Seminar Presentation in the research article entitled:

Bioenergy from wastewater resources: Nutrient removal,

productivity and settleability of indigenous algal-bacteria
polyculture, and effect of biomass composition variability on
methane production kinetics and anaerobic digestion energy PRESENTED BY:
balance
Suvranil Banerjee
ROLL NO: 002230601001
Article Detail: Academic Session : 2022-2023
Authors - Pavlo Bohutskyia,Ruth E. Spierlingb, Duc Phand, Anatoliy M.
Kopachevsky,Yuting Tangi, Michael J. Betenbaughi, Edward J. Bouwerd, Trygve Semester :1st
J. Lundquist
Name of Journal- Algal Reserach, Master of Technology – Energy Science and
Volume - 36, Technology
Period of Publication –December 2018,
Pages- 217-228 School of Energy Studies
Web Link: https://doi.org/10.1016/j.algal.2018.10.020
Jadavpur University
7th November, 2022
Abstract
 Algal-bacteria high-rate ponds represent an energy-efficient wastewater treatment approach and a source for affordable
and sustainable biomass feedstock for production of renewable energy through anaerobic digestion (AD). However, there
is still a need for more data on wastewater treatment efficiency, biomass productivity and settleability from outdoor
treatment facilities, as well as on impact of variability in biomass composition and digestibility on methane yield and
energy output.
 Hydraulic retention time (HRT) and wastewater quality fed into 30 m 2 raceway ponds had a major effect on algal-
bacteria polyculture productivity, settleability, phylogenetic and biochemical compositions, digestibility and methane
yield. While Micractinium, Scenedesmus, Chlorella, and pennate diatoms were always among the key species observed,
the gross productivity and 2-hour settleability during summer cultivation varied in the first-stage ponds treating primary
wastewater from 29 ± 5 to 54 ± 12 g AFDW/m2 /d and from 88 ± 8 to 94 ± 4% for HRT of 3 and 2 days, respectively. For
these conditions, the effluent had carbonaceous BOD of 3.4 or 3.3 mg/L and N total (mostly NO3 −-N) of 9.2 or 8.2 mg/L,
respectively.
 The second-stage algal ponds (HRT 3 days) showed lower productivity of 16 ± 6 g AFDW/m2 /d, settleability of 84 ± 11%,
and effluent csBOD 3.7 mg/L and Ntotal 0.8 mg/L. Biomass composition from different ponds was 34–38% protein, 18–
28% total lipids and 6–14% FAME. The methane yield varied about 30% with largest value of 0.34 ± 0.01 L/g VS and
showed a positive correlation with biomass lipid content (R2 = 0.93).
 First-order and pseudo-parallel first-order rate kinetic models exhibited a better fit for methane production (most R 2 >
0.993) than the modified Gompertz model. The variation in biomass composition led to significant differences in energy
output (varied by about 60%), Net Energy Ratios (ranged from 1.6 to 2.2) and Net Energy Efficiency (from 60% to 70%)
when projecting the energy balance for a large-scale continuous AD process with an optimal HRT of 20–30 days.

2
Renewable Biofuels:
• Renewable biofuels from algae are a promising alternative to fossil fuels. Although there has been considerable progress in
understanding of algal cell biology and advances in algae cultivation and processing techniques, algal biofuels are not yet
economical compared to fuels.
• large-scale production of algal-based biofuels requires massive amounts of fertilizers and water resources discrediting their
sustainability
• One potential solution to improve process economics could be the co-production of algal biofuels with high-value chemicals
biosynthesized by some algae
• Alternatively, treating wastewater with algae allows for the utilization of waste nutrients and water resources which improves
the sustainability, while producing the co-product of treated wastewater, improving economics.
• The other advantages of this technology include less complicated and costly process control, lower energy intensity of
wastewater treatment, and the generation of treated wastewater and algal-bacteria biomass as a feedstock for biofuel
production.

3
Algal-bacteria Biomass Generation with Wastewater Treatment :

• The coupled process of algal-bacteria biomass generation with wastewater treatment allows significant reduction in energy
input compared to independently operated conventional treatment processes .
• In situ exchange of O2 and CO2 between algae and heterotrophic bacteria enable degradation of organic contaminants by
bacteria and fixing of CO2 by algal cells, and algal growth allows for efficient removal of nutrients (N and P).
• Importantly, such processes avoid application of energy-intensive mechanical or bubble diffuser aeration processes that require
> 50% of the energy used in conventional Waste Water Treatment process .
• It has been estimated that algae-based wastewater treatment can provide a second-order energy return on investment of 1.44, or
a net energy saving (or production) of ~7 × 10 6 kW per year if applying to wastewater generated.

