiles were carried out on an Agilent 7890 GC with flame ionisation detector (FID) and Electron Ionisation (EI) Turbo Mass Spectrometer (MS). A DB-23 column with cyanopropyl stationary phase (60 m × 0.55 mm id × 0.15 µm) with He2 (g) injection (33 cm s−1 at 50 °C) at 230 kPa was used for sample separation. Constant inlet temperatures for injector and FID were maintained at 150 °C and 250 °C with split injection of 1/50, respectively. Oven and column temperature settings were based on instrumental protocols by the manufacturer. Unknown FAME profiles were determined via comparison of peaks and retention times of pure external standards, whilst the recovery potential was corrected using a factor derived from the known concentrations of nonadecanoic acid (C19:0) used as internal standards.

Characterization of CRFW

25 ± 2 g CRFW and RS were freeze-dried over 48 h. The subsequent lyophilised products were then homogenised in pre-dried (105 ± 2 °C for 4 h) porcelain mortars into a fine powder, which were passed through a 1 mm2 stainless steel mesh to exclude large fragments for CHNS-O analysis. Total and volatile solids (TS and VS, [25]) for CRFW and RS were measured and moisture contents were back calculated (moisture% = 100−TS).

Calculating Bio-diesel and Bio-energy Potential of Bio-refined CRFW

The potential physicochemical properties of biodiesel were calculated based on levels of (un)saturation and carbon length of the individual FAMEs using established models. For this study, the values of cetane (CN), kinematic viscosity (υ), density (ρ), and higher heating values (HHVB) of biodiesel were calculated using Equations 1–4, respectively.

CN= ∑i(−7.8+0.302 ×Mi−20 ×N)                           (1)

ln(υi)= −12.503+2.496 ×ln(Mi)−0.178×N              (2)

ρi=0.8463+4.9Mi+0.0118×N                                  (3)

HHVB(i)=46.19+1794Mi−0.21×N                          (4)

where Ni, Mi, and Di represent the percentage, molecular weight and number of double bonds in the respective ith FAME.

Based on the elemental analysis, theoretical biogas (Bth) yields of RS were calculated.

Results

Characteristics of Hydrolysates from Pre-treated CRFW

Hydrolysed CRFW had a pH range of 3.77–5.79 based on the different pre-treatments. Therefore, HCl or NaOH were used to reduce/increase the pH of hydrolysates for cultivation of R. glutinis. Buffering capacities of the hydrolysates differed, requiring variable volumes and concentrations of acids/bases. Compared to Pcon leachate total carbohydrate content (~45 mg eq-Gluc.g−1), carbohydrate release was higher (~25%; i.e., ~65 mg eq-Gluc.g−1) in the thermal hydrolysis leading to a change in leachate pH from 4.01 to 5.79, whereas, the Ch or TCh pre-treatments did not improve total carbohydrate release from CRFW. These results are in contrast to acid-hydrolysed fruit and vegetable waste which released more carbohydrates than when subjected to alkali or Th hydrolysis.

Growth of R. glutinis in CRFW Hydrolysates

Whilst final cell concentrations were similar for Th-, Ch- and Pcon-treated CRFW, growth responses of R. glutinis in the early stages of cultivation varied. Foam formation occurred in the culture based on TCh-hydrolysate causing >80% loss of of R. glutinis cells and leading to termination on day 4. Foaming could be due to high aeration and inhibited fermentation processes led to protein degradation in the systems. Culture growth was accompanied by a shift in system pH from 4 to 7.62 ± 0.17 and 6.36 ± 0.02 in Pcon and Th cultures, respectively. Growth of R. glutinis was comparable between Th and Ch hydrolysates, peaking within 2 days. However, Ch hydrolysates better supported the growth of R. glutinis and achieved maximum cell counts within 24 h. On the other hand, Th hydrolysates contained more carbohydrates, which were expected to provide higher R. glutinis biomass.

