Semi-closed Cycle Process For Hydrothermal Transformation Of Filamentous Fungi And Microalgae

Microalgae-based bio-refinery systems are facing a series of major challenges that have hindered their industrialization of microalgae-based bioenergy.Microalgae biomass of hydrothermal conversion technology,such as hydrothermal carbonization（HTC）,hydrothermal liquefaction（HTL）,and sequential hydrothermal liquefaction（SEQHTL）has been intensity investigated.The advantages of hydrothermal conversion technology are that it uses wet biomass directly,has a relatively high production yield,requires no additional catalyst,and convert the whole microalgal biomass（lipid,protein,carbohydrate,and pigment）into different products by finely-tuned operating conditions,which broadens the potential industrial applications of microalgae.However,hydrothermal conversion process produces too much byproduct aqueous phase（AP）,aggravating the processing burden because of requiring high cost of wastewater treatment.In this study,a novel fungi-assisted microalgae-based hydrothermal conversion biofuel system was developed,in which to harvest microalgae,product biofuel,treat AP by microalgae and fungi cultivation and recovery nutrients in sustainable way.（1）The study has systematically evaluated and compared fungal spore-assisted（FSA）and fungal pellet-assisted（FPA）microalgal harvesting methods.In this study,harvesting Chlorella sp.cells by co-culture with Aspergillus fumigatus spores or pellets was compared.Temperature,glucose concentration,p H and fungi:algae ratio were the critical parameters for harvesting efficiency.The highest flocculation efficiency（99%）of FSA method was achieved in 28 h at 40?C,160 rpm,5 g glucose/L and 1.1×10⁴cells/m L（spore）.FPA method can harvest 98.26%algae cells in 2.5 h at 34?C,160 rpm,p H 4.0 with the fungi:algae ratio of 1:2.The carbon input for FPA is only half of that for FSA.FPA takes less time and needs less glucose input compared with FSA and may be more promising to be further developed as an effective microalgae bioflocculation method.（2）In this study,mono-,co-,and stepwise-culture of fungi（Aspergillus fumigatus）and microalgae（Chlorella sp.）on AP from HTC of the same fungi-microalgae strains was investigated.The results showed that the stepwise culture for six days（first fungi and then microalgae cultivation）in 5%AP achieved the biomass yield of 0.63 g/L and best performance for the removal of TN（32.91%）,TP（95.3%）,COD（64.38%）,NH₄-N（26.97%）,and organic components,probably due to the strong detoxification capability of the fungi,which improved microalgae growth.The microalgae and fungi biomass harvested from 5%AP can be sustainable feedstocks for fuel production.This study demonstrated that utilizing the AP for the cultivation of fungi and microalgae has the potential to close the loop in the biomass hydrothermal carbonization approach.（3）In this study,filamentous fungus was used to treat organics of AP produced from the HTL process（temperature of 300°C with a residence time of 30 mins）of chlorella powder.Our study shows that the culture of filamentous fungus（Trichoderma atrophy and Aspergillus fumigatus）had some biomass growth and COD removal when the concentration of HTL-AP increased from 1%to 10%.The optimal culture conditions of fungi were 1*10³ spores/m L,1g/L glucose,40mg/L Mg²⁺and initial p H5.0.At the optimal culture conditions,fungal biomass was improved.In combination with Excitation Emission Matrix Spectra（EEMS）and parallel factor analysis,it was found that the organic compounds in HTL-AP distributed in the region of Marine acid and humic acid,with a total of four components:C1-C3 was humic acid,and C4 was Marine humic acid.Order of removal rate of different components in 10%HTL-AP with glucose and Mg²⁺by Trichoderma atrophy culture:C4（54.00%）>C1（40.62%）>C2（33.60%）>C3（25.04%）.In the 10%HTL-AP,removal rates of C4 and C1 reduced from 54%to 26.41%and 40.64%to 33.67%,respectively.GC/MS analysis showed that Trichoderma atrophia culture could completely remove amines,pyridine derivatives and organic acids from HTL-AP with or without additional glucose and Mg²⁺,while alcohol amines and alcohols were partially utilized.In 10%HTL-AP(with glucose and Mg²⁺,Trichoderma atrophia almost completely utilized glucose and its derivatives,amines,and other substances,which explained the better growth of Trichoderma atrophia,but the addition of glucose and Mg²⁺substances in the wastewater did not have a significant effect on the removal of organic matter.（4）The objective of this study was to integrate AP recirculation and fungal-microalgal cultivation with the SEQ-HTL to simultaneously improve bio-oil yield,energy recovery,and nutrient recovery.Interestingly,the SI-AP showed excellent biodegradability,and the complementary cultivation of fungi and algae has tremendous potential to recover nutrients.For example,0.96 g/L fungi biomass and 87.71%chemical oxygen demand（COD）removal rate were obtained by fungi cultivation in SI-AP,and 1.35 g/L biomass and 63.80%ammonia nitrogen（NH₄-N）removal rate were received by microalgae cultivation in SI-AP.The difference of dissolved organic matter in SI-AP and the AP from conventional HTL（HTL-AP,inhibited microalgae significantly）pointed out unsaturated substances and lignins/carboxylic-rich alicyclic molecules substances might be the primary factor inhibiting microalgae growth.During SII-AP recirculation,the yields of bio-oil（SII-Oil）and hydrochar（SII-Char）increased from 16.00%to 28.65%and 2.93%to 5.66%.32.96～53.08%C and 15.93～24.00%N in SII-AP were recovered and transferred into SII-Oil and SII-Char.A series of characterizations showed that the increased bio-oil formation was mainly the transformation of amines,glycerol,alcohols,acetic acid,and amino acid fragments from the AP into the oil phase via Maillard reaction,Michael addition reaction and acylation.Overall,the novel strategy could improve the economic feasibility of processing high-protein algae biomass using the SEQ-HTL technique and is promising to the industrial applications of microalgae.This study brings to light a new potential and efficient microalgae biofuel system that combines fuel production,wastewater treatment,nutrients recovery,and microalgae harvesting,which worth further investigation.