4
High Rate Wastewater Ponds :
• High-rate wastewater ponds support a robust algal-bacterial polyculture of indigenous species that constantly adapt to
fluctuating environmental parameters and wastewater quality as well as has demonstrated superior settleability characteristics .
• However, the unpredictable growth conditions (e.g. changing irradiance, hydraulic retention time, organic loading rate) may
lead to unreliable biomass productivity and reduced settleability, which are critically important for quality of treated wastewater
and a major factor for biomass production cost. Application of chemical coagulants or expensive polymeric flocculants is
required in the case of algal-bacterial polyculture with poor settleability, which may increase biomass ash content or drive up
treatment cost significantly.
• Also, the harvested biomass has inconsistent biochemical composition and do not accumulate the significant amounts of lipid
required for the production of biodiesel. Processing of such low-cost biomass requires a technology, like anaerobic digestion,
that does not rely on specific biomass composition but can convert all fractions of algal biomass including proteins,
carbohydrates and lipids to biofuel. digestion

5
Challenges of Algal-bacteria Wastewater Treatment followed by
Anaerobic Digestion:
• Uncertainty in performance of the process including biomass productivity, settleability and effluent quality. Importantly, there
is still insufficient data from outdoor pilot-scale cultivation facilities processing real wastewater, which makes process
optimization and analysis difficult.
• Inability to predict variation in methane yield and energy output from digestion of the algal-bacteria biomass due the high
variability in production and composition.
• Therefore, investigation into increasing polyculture productivity and settleability, and comprehension of the effect of biomass
variability on energy output would be highly valuable for advancing the technology of algal-bacteria WWT and bioenergy
production through anaerobic

6
Objective of the Study:
• The first objective of this study is to investigate the effect of pond hydraulic retention time (HRT) and operational scheme (one-
stage vs two-stage treatment) on biomass productivity, settleability and nutrient removal capability of the algal-bacterial
polyculture cultivated in hig hrate ponds fed with primary-treated municipal wastewater.
• The second objective is to evaluate how variability in the polyculture composition effects methane yield and methane
production rate using various kinetic models.
• Finally, the third objective is to examine the feasibility of anaerobic digestion for conversion of this biomass into bioenergy,
and to assess the importance of the composition variation on energy output through estimation of the energy balance for
continuous large-scale anaerobic digestion process.

7
Materials and Methods – Algae Cultivation Facility:
• Algal-bacteria biomass was generated using raceway algal ponds at California Polytechnic State University's Algae Field
Station located at the Water Resource Recovery Facility (WRFF) in San Luis Obispo California (35.2541° N, 120.6744° W).
• Algal ponds were supplied with clarified municipal primary wastewater from the City of San Luis Obispo (population 46,400 at
2014, 6 MGD WRFF).
• The influent wastewater quality is shown in this table:

8
Materials and Methods – Algae Cultivation Facility:
• Nine paddle-wheel mixed (average channel velocity 27 cm/s) open ponds (32.3 m 2 area, 0.3 m depth) were operated in triplicate as
follows. During the period of biomass cultivation and harvest, the ponds were operated as follows:

9
Materials and Methods – Algae Cultivation Facility:
• The Gamma ponds were operated on a 2-day hydraulic residence time (HRT) and diluted continuously with clarified municipal
wastewater; the Beta ponds were operated on a 3-day HRT and diluted continuously with clarified municipal wastewater, and
the Alpha ponds were operated on a 3-day HRT and diluted continuously with clarified effluent from the Beta ponds.
• Pond depth was maintained using 4-inch overflow standpipes. pH in the ponds was maintained between a pH of 8.3 and 8.5 by
introducing CO2 using an automated system with pH probe, control system and data logger solenoid, and a 50 lb. compressed
gas cylinder (CO2 99.5% purity, Airgas).
• All ponds contained a spontaneous native algal polyculture with natural bioflocculation properties. Biomass was harvested by
gravity from the bottom of 60° up-flow tube settlers (0.085 m 2 cross-section area) receiving a flow of 2.25 L/min for the Alpha
and Beta ponds and 0.8 L/min for the Gamma ponds.
• Algal biomass used for anaerobic digestion experiments was collected on a single day (07/13/ 2012) from the bottom of the
tube settlers; therefore, data from 6/20/ 12–07/27/12 was used to evaluate biomass productivity and to characterize composition
of the algal polyculture. The samples of harvested biomass were centrifuged at 4500 rpm for 5 min. Then, the liquid phase was
separated, and samples were stored in a −20 °C freezer until use.

10
Biomethane potential (BMP) test and theoretical methane
production:
• The measurement of biogas and methane were performed in two biological replicates. The error bars represent the measurement
standard deviation. The minor modifications to the BMP test and procedure for biogas analysis were described earlier [25]. The
theoretical yield of biogas was estimated using the modified Buswell equation:

11
Kinetic Model and Statistical Indicators:
• 1st order model: This simplistic model may provide a proper description for exponential production of biogas and methane.