All systems reached R. glutinis densities of 2.60–2.67 × 108 cells within 4 days are remained stable during stationary phase until day 7. In terms of biomass yield, ~3.42–3.61 g DWbiomass L−1 was measured from Pcon (3.61 ± 0.25 g DWbiomass L−1), Ch (3.42 ± 0.41g DWbiomass L−1) and Th (3.53 ± 0.94 g DWbiomass L−1) hydrolysates and no significant differences were observed between the cultivation media. Carbohydrate analysis at harvest indicated that secondary or continuous cultivation of R. glutinis over a longer period may be possible, as ~29% (from Pcon and TCh hydrolysates) and ~24% (from Ch hydrolysate) of carbohydrates were assimilated within 7 days.

FAME Profile of R. glutinis Cultivated in CRFW Hydrolysates

The total fatty acid (TotFA) contents and FAME profiles of R. glutinis enriched in CRFW hydrolysates. TotFA contents were 41.79 ± 12.6, 38.05 ± 8.2 and 65.56 ± 30.9 mgTotFA.g−1DWbiomass for Pcon-, Ch- and Th-cultivated R. glutinis, respectively. The FAME profiles of CRFW hydrolysate-cultivated R. glutinis. Mono- and poly-unsaturated fatty acids (MUFA and PUFA, respectively) accounted for 61%–67%, while saturated fatty acids (SFA) were only 32%–39% of the biomass. While general fatty acid profiles were similar, the percentage distributions of fatty acids differed. Highest MUFA and PUFA contents were achieved in R. glutinis cultivated in Th and PCon hydrolysates, respectively. Overall, the FAME profiles obtained from all R. glutinis cultures are considered to be ideal precursors for the biofuel industry.

Biodiesel Potential of R. glutinis Enriched from CRFW Hydrolysates

Biodiesel properties calculated based on FAME profiles of R. glutinis biomass cultivated using CRFW-hydrolysates yielded a density of ρ ~ 0.79 to 0.85 g cm−3 and mean HHVs of 35.79–39.36 MJ.kg−1. Calculated CNs were comparable with the biodiesel standard (ASTM D6751-02), i.e., CN > 51, but higher than demanded standards for fossil fuel-derived diesel (ASTM D975; CN ~ 40–50). In addition, the calculated viscosity (υ) for the biomass was within the standard limits (υ −1.3–4.1 mm2S−1) for fossil fuel-derived diesel.

Characteristics of Residual Solids and Bioenergy Potential

The elemental composition of RS from hydrolysis pre-treatments. Hydrolysis pre-treatment had no large effect on C, O, N, H, and S contents. Also, high VS (~96%–97%) and TS contents suggest that the RS is suitable for bioconversion. For anaerobic digestion, optimal C/N ratios of 27–32 were recommended in order to avoid any build-up of ammonia and associated toxicity effects in reactors that subsequently affect CH4 yield. In the context of the proposed bio-refinery concept, the actual CH₄ potential of recycled RS from the hydrolysates of CRFW will require experimental validation.

Conclusions

The proposed integrated yeast fermentation and anaerobic digestion process appears to be a promising approach for the bio-refining of CRFW for biolipids and bioenergy production. Biomass yields, total fatty acid content and profile, as well as calculated important diesel characteristics, render R. glutinis a suitable alternative to green microalgal biodiesel when cultivated on CRFW hydrolysates under controlled conditions, potentially requiring a fraction of the cultivation foot print. Ch hydrolysates provided better biomass yields, however biodiesel properties and FAME yields were higher with the Th hyrolysate cultivates. In addition, solid residue from the Th pre-treatment was estimated with higher methane potential. Therefore, Th hydrolysis of CRFW followed by R. glutinis cultivation under buffered condition should be recommended for further investigation.

Further research is required with regards to outdoor cultivation suitability and competitiveness for CRFW recycling. In addition, unutilized sugars from R. glutinis fermentation could be potentially re-cycled within the fermentation system or anaerobically digested for making this technology more energy efficient and economically viable. Furthermore, heat and power generated from biogas combustion could be potentially re-routed for hydrolysis pre-treatments which would benefit the proposed integrated bio-refining approach.

Citation:

Nguyen Hao, H.T., Karthikeyan, O.P. and Heimann, K., 2015. Bio-refining of carbohydrate-rich food waste for biofuels. Energies, 8(7), pp.6350-6364.