• Pseudo-parallel 1st-order kinetic model:

This model may provide a better fit for experimental data for biogas and methane production from biomass
containing a readily biodegradable portion and a portion resistant to biodegradation like an algal cell wall

12
Kinetic Model and Statistical Indicators:
• Modified Gompertz model:
This model is superior for fitting experimental biogas and methane production data with significant lag phase due to presence of a
significant amount of biologically resistant or toxic materials.

The model's fitting performance was evaluated through visual comparison of observed data and model predictions as well as through
calculation of Root Mean Square Deviation (RMSD) and coefficient of determination R 2.

13
Analytic Technique used:
• During the experimental period (June 2013 to April 2014) data were collected on productivity, and settleability of the algae
biomass as well as the influent and effluent water quality to measure the effectiveness of wastewater treatment. Grab samples
were collected weekly between the hours of 7–8 am upstream of the overflow standpipes.
• Gross and net algal areal productivity, as gram of biomass ash free dry weight per m2 per day (AFDW/m 2 /d), were determined
weekly using APHA standard methods 2540 D and 2540 E . Biomass net productivity was determined by subtracting the
influent solids from the ponds solids, while the gross productivity was determined from the ponds solids alone.
• Alkalinity, ammonia, nitrate, nitrite, total Kjeldahl nitrogen (TKN) and biochemical oxygen demand (BOD) were also
measured weekly using APHA standard methods. Microscopy was performed weekly using an Olympus Model CX41RF phase
contrast microscope and Infinity 2-1C cameras with Infinity Analyze 2.0 software to identify algal genera, and perform grazer
counts as well as to microscopically analyze the floc structure.
• Settling was measured weekly using gravity settling in Imhoff cones and measuring supernatant AFDW at 2 and 24 h of
settling.

14
Analytic Technique used:
• Pond water temperature, dissolved oxygen, and pH were logged and stored hourly in data loggers (Apex, Neptune Systems).
• All probes were calibrated weekly.
• Hourly weather data were obtained through the California Irrigation Management Information System (CIMIS) weather station
52 (located approximately 3 miles from the growing location) on the Cal Poly Campus.
• The algal biomass for the BMP test was assayed for total solids (TS) and volatile solids (VS) according to Standard Methods
[28]; the COD was measured using a HACH COD TNT plus Kit; the total lipid content was determined using the Bligh and
Dyer method [29].
• The protocol for fatty acid methyl ester (FAME) profiling of algal lipids was previously reported . The CHN elemental
composition of lyophilized algal biomass was determined by Micro Analysis, Inc. with a %CHN Analyzer.

15
Results & Discussions:
Wastewater polyculture cultivation parameters:

16
Results & Discussions:

17
Results & Discussions:

18
Results & Discussions:

Specific biogas (A) and methane (B) yields from algal-bacterial

biomass from different ponds (All data is shown as mean of two
biological replicates ± SD). Lines show predicted yields derived
from the 1st-order rate (dotted), pseudo-parallel 1st-order rate
(dash-dot) and the modified Gompertz kinetic models (dash-dot-
dot).

19
Results & Discussions:

20
Results & Discussions:

21
Results & Discussions:

Theoretically predicted (A) and experimentally observed (B)

methane yields from algal-bacterial biomass with various lipid
content (methane yield is shown as mean of two replicates ± SD
and lipid content as mean of three replicates ± SD)

22
Results & Discussions:

Energy analysis of Anaerobic Digestion process. (A)

Energy Output; (B) Specific energy output per m3 of
AD bioreactor; (C) Specific energy output per ton of
algal biomass volatile solids; (D) Net Energy Ratio;
(E) AD process Net Energy Efficiency; (E) Net
Energy Recovery as energy output per high heating
value of processed biomass.

23
Conclusions:
• Wastewater treatment efficiency, biomass productivity, settleability, composition, digestibility and methane potential vary in
response to environmental conditions. However, these process characteristics may be somewhat controlled and improved
through optimization of the high-rate algal pond operation parameters such as hydraulic retention time (HRT) and process
scheme (one-stage vs. twostage process).

• A short HRT of 2 days resulted in the highest biomass productivity (54 ± 12 g AFDW/m 2 /d) and better settling properties (2-
hour solid removal of 94 ± 3%) of the algal-bacteria biomass compared to a HRT of 3 days. The two-stage process provides
higher nitrogen removal efficiency than the one-stage treatment. Variability in biomass composition caused a significant
variation in methane yield (up to 30%, from 267 to 340 mL CH 4 gVS−1) and strongly impacted energy output and energy
production efficiency.

• Indeed, the total energy output may vary nearly 40% in response to changing compositions of algal-bacteria biomass.
Importantly, the energy balance showed Net Energy Ratios ranging from 1.5 to 2.2 and Net Energy Efficiencies ranging from
about 60% to 68% (at optimal HRT of 20–30 days) for all biomass harvests despite variations in biomass composition and
energy output.

• However, at this HRT the energy output represented only 15–20% of the biomass energy (as HHV). These low percentages of
accessed energy output indicates that future research should address the low digestibility of algal biomass (methane yields was
only 40–50% of theoretical) to improve energy recovery from algal-bacteria biomass and enhance i

24
References:
1. Y. Tang, J.N. Rosenberg, P. Bohutskyi, G. Yu, M.J. Betenbaugh, F. Wang, Microalgae as a feedstock for biofuel precursors and value-added products: green fuels and
golden opportunities, BioResources 11 (2015).
2. T.J. Lundquist, I.C. Woertz, N.W.T. Quinn, J.R. Benemann, A Realistic Technology and Engineering Assessment of Algae Biofuel Production, UC Berkeley, CA, 2010, p.
178.
3. P. Bohutskyi, D.C. Kligerman, N. Byers, L.K. Nasr, C. Cua, S. Chow, C. Su, Y. Tang, M.J. Betenbaugh, E.J. Bouwer, Effects of inoculum size, light intensity, and dose of
anaerobic digestion centrate on growth and productivity of Chlorella and Scenedesmus microalgae and their poly-culture in primary and secondary wastewater, Algal Res.
19 (2016) 278–290.
4. P. Bohutskyi, K. Liu, L.K. Nasr, N. Byers, J.N. Rosenberg, G.A. Oyler, M.J. Betenbaugh, E.J. Bouwer, Bioprospecting of microalgae for integrated biomass production
and phytoremediation of unsterilized wastewater and anaerobic digestion centrate, Appl. Microbiol. Biotechnol. 99 (2015) 6139–6154.
5. I. Woertz, A. Feffer, T. Lundquist, Y. Nelson, Algae grown on dairy and municipal wastewater for simultaneous nutrient removal and lipid production for biofuel
feedstock, J. Environ. Eng. 135 (2009) 1115–1122.
6. P. Bohutskyi, L.A. Kucek, E. Hill, G.E. Pinchuk, S.G. Mundree, A.S. Beliaev, Conversion of stranded waste-stream carbon and nutrients into value-added products via
metabolically coupled binary heterotroph-photoautotroph system, Bioresour. Technol. 269 (2018) 210–220.
7. P. Bohutskyi, S. Chow, B. Ketter, C. Fung Shek, D. Yacar, Y. Tang, M. Zivojnovich, M.J. Betenbaugh, E.J. Bouwer, Phytoremediation of agriculture runoff by filamentous
algae poly-culture for biomethane production, and nutrient recovery for secondary cultivation of lipid generating microalgae, Bioresour. Technol. 222 (2016) 294–308.
8. R.J. Craggs, S. Heubeck, T.J. Lundquist, J.R. Benemann, Algal biofuels from wastewater treatment high rate algal ponds, Water Sci. Technol. 63 (2011).
9. F. Passos, R. Gutiérrez, E. Uggetti, M. Garfí, J. García, I. Ferrer, Towards energy neutral microalgae-based wastewater treatment plants, Algal Res. 28 (2017) 235–243.
10. G. Tchobanoglous, F.L. Burton, H.D. Stensel, Wastewater Engineering: Treatment and Reuse, Metcalf & Eddy Inc. 4 ed., McGraw-Hill Science, Boston, 2003.
11. M. Beal, A.S. Stillwell, C.W. King, S.M. Cohen, H. Berberoglu, R.P. Bhattarai, R.L. Connelly, M.E. Webber, R.E. Hebner, Energy return on investment for algal biofuel
production coupled with wastewater treatment, Water Environ. Res. 84 (2012) 692–710.
12. F. Passos, R. Gutiérrez, D. Brockmann, J.-P. Steyer, J. García, I. Ferrer, Microalgae production in wastewater treatment systems, anaerobic digestion and modelling using
ADM1, Algal Res. 10 (2015) 55–63.
13. R. Gutiérrez, I. Ferrer, A. González-Molina, H. Salvadó, J. García, E. Uggetti, Microalgae recycling improves biomass recovery from wastewater treatment high rate algal
ponds, Water Res. 106 (2016) 539–549.
14. G. Quijano, J.S. Arcila, G. Buitrón, Microalgal-bacterial aggregates: applications and perspectives for wastewater treatment, Biotechnol. Adv. 35 (2017) 772–781.

25
Thank you
Suvranil Banerjee

suvranil.banerjee@gmail